WorldWideScience

Sample records for reveals high sequence

  1. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

    2017-11-06

    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

  2. Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus.

    Directory of Open Access Journals (Sweden)

    Kui Lin

    2014-01-01

    Full Text Available Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

  3. Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

    Science.gov (United States)

    Conway, Tyrrell; Creecy, James P; Maddox, Scott M; Grissom, Joe E; Conkle, Trevor L; Shadid, Tyler M; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada; Wanner, Barry L

    2014-07-08

    We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are

  4. Sequencing of 50 human exomes reveals adaptation to high altitude

    DEFF Research Database (Denmark)

    Yi, Xin; Liang, Yu; Huerta-Sanchez, Emilia

    2010-01-01

    Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which repres...... in genetic adaptation to high altitude.......Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which...... represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency...

  5. Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

    Directory of Open Access Journals (Sweden)

    Ouyang Shu

    2005-09-01

    Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

  6. High-throughput sequencing of the B-cell receptor in African Burkitt lymphoma reveals clues to pathogenesis.

    Science.gov (United States)

    Lombardo, Katharine A; Coffey, David G; Morales, Alicia J; Carlson, Christopher S; Towlerton, Andrea M H; Gerdts, Sarah E; Nkrumah, Francis K; Neequaye, Janet; Biggar, Robert J; Orem, Jackson; Casper, Corey; Mbulaiteye, Sam M; Bhatia, Kishor G; Warren, Edus H

    2017-03-28

    Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy ( IGH ) and light chain ( IGK / IGL ) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK / IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK / IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

  7. High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    Directory of Open Access Journals (Sweden)

    Weiguo Hou

    2018-05-01

    Full Text Available Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length encoding the cyanophage gp23 major capsid protein (MCP. Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92% belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

  8. Accurate and High-Coverage Immune Repertoire Sequencing Reveals Characteristics of Antibody Repertoire Diversification in Young Children with Malaria

    Science.gov (United States)

    Jiang, Ning

    Accurately measuring the immune repertoire sequence composition, diversity, and abundance is important in studying repertoire response in infections, vaccinations, and cancer immunology. Using molecular identifiers (MIDs) to tag mRNA molecules is an effective method in improving the accuracy of immune repertoire sequencing (IR-seq). However, it is still difficult to use IR-seq on small amount of clinical samples to achieve a high coverage of the repertoire diversities. This is especially challenging in studying infections and vaccinations where B cell subpopulations with fewer cells, such as memory B cells or plasmablasts, are often of great interest to study somatic mutation patterns and diversity changes. Here, we describe an approach of IR-seq based on the use of MIDs in combination with a clustering method that can reveal more than 80% of the antibody diversity in a sample and can be applied to as few as 1,000 B cells. We applied this to study the antibody repertoires of young children before and during an acute malaria infection. We discovered unexpectedly high levels of somatic hypermutation (SHM) in infants and revealed characteristics of antibody repertoire development in young children that would have a profound impact on immunization in children.

  9. Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

    DEFF Research Database (Denmark)

    Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis

    2018-01-01

    In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis...... specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that......, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing...

  10. Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing.

    Science.gov (United States)

    Zhang, Yanying; Yang, Qingsong; Ling, Juan; Van Nostrand, Joy D; Shi, Zhou; Zhou, Jizhong; Dong, Junde

    2017-01-01

    Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata , Avicennia marina , and Ceriops tagal , was undertaken using high - throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

  11. Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Yanying Zhang

    2017-10-01

    Full Text Available Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata, Avicennia marina, and Ceriops tagal, was undertaken using high-throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

  12. Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

    Science.gov (United States)

    Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

  13. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

    Science.gov (United States)

    Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

  14. MicroRNA sequence motifs reveal asymmetry between the stem arms

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Havgaard, Jakob Hull; Ensterö, M.

    2006-01-01

    The processing of micro RNAs (miRNAs) from their stemloop precursor have revealed asymmetry in the processing of the mature and its star sequence. Furthermore, the miRNA processing system between organism differ. To assess this at the sequence level we have investigated mature miRNAs in their gen......The processing of micro RNAs (miRNAs) from their stemloop precursor have revealed asymmetry in the processing of the mature and its star sequence. Furthermore, the miRNA processing system between organism differ. To assess this at the sequence level we have investigated mature mi...

  15. Bacterial Pathogens and Community Composition in Advanced Sewage Treatment Systems Revealed by Metagenomics Analysis Based on High-Throughput Sequencing

    Science.gov (United States)

    Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

    2015-01-01

    This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence. PMID:25938416

  16. High quality maize centromere 10 sequence reveals evidence of frequent recombination events

    Directory of Open Access Journals (Sweden)

    Thomas Kai Wolfgruber

    2016-03-01

    Full Text Available The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR have presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 x 10-6 and 5 x 10-5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb of the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length centromeric retrotransposons from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. This repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to facilitate the repair of frequent DSBs in centromeres.

  17. Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

    Directory of Open Access Journals (Sweden)

    Wesley Morovic

    2016-11-01

    Full Text Available Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation well defined strains, encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species, and determined their content using plate counting, targeted genotyping. Additionally, strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS of the 16S rRNA sequence using paired-end 2x250bp Illumina MiSeq technology. Using multiple methods, we tested the hypothesis that products do contain the quantitative amount of labeled bacteria, and qualitative list of labeled microbial species. We found that 17 samples (33% were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58% of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to qualitatively determine the correct taxonomy and quantitatively ascertain the relative amounts of mixed microbial populations in commercial probiotic products.

  18. Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

    Science.gov (United States)

    Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it.

  19. Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

    Science.gov (United States)

    Calmels, Nadège; Greff, Géraldine; Obringer, Cathy; Kempf, Nadine; Gasnier, Claire; Tarabeux, Julien; Miguet, Marguerite; Baujat, Geneviève; Bessis, Didier; Bretones, Patricia; Cavau, Anne; Digeon, Béatrice; Doco-Fenzy, Martine; Doray, Bérénice; Feillet, François; Gardeazabal, Jesus; Gener, Blanca; Julia, Sophie; Llano-Rivas, Isabel; Mazur, Artur; Michot, Caroline; Renaldo-Robin, Florence; Rossi, Massimiliano; Sabouraud, Pascal; Keren, Boris; Depienne, Christel; Muller, Jean; Mandel, Jean-Louis; Laugel, Vincent

    2016-03-22

    Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

  20. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Science.gov (United States)

    Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

    2012-01-01

    RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  1. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Directory of Open Access Journals (Sweden)

    Sara Kangaspeska

    Full Text Available RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60% of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  2. Next generation sequencing reveals the hidden diversity of zooplankton assemblages.

    Directory of Open Access Journals (Sweden)

    Penelope K Lindeque

    Full Text Available BACKGROUND: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. METHODOLOGY/PRINCIPLE FINDINGS: Plankton net hauls (200 µm were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. CONCLUSIONS: Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may

  3. High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

    Science.gov (United States)

    van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

    2010-11-01

    The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

  4. Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

    Science.gov (United States)

    Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-Suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B; Nauck, Markus; Kaminski, Wolfgang E

    2017-01-01

    The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

  5. Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

    Science.gov (United States)

    Holcomb, C L; Rastrou, M; Williams, T C; Goodridge, D; Lazaro, A M; Tilanus, M; Erlich, H A

    2014-01-01

    The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

    Science.gov (United States)

    Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

    2007-04-13

    Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

  7. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r......RNA gene amplicon sequencing can be used to reveal factors of importance for the operation of full-scale nutrient removal plants related to settling problems and floc properties. Using optimized DNA extraction protocols, indexed primers and our in-house Illumina platform, we prepared multiple samples...... be correlated to the presence of the species that are regarded as “strong” and “weak” floc formers. In conclusion, 16S rRNA gene amplicon sequencing provides a high throughput approach for a rapid and cheap community profiling of activated sludge that in combination with multivariate statistics can be used...

  8. Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

    Science.gov (United States)

    Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

    2012-01-01

    Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDRschizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

  9. Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing.

    Science.gov (United States)

    Ma, Qiao; Qu, Yuanyuan; Shen, Wenli; Zhang, Zhaojing; Wang, Jingwei; Liu, Ziyan; Li, Duanxing; Li, Huijie; Zhou, Jiti

    2015-03-01

    In this study, Illumina high-throughput sequencing was used to reveal the community structures of nine coking wastewater treatment plants (CWWTPs) in China for the first time. The sludge systems exhibited a similar community composition at each taxonomic level. Compared to previous studies, some of the core genera in municipal wastewater treatment plants such as Zoogloea, Prosthecobacter and Gp6 were detected as minor species. Thiobacillus (20.83%), Comamonas (6.58%), Thauera (4.02%), Azoarcus (7.78%) and Rhodoplanes (1.42%) were the dominant genera shared by at least six CWWTPs. The percentages of autotrophic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were unexpectedly low, which were verified by both real-time PCR and fluorescence in situ hybridization analyses. Hierarchical clustering and canonical correspondence analysis indicated that operation mode, flow rate and temperature might be the key factors in community formation. This study provides new insights into our understanding of microbial community compositions and structures of CWWTPs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE and high human DNA contamination (up to ~90% we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

  11. Sequence analysis of serum albumins reveals the molecular evolution of ligand recognition properties.

    Science.gov (United States)

    Fanali, Gabriella; Ascenzi, Paolo; Bernardi, Giorgio; Fasano, Mauro

    2012-01-01

    Serum albumin (SA) is a circulating protein providing a depot and carrier for many endogenous and exogenous compounds. At least seven major binding sites have been identified by structural and functional investigations mainly in human SA. SA is conserved in vertebrates, with at least 49 entries in protein sequence databases. The multiple sequence analysis of this set of entries leads to the definition of a cladistic tree for the molecular evolution of SA orthologs in vertebrates, thus showing the clustering of the considered species, with lamprey SAs (Lethenteron japonicum and Petromyzon marinus) in a separate outgroup. Sequence analysis aimed at searching conserved domains revealed that most SA sequences are made up by three repeated domains (about 600 residues), as extensively characterized for human SA. On the contrary, lamprey SAs are giant proteins (about 1400 residues) comprising seven repeated domains. The phylogenetic analysis of the SA family reveals a stringent correlation with the taxonomic classification of the species available in sequence databases. A focused inspection of the sequences of ligand binding sites in SA revealed that in all sites most residues involved in ligand binding are conserved, although the versatility towards different ligands could be peculiar of higher organisms. Moreover, the analysis of molecular links between the different sites suggests that allosteric modulation mechanisms could be restricted to higher vertebrates.

  12. Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4.

    Science.gov (United States)

    Khamrin, Pattara; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

    2013-07-01

    Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1-4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011-11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles.

    Science.gov (United States)

    Gadala-Maria, Daniel; Yaari, Gur; Uduman, Mohamed; Kleinstein, Steven H

    2015-02-24

    Individual variation in germline and expressed B-cell immunoglobulin (Ig) repertoires has been associated with aging, disease susceptibility, and differential response to infection and vaccination. Repertoire properties can now be studied at large-scale through next-generation sequencing of rearranged Ig genes. Accurate analysis of these repertoire-sequencing (Rep-Seq) data requires identifying the germline variable (V), diversity (D), and joining (J) gene segments used by each Ig sequence. Current V(D)J assignment methods work by aligning sequences to a database of known germline V(D)J segment alleles. However, existing databases are likely to be incomplete and novel polymorphisms are hard to differentiate from the frequent occurrence of somatic hypermutations in Ig sequences. Here we develop a Tool for Ig Genotype Elucidation via Rep-Seq (TIgGER). TIgGER analyzes mutation patterns in Rep-Seq data to identify novel V segment alleles, and also constructs a personalized germline database containing the specific set of alleles carried by a subject. This information is then used to improve the initial V segment assignments from existing tools, like IMGT/HighV-QUEST. The application of TIgGER to Rep-Seq data from seven subjects identified 11 novel V segment alleles, including at least one in every subject examined. These novel alleles constituted 13% of the total number of unique alleles in these subjects, and impacted 3% of V(D)J segment assignments. These results reinforce the highly polymorphic nature of human Ig V genes, and suggest that many novel alleles remain to be discovered. The integration of TIgGER into Rep-Seq processing pipelines will increase the accuracy of V segment assignments, thus improving B-cell repertoire analyses.

  14. Transcriptome sequencing revealed significant alteration of cortical promoter usage and splicing in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Jing Qin Wu

    Full Text Available While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression.The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22 from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05. Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1 gene.This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia.

  15. Sequence tagging reveals unexpected modifications in toxicoproteomics

    Science.gov (United States)

    Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

    2010-01-01

    Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

  16. Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry

    Directory of Open Access Journals (Sweden)

    Javier Villacreses

    2015-04-01

    Full Text Available Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1. High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs: ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV, Petuvirus genus. ORF1 encodes a movement protein (MP; ORF2 a Reverse Transcriptase (RT and a Ribonuclease H (RNase H domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs, AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq. Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.

  17. A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing.

    Science.gov (United States)

    Chen, Shi-Yi; Deng, Feilong; Jia, Xianbo; Li, Cao; Lai, Song-Jia

    2017-08-09

    It is widely acknowledged that transcriptional diversity largely contributes to biological regulation in eukaryotes. Since the advent of second-generation sequencing technologies, a large number of RNA sequencing studies have considerably improved our understanding of transcriptome complexity. However, it still remains a huge challenge for obtaining full-length transcripts because of difficulties in the short read-based assembly. In the present study we employ PacBio single-molecule long-read sequencing technology for whole-transcriptome profiling in rabbit (Oryctolagus cuniculus). We totally obtain 36,186 high-confidence transcripts from 14,474 genic loci, among which more than 23% of genic loci and 66% of isoforms have not been annotated yet within the current reference genome. Furthermore, about 17% of transcripts are computationally revealed to be non-coding RNAs. Up to 24,797 alternative splicing (AS) and 11,184 alternative polyadenylation (APA) events are detected within this de novo constructed transcriptome, respectively. The results provide a comprehensive set of reference transcripts and hence contribute to the improved annotation of rabbit genome.

  18. Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

    DEFF Research Database (Denmark)

    Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens

    2015-01-01

    -stringency in-solution hybridization method enables detection of discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral...... sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer...

  19. Re-Analysis of Metagenomic Sequences from Acute Flaccidmyelitis Patients Reveals Alternatives to Enterovirus D68 Infection

    Science.gov (United States)

    2015-07-13

    caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68...distribution is unlimited. Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus D68...Street Baltimore, MD 21218 -2685 ABSTRACT Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus

  20. Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

    Science.gov (United States)

    Cohrs, Randall J; Lee, Katherine S; Beach, Addilynn; Sanford, Bridget; Baird, Nicholas L; Como, Christina; Graybill, Chiharu; Jones, Dallas; Tekeste, Eden; Ballard, Mitchell; Chen, Xiaomi; Yalacki, David; Frietze, Seth; Jones, Kenneth; Lenac Rovis, Tihana; Jonjić, Stipan; Haas, Jürgen; Gilden, Don

    2017-10-15

    The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture. IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture. Copyright © 2017 American Society for Microbiology.

  1. High-resolution deep sequencing reveals biodiversity, population structure, and persistence of HIV-1 quasispecies within host ecosystems

    Directory of Open Access Journals (Sweden)

    Yin Li

    2012-12-01

    Full Text Available Abstract Background Deep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3 within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children. Results Biodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later. Conclusions Deep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

  2. High resolution sequence stratigraphy in China

    International Nuclear Information System (INIS)

    Zhang Shangfeng; Zhang Changmin; Yin Yanshi; Yin Taiju

    2008-01-01

    Since high resolution sequence stratigraphy was introduced into China by DENG Hong-wen in 1995, it has been experienced two development stages in China which are the beginning stage of theory research and development of theory research and application, and the stage of theoretical maturity and widely application that is going into. It is proved by practices that high resolution sequence stratigraphy plays more and more important roles in the exploration and development of oil and gas in Chinese continental oil-bearing basin and the research field spreads to the exploration of coal mine, uranium mine and other strata deposits. However, the theory of high resolution sequence stratigraphy still has some shortages, it should be improved in many aspects. The authors point out that high resolution sequence stratigraphy should be characterized quantitatively and modelized by computer techniques. (authors)

  3. Whole Exome Sequencing Reveals Genetic Predisposition in a Large Family with Retinitis Pigmentosa

    Directory of Open Access Journals (Sweden)

    Juan Wu

    2014-01-01

    Full Text Available Next-generation sequencing has become more widely used to reveal genetic defect in monogenic disorders. Retinitis pigmentosa (RP, the leading cause of hereditary blindness worldwide, has been attributed to more than 67 disease-causing genes. Due to the extreme genetic heterogeneity, using general molecular screening alone is inadequate for identifying genetic predispositions in susceptible individuals. In order to identify underlying mutation rapidly, we utilized next-generation sequencing in a four-generation Chinese family with RP. Two affected patients and an unaffected sibling were subjected to whole exome sequencing. Through bioinformatics analysis and direct sequencing confirmation, we identified p.R135W transition in the rhodopsin gene. The mutation was subsequently confirmed to cosegregate with the disease in the family. In this study, our results suggest that whole exome sequencing is a robust method in diagnosing familial hereditary disease.

  4. Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

    Science.gov (United States)

    Zhang, Yunxia; Cheng, Chunyan; Li, Ji; Yang, Shuqiong; Wang, Yunzhu; Li, Ziang; Chen, Jinfeng; Lou, Qunfeng

    2015-09-25

    Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in

  5. Whole-Exome Sequencing Reveals Clinically Relevant Variants in Family Affected with Autism Spectrum Disorder

    Directory of Open Access Journals (Sweden)

    Jiaxiu Zhou

    2016-10-01

    Full Text Available Chromosomal microarray (CMA has been suggested as a first tier clinical diagnostic test for ASD. High-throughput sequencing (HTS has associated hundreds of genes associated with ASD. Whole Exome Sequencing (WES was used in combination with CMA to identify clinically-relevant ASD variants. In prior work, a trio-based (father, mother, and proband WGS (Whole Genome Sequencing was used to reveal clinically-relevant de novo, or inherited, rare variants in half (16 / 32 of the ASD families in which all probands had normal, or VOUS (Variant of Uncertain Clinical Significance, CMA results. In this study, after CMA screening chromosome structural abnormalities of a proband affected with ASD, a WES was performed on the patient and parents. Some rare de novo, and inherited, variants were detected using trio-based bioinformatics analysis. ASD variants were ranked by SFARI Gene score, HPO (human phenotype ontology, protein function damage, and manual searching PubMed. Sanger sequencing was used to validated some candidate variants in family members. A de novo homozygous mutation in SPG11 (p.C209F, two inherited, compound-heterozygote mutations in SCN9A (p.Q10R and p.R1893H and BEST1 (p.A135V and p.A297V were confirmed. Heterozygous mutations in TSC1 (p.S487C and SHANK2 (p.Arg569His inherited from mother were also confirmed.

  6. Evolutionary growth process of highly conserved sequences in vertebrate genomes.

    Science.gov (United States)

    Ishibashi, Minaka; Noda, Akiko Ogura; Sakate, Ryuichi; Imanishi, Tadashi

    2012-08-01

    Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation

    DEFF Research Database (Denmark)

    Czaban, Adrian; Sharma, Sapna; Byrne, Stephen

    2015-01-01

    species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family...... clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity...... enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some...

  8. Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

    Directory of Open Access Journals (Sweden)

    Heike Hadrys

    Full Text Available Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera. We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.

  9. Whole-exome sequencing reveals GPIHBP1 mutations in infantile colitis with severe hypertriglyceridemia.

    Science.gov (United States)

    Gonzaga-Jauregui, Claudia; Mir, Sabina; Penney, Samantha; Jhangiani, Shalini; Midgen, Craig; Finegold, Milton; Muzny, Donna M; Wang, Min; Bacino, Carlos A; Gibbs, Richard A; Lupski, James R; Kellermayer, Richard; Hanchard, Neil A

    2014-07-01

    Severe congenital hypertriglyceridemia (HTG) is a rare disorder caused by mutations in genes affecting lipoprotein lipase (LPL) activity. Here we report a 5-week-old Hispanic girl with severe HTG (12,031 mg/dL, normal limit 150 mg/dL) who presented with the unusual combination of lower gastrointestinal bleeding and milky plasma. Initial colonoscopy was consistent with colitis, which resolved with reduction of triglycerides. After negative sequencing of the LPL gene, whole-exome sequencing revealed novel compound heterozygous mutations in GPIHBP1. Our study broadens the phenotype of GPIHBP1-associated HTG, reinforces the effectiveness of whole-exome sequencing in Mendelian diagnoses, and implicates triglycerides in gastrointestinal mucosal injury.

  10. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    Science.gov (United States)

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  11. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

    Directory of Open Access Journals (Sweden)

    Fabian König

    Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

  12. Full Genome Sequencing Reveals New Southern African Territories Genotypes Bringing Us Closer to Understanding True Variability of Foot-and-Mouth Disease Virus in Africa

    Science.gov (United States)

    Lasecka-Dykes, Lidia; Wright, Caroline F.; Di Nardo, Antonello; Logan, Grace; Mioulet, Valerie; Jackson, Terry; Tuthill, Tobias J.; Knowles, Nick J.; King, Donald P.

    2018-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that poses a constant burden on farmers in endemic regions and threatens the livestock industries in disease-free countries. Despite the increased number of publicly available whole genome sequences, FMDV data are biased by the opportunistic nature of sampling. Since whole genomic sequences of Southern African Territories (SAT) are particularly underrepresented, this study sequenced 34 isolates from eastern and southern Africa. Phylogenetic analyses revealed two novel genotypes (that comprised 8/34 of these SAT isolates) which contained unusual 5′ untranslated and non-structural encoding regions. While recombination has occurred between these sequences, phylogeny violation analyses indicated that the high degree of sequence diversity for the novel SAT genotypes has not solely arisen from recombination events. Based on estimates of the timing of ancestral divergence, these data are interpreted as being representative of un-sampled FMDV isolates that have been subjected to geographical isolation within Africa by the effects of the Great African Rinderpest Pandemic (1887–1897), which caused a mass die-out of FMDV-susceptible hosts. These findings demonstrate that further sequencing of African FMDV isolates is likely to reveal more unusual genotypes and will allow for better understanding of natural variability and evolution of FMDV. PMID:29652800

  13. Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma.

    Science.gov (United States)

    Lim, Weng Khong; Ong, Choon Kiat; Tan, Jing; Thike, Aye Aye; Ng, Cedric Chuan Young; Rajasegaran, Vikneswari; Myint, Swe Swe; Nagarajan, Sanjanaa; Nasir, Nur Diyana Md; McPherson, John R; Cutcutache, Ioana; Poore, Gregory; Tay, Su Ting; Ooi, Wei Siong; Tan, Veronique Kiak Mien; Hartman, Mikael; Ong, Kong Wee; Tan, Benita K T; Rozen, Steven G; Tan, Puay Hoon; Tan, Patrick; Teh, Bin Tean

    2014-08-01

    Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.

  14. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea

    KAUST Repository

    Pearman, John K.

    2015-11-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore–offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  15. Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

    Science.gov (United States)

    Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica; Nielsen, Jens; Nielsen, Kristian Fog; Workman, Mhairi; Frisvad, Jens Christian

    2016-01-01

    A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species. PMID:27739446

  16. High-throughput sequence alignment using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    Trapnell Cole

    2007-12-01

    Full Text Available Abstract Background The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. Results This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. Conclusion MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

  17. FRESCO: Referential compression of highly similar sequences.

    Science.gov (United States)

    Wandelt, Sebastian; Leser, Ulf

    2013-01-01

    In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware.

  18. The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist.

    Directory of Open Access Journals (Sweden)

    Garret Suen

    Full Text Available Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs, carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.

  19. Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing

    Directory of Open Access Journals (Sweden)

    Jacqueline Morris

    2017-12-01

    Full Text Available Summary: A number of mitochondrial diseases arise from single-nucleotide variant (SNV accumulation in multiple mitochondria. Here, we present a method for identification of variants present at the single-mitochondrion level in individual mouse and human neuronal cells, allowing for extremely high-resolution study of mitochondrial mutation dynamics. We identified extensive heteroplasmy between individual mitochondrion, along with three high-confidence variants in mouse and one in human that were present in multiple mitochondria across cells. The pattern of variation revealed by single-mitochondrion data shows surprisingly pervasive levels of heteroplasmy in inbred mice. Distribution of SNV loci suggests inheritance of variants across generations, resulting in Poisson jackpot lines with large SNV load. Comparison of human and mouse variants suggests that the two species might employ distinct modes of somatic segregation. Single-mitochondrion resolution revealed mitochondria mutational dynamics that we hypothesize to affect risk probabilities for mutations reaching disease thresholds. : Morris et al. use independent sequencing of multiple individual mitochondria from mouse and human brain cells to show high pervasiveness of mutations. The mutations are heteroplasmic within single mitochondria and within and between cells. These findings suggest mechanisms by which mutations accumulate over time, resulting in mitochondrial dysfunction and disease. Keywords: single mitochondrion, single cell, human neuron, mouse neuron, single-nucleotide variation

  20. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

    Directory of Open Access Journals (Sweden)

    Wang Nian

    2012-08-01

    Full Text Available Abstract Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

  1. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  2. Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Miri eMichaeli

    2012-12-01

    Full Text Available High throughput sequencing (HTS yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig genes, which are variable and often highly mutated. This paper describes Ig-HTS-Cleaner (Ig High Throughput Sequencing Cleaner, a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig-Indel-Identifier (Ig Insertion – Deletion Identifier, a program for identifying legitimate and artifact insertions and/or deletions (indels. Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.

  3. CoLIde: A bioinformatics tool for CO-expression based small RNA Loci Identification using high-throughput sequencing data

    OpenAIRE

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-01-01

    Small RNAs (sRNAs) are 20–25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the...

  4. High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

    Science.gov (United States)

    Li, Ruixue; Chen, Dandan; Wang, Taichu; Wan, Yizhen; Li, Rongfang; Fang, Rongjun; Wang, Yuting; Hu, Fei; Zhou, Hong; Li, Long; Zhao, Weiguo

    2017-01-01

    MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding

  5. High diversity of picornaviruses in rats from different continents revealed by deep sequencing.

    Science.gov (United States)

    Hansen, Thomas Arn; Mollerup, Sarah; Nguyen, Nam-Phuong; White, Nicole E; Coghlan, Megan; Alquezar-Planas, David E; Joshi, Tejal; Jensen, Randi Holm; Fridholm, Helena; Kjartansdóttir, Kristín Rós; Mourier, Tobias; Warnow, Tandy; Belsham, Graham J; Bunce, Michael; Willerslev, Eske; Nielsen, Lars Peter; Vinner, Lasse; Hansen, Anders Johannes

    2016-08-17

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

  6. Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony.

    Science.gov (United States)

    Metzger, Julia; Gast, Alana Christina; Schrimpf, Rahel; Rau, Janina; Eikelberg, Deborah; Beineke, Andreas; Hellige, Maren; Distl, Ottmar

    2017-04-01

    The Miniature Shetland pony represents a horse breed with an extremely small body size. Clinical examination of a dwarf Miniature Shetland pony revealed a lowered size at the withers, malformed skull and brachygnathia superior. Computed tomography (CT) showed a shortened maxilla and a cleft of the hard and soft palate which protruded into the nasal passage leading to breathing difficulties. Pathological examination confirmed these findings but did not reveal histopathological signs of premature ossification in limbs or cranial sutures. Whole-genome sequencing of this dwarf Miniature Shetland pony and comparative sequence analysis using 26 reference equids from NCBI Sequence Read Archive revealed three probably damaging missense variants which could be exclusively found in the affected foal. Validation of these three missense mutations in 159 control horses from different horse breeds and five donkeys revealed only the aggrecan (ACAN)-associated g.94370258G>C variant as homozygous wild-type in all control samples. The dwarf Miniature Shetland pony had the homozygous mutant genotype C/C of the ACAN:g.94370258G>C variant and the normal parents were heterozygous G/C. An unaffected full sib and 3/5 unaffected half-sibs were heterozygous G/C for the ACAN:g.94370258G>C variant. In summary, we could demonstrate a dwarf phenotype in a miniature pony breed perfectly associated with a missense mutation within the ACAN gene.

  7. Small RNA sequencing reveals a comprehensive miRNA signature of BRCA1-associated high-grade serous ovarian cancer

    NARCIS (Netherlands)

    Brouwer, Jan; Kluiver, Joost; de Almeida, Rodrigo C.; Modderman, Rutger; Terpstra, Martijn; Kok, Klaas; Withoff, Sebo; Hollema, Harry; Reitsma, Welmoed; de Bock, Geertruida H.; Mourits, Marian J. E.; van den Berg, Anke

    2016-01-01

    AimsBRCA1 mutation carriers are at increased risk of developing high-grade serous ovarian cancer (HGSOC), a malignancy that originates from fallopian tube epithelium. We aimed to identify differentially expressed known and novel miRNAs in BRCA1-associated HGSOC. Methods Small RNA sequencing was

  8. Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing.

    Science.gov (United States)

    Lalonde, Emilie; Albrecht, Steffen; Ha, Kevin C H; Jacob, Karine; Bolduc, Nathalie; Polychronakos, Constantin; Dechelotte, Pierre; Majewski, Jacek; Jabado, Nada

    2010-08-01

    Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity.

  9. Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

    Science.gov (United States)

    Oberstrass, Florian C.; Fernandes, Louis E.; Bryant, Zev

    2012-01-01

    B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. “Bubble” templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context. PMID:22474350

  10. Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing.

    Science.gov (United States)

    Jo, Yeong Deuk; Choi, Yoomi; Kim, Dong-Hwan; Kim, Byung-Dong; Kang, Byoung-Cheorl

    2014-07-04

    Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. Although large portion of sequence context was

  11. Salmon louse (Lepeophtheirus salmonis transcriptomes during post molting maturation and egg production, revealed using EST-sequencing and microarray analysis

    Directory of Open Access Journals (Sweden)

    Jonassen Inge

    2008-03-01

    Full Text Available Abstract Background Lepeophtheirus salmonis is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of L. salmonis revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production. Results EST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 L. salmonis proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species. Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins. Conclusion The copepod L.salmonis contains high a level of novel genes. Among analyzed L.salmonis proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated

  12. Communicating the Benefits of a Full Sequence of High School Science Courses

    Science.gov (United States)

    Nicholas, Catherine Marie

    High school students are generally uninformed about the benefits of enrolling in a full sequence of science courses, therefore only about a third of our nation's high school graduates have completed the science sequence of Biology, Chemistry and Physics. The lack of students completing a full sequence of science courses contributes to the deficit in the STEM degree production rate needed to fill the demand of the current job market and remain competitive as a nation. The purpose of the study was to make a difference in the number of students who have access to information about the benefits of completing a full sequence of science courses. This dissertation study employed qualitative research methodology to gain a broad perspective of staff through a questionnaire and document review and then a deeper understanding through semi-structured interview protocol. The data revealed that a universal sequence of science courses in the high school district did not exist. It also showed that not all students had access to all science courses; students were sorted and tracked according to prerequisites that did not necessarily match the skill set needed for the courses. In addition, the study showed a desire for more support and direction from the district office. It was also apparent that there was a disconnect that existed between who staff members believed should enroll in a full sequence of science courses and who actually enrolled. Finally, communication about science was shown to occur mainly through counseling and peers. A common science sequence, detracking of science courses, increased communication about the postsecondary and academic benefits of a science education, increased district direction and realistic mathematics alignment were all discussed as solutions to the problem.

  13. Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity.

    Science.gov (United States)

    Leisner, Courtney P; Hamilton, John P; Crisovan, Emily; Manrique-Carpintero, Norma C; Marand, Alexandre P; Newton, Linsey; Pham, Gina M; Jiang, Jiming; Douches, David S; Jansky, Shelley H; Buell, C Robin

    2018-05-01

    Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self-compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high-confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome-enabled development of inbred diploid potatoes with the potential to accelerate potato breeding. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  14. The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae

    Directory of Open Access Journals (Sweden)

    David B. Neale

    2017-09-01

    Full Text Available A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb. Franco (Coastal Douglas-fir is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp. Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms.

  15. High-Throughput Block Optical DNA Sequence Identification.

    Science.gov (United States)

    Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

    2018-01-01

    Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition

    Directory of Open Access Journals (Sweden)

    O'Brien Kimberly

    2008-06-01

    Full Text Available Abstract Background The Solanaceae family contains a number of important crop species including potato (Solanum tuberosum which is grown for its underground storage organ known as a tuber. Albeit the 4th most important food crop in the world, other than a collection of ~220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses. Results In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC clone library (87 Mb and sequencing of 22 potato BAC clones (2.9 Mb. The GC content of potato is very similar to Solanum lycopersicon (tomato and other dicotyledonous species yet distinct from the monocotyledonous grass species, Oryza sativa. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two Solanum species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed. Conclusion Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that

  17. Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

    Directory of Open Access Journals (Sweden)

    Piñeyro David

    2011-11-01

    Full Text Available Abstract Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the HeT-A retrotransposon. The sequence of the HeT-A retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the HeT-A is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different HeT-A scenarios. Results A detailed study on the variability of HeT-A reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of HeT-A subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the HeT-A RNA. Interestingly, our results also suggest that most HeT-A copies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the HeT-A sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.

  18. Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

    DEFF Research Database (Denmark)

    Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica

    2016-01-01

    A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes...... confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted...... of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential...

  19. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

    Directory of Open Access Journals (Sweden)

    Jie Qiu

    Full Text Available Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou and a wild line (Lanxi 1 collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1 no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2 besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3 high heterozygous rates (0.19-0.49 were observed in several semi-wild lines; and (4 over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

  20. High-Throughput Next-Generation Sequencing of Polioviruses

    Science.gov (United States)

    Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

    2016-01-01

    ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

  1. [Exome sequencing revealed Allan-Herndon-Dudley syndrome underlying multiple disabilities].

    Science.gov (United States)

    Arvio, Maria; Philips, Anju K; Ahvenainen, Minna; Somer, Mirja; Kalscheuer, Vera; Järvelä, Irma

    2014-01-01

    Normal function of the thyroid gland is the cornerstone of a child's mental development and physical growth. We describe a Finnish family, in which the diagnosis of three brothers became clear after investigations that lasted for more than 30 years. Two of the sons have already died. DNA analysis of the third one, a 16-year-old boy, revealed in exome sequencing of the complete X chromosome a mutation in the SLC16A2 gene, i.e. MCT8, coding for a thyroid hormone transport protein. Allan-Herndon-Dudley syndrome was thus shown to be the cause of multiple disabilities.

  2. Whole-exome sequencing revealed two novel mutations in Usher syndrome.

    Science.gov (United States)

    Koparir, Asuman; Karatas, Omer Faruk; Atayoglu, Ali Timucin; Yuksel, Bayram; Sagiroglu, Mahmut Samil; Seven, Mehmet; Ulucan, Hakan; Yuksel, Adnan; Ozen, Mustafa

    2015-06-01

    Usher syndrome is a clinically and genetically heterogeneous autosomal recessive inherited disorder accompanied by hearing loss and retinitis pigmentosa (RP). Since the associated genes are various and quite large, we utilized whole-exome sequencing (WES) as a diagnostic tool to identify the molecular basis of Usher syndrome. DNA from a 12-year-old male diagnosed with Usher syndrome was analyzed by WES. Mutations detected were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined by in silico analysis. A maternally inherited deleterious frameshift mutation, c.14439_14454del in exon 66 and a paternally inherited non-sense c.10830G>A stop-gain SNV in exon 55 of USH2A were found as two novel compound heterozygous mutations. Both of these mutations disrupt the C terminal of USH2A protein. As a result, WES revealed two novel compound heterozygous mutations in a Turkish USH2A patient. This approach gave us an opportunity to have an appropriate diagnosis and provide genetic counseling to the family within a reasonable time. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  4. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    Directory of Open Access Journals (Sweden)

    Gustafsson Lars

    2010-12-01

    Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

  5. Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Bastiaan A van den Berg

    Full Text Available Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

  6. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus...

  7. Comparative sequence analyses of the major quantitative trait locus phosphorus uptake 1 (Pup1) reveal a complex genetic structure.

    Science.gov (United States)

    Heuer, Sigrid; Lu, Xiaochun; Chin, Joong Hyoun; Tanaka, Juan Pariasca; Kanamori, Hiroyuki; Matsumoto, Takashi; De Leon, Teresa; Ulat, Victor Jun; Ismail, Abdelbagi M; Yano, Masahiro; Wissuwa, Matthias

    2009-06-01

    The phosphorus uptake 1 (Pup1) locus was identified as a major quantitative trait locus (QTL) for tolerance of phosphorus deficiency in rice. Near-isogenic lines with the Pup1 region from tolerant donor parent Kasalath typically show threefold higher phosphorus uptake and grain yield in phosphorus-deficient field trials than the intolerant parent Nipponbare. In this study, we report the fine mapping of the Pup1 locus to the long arm of chromosome 12 (15.31-15.47 Mb). Genes in the region were initially identified on the basis of the Nipponbare reference genome, but did not reveal any obvious candidate genes related to phosphorus uptake. Kasalath BAC clones were therefore sequenced and revealed a 278-kbp sequence significantly different from the syntenic regions in Nipponbare (145 kb) and in the indica reference genome of 93-11 (742 kbp). Size differences are caused by large insertions or deletions (INDELs), and an exceptionally large number of retrotransposon and transposon-related elements (TEs) present in all three sequences (45%-54%). About 46 kb of the Kasalath sequence did not align with the entire Nipponbare genome, and only three Nipponbare genes (fatty acid alpha-dioxygenase, dirigent protein and aspartic proteinase) are highly conserved in Kasalath. Two Nipponbare genes (expressed proteins) might have evolved by at least three TE integrations in an ancestor gene that is still present in Kasalath. Several predicted Kasalath genes are novel or unknown genes that are mainly located within INDEL regions. Our results highlight the importance of sequencing QTL regions in the respective donor parent, as important genes might not be present in the current reference genomes.

  8. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norv...

  9. Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Zhu Wang

    Full Text Available Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.

  10. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Kristensen, Claus; Betenbaugh, Michael J.

    2015-01-01

    Background: The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins.  Results: Here we present the genomic sequence...... of the CHO DXB11 genome sequenced to a depth of 33x. Overall a significant genomic drift was seen favoring GC -> AT point mutations in line with the chemical mutagenesis strategy used for generation of the cell line. The sequencing depth for each gene in the genome revealed distinct peaks at sequencing...... in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find similar to 2...

  11. A high-speed on-chip pseudo-random binary sequence generator for multi-tone phase calibration

    Science.gov (United States)

    Gommé, Liesbeth; Vandersteen, Gerd; Rolain, Yves

    2011-07-01

    An on-chip reference generator is conceived by adopting the technique of decimating a pseudo-random binary sequence (PRBS) signal in parallel sequences. This is of great benefit when high-speed generation of PRBS and PRBS-derived signals is the objective. The design implemented standard CMOS logic is available in commercial libraries to provide the logic functions for the generator. The design allows the user to select the periodicity of the PRBS and the PRBS-derived signals. The characterization of the on-chip generator marks its performance and reveals promising specifications.

  12. A high-speed on-chip pseudo-random binary sequence generator for multi-tone phase calibration

    International Nuclear Information System (INIS)

    Gommé, Liesbeth; Vandersteen, Gerd; Rolain, Yves

    2011-01-01

    An on-chip reference generator is conceived by adopting the technique of decimating a pseudo-random binary sequence (PRBS) signal in parallel sequences. This is of great benefit when high-speed generation of PRBS and PRBS-derived signals is the objective. The design implemented standard CMOS logic is available in commercial libraries to provide the logic functions for the generator. The design allows the user to select the periodicity of the PRBS and the PRBS-derived signals. The characterization of the on-chip generator marks its performance and reveals promising specifications

  13. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    Science.gov (United States)

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.

  14. Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.

    Science.gov (United States)

    Liu, Mingshan; Liu, Yang; Di, Jiabo; Su, Zhe; Yang, Hong; Jiang, Beihai; Wang, Zaozao; Zhuang, Meng; Bai, Fan; Su, Xiangqian

    2017-11-23

    Colorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors. We sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels. A variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different. Our results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

  15. Determination of 5 '-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Nielsen, Jens

    1999-01-01

    We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length...... (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide...

  16. Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jared W Wenger

    2010-05-01

    Full Text Available Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

  17. Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

    Directory of Open Access Journals (Sweden)

    Karin Voordeckers

    2015-11-01

    Full Text Available Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

  18. Peripheral blood transcriptome sequencing reveals rejection-relevant genes in long-term heart transplantation.

    Science.gov (United States)

    Chen, Yan; Zhang, Haibo; Xiao, Xue; Jia, Yixin; Wu, Weili; Liu, Licheng; Jiang, Jun; Zhu, Baoli; Meng, Xu; Chen, Weijun

    2013-10-03

    Peripheral blood-based gene expression patterns have been investigated as biomarkers to monitor the immune system and rule out rejection after heart transplantation. Recent advances in the high-throughput deep sequencing (HTS) technologies provide new leads in transcriptome analysis. By performing Solexa/Illumina's digital gene expression (DGE) profiling, we analyzed gene expression profiles of PBMCs from 6 quiescent (grade 0) and 6 rejection (grade 2R&3R) heart transplant recipients at more than 6 months after transplantation. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an independent validation cohort of 47 individuals from three rejection groups (ISHLT, grade 0,1R, 2R&3R). Through DGE sequencing and qPCR validation, 10 genes were identified as informative genes for detection of cardiac transplant rejection. A further clustering analysis showed that the 10 genes were not only effective for distinguishing patients with acute cardiac allograft rejection, but also informative for discriminating patients with renal allograft rejection based on both blood and biopsy samples. Moreover, PPI network analysis revealed that the 10 genes were connected to each other within a short interaction distance. We proposed a 10-gene signature for heart transplant patients at high-risk of developing severe rejection, which was found to be effective as well in other organ transplant. Moreover, we supposed that these genes function systematically as biomarkers in long-time allograft rejection. Further validation in broad transplant population would be required before the non-invasive biomarkers can be generally utilized to predict the risk of transplant rejection. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

    Science.gov (United States)

    Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

    2017-05-15

    B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

  20. Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe.

    Science.gov (United States)

    Gonzalez, P; Labarère, J

    1998-11-01

    A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The

  1. Computational Analysis of G-Quadruplex Forming Sequences across Chromosomes Reveals High Density Patterns Near the Terminal Ends.

    Directory of Open Access Journals (Sweden)

    Julia H Chariker

    Full Text Available G-quadruplex structures (G4 are found throughout the human genome and are known to play a regulatory role in a variety of molecular processes. Structurally, they have many configurations and can form from one or more DNA strands. At the gene level, they regulate gene expression and protein synthesis. In this paper, chromosomal-level patterns of distribution are analyzed on the human genome to identify high-level distribution patterns potentially related to global functional processes. Here we show unique high density banding patterns on individual chromosomes that are highly correlated, appearing in a mirror pattern, across forward and reverse DNA strands. The highest density of G4 sequences occurs within four megabases of one end of most chromosomes and contains G4 motifs that bind with zinc finger proteins. These findings suggest that G4 may play a role in global chromosomal processes such as those found in meiosis.

  2. Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

    Science.gov (United States)

    Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

    2003-08-14

    The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

  3. Highly conserved non-coding sequences are associated with vertebrate development.

    Directory of Open Access Journals (Sweden)

    Adam Woolfe

    2005-01-01

    Full Text Available In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH, in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development

  4. Phylogenetic and functional analysis of metagenome sequence from high-temperature archaeal habitats demonstrate linkages between metabolic potential and geochemistry

    Directory of Open Access Journals (Sweden)

    William P. Inskeep

    2013-05-01

    Full Text Available Geothermal habitats in Yellowstone National Park (YNP provide an unparalled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (~40-45 Mbase Sanger sequencing per site was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G+C content and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH. These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high temperature systems of YNP.

  5. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  6. Sequencing of BAC pools by different next generation sequencing platforms and strategies

    Directory of Open Access Journals (Sweden)

    Scholz Uwe

    2011-10-01

    Full Text Available Abstract Background Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs improve the assemblies by scaffolding and whether barcoding of BACs is dispensable. Results Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library. Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%. Conclusion Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.

  7. Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus and other eukaryote species revealed by nucleotide and amino acid sequence analyses

    Directory of Open Access Journals (Sweden)

    Andréia B. Poletto

    2008-01-01

    Full Text Available Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

  8. Private selective sweeps identified from next-generation pool-sequencing reveal convergent pathways under selection in two inbred Schistosoma mansoni strains.

    Directory of Open Access Journals (Sweden)

    Julie A J Clément

    Full Text Available BACKGROUND: The trematode flatworms of the genus Schistosoma, the causative agents of schistosomiasis, are among the most prevalent parasites in humans, affecting more than 200 million people worldwide. In this study, we focused on two well-characterized strains of S. mansoni, to explore signatures of selection. Both strains are highly inbred and exhibit differences in life history traits, in particular in their compatibility with the intermediate host Biomphalaria glabrata. METHODOLOGY/PRINCIPAL FINDINGS: We performed high throughput sequencing of DNA from pools of individuals of each strain using Illumina technology and identified single nucleotide polymorphisms (SNP and copy number variations (CNV. In total, 708,898 SNPs were identified and roughly 2,000 CNVs. The SNPs revealed low nucleotide diversity (π = 2 × 10(-4 within each strain and a high differentiation level (Fst = 0.73 between them. Based on a recently developed in-silico approach, we further detected 12 and 19 private (i.e. specific non-overlapping selective sweeps among the 121 and 151 sweeps found in total for each strain. CONCLUSIONS/SIGNIFICANCE: Functional annotation of transcripts lying in the private selective sweeps revealed specific selection for functions related to parasitic interaction (e.g. cell-cell adhesion or redox reactions. Despite high differentiation between strains, we identified evolutionary convergence of genes related to proteolysis, known as a key virulence factor and a potential target of drug and vaccine development. Our data show that pool-sequencing can be used for the detection of selective sweeps in parasite populations and enables one to identify biological functions under selection.

  9. Screening of whole genome sequences identified high-impact variants for stallion fertility.

    Science.gov (United States)

    Schrimpf, Rahel; Gottschalk, Maren; Metzger, Julia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

    2016-04-14

    Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant

  10. Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

    Science.gov (United States)

    Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

    2016-10-01

    The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

  11. First full-length genome sequence of the polerovirus luffa aphid-borne yellows virus (LABYV) reveals the presence of at least two consensus sequences in an isolate from Thailand.

    Science.gov (United States)

    Knierim, Dennis; Maiss, Edgar; Kenyon, Lawrence; Winter, Stephan; Menzel, Wulf

    2015-10-01

    Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.

  12. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure...... that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred...

  13. High levels of diversity characterize mandrill (Mandrillus sphinx) Mhc-DRB sequences.

    Science.gov (United States)

    Abbott, Kristin M; Wickings, E Jean; Knapp, Leslie A

    2006-08-01

    The major histocompatibility complex (MHC) is highly polymorphic in most primate species studied thus far. The rhesus macaque (Macaca mulatta) has been studied extensively and the Mhc-DRB region demonstrates variability similar to humans. The extent of MHC diversity is relatively unknown for other Old World monkeys (OWM), especially among genera other than Macaca. A molecular survey of the Mhc-DRB region in mandrills (Mandrillus sphinx) revealed extensive variability, suggesting that other OWMs may also possess high levels of Mhc-DRB polymorphism. In the present study, 33 Mhc-DRB loci were identified from only 13 animals. Eleven were wild-born and presumed to be unrelated and two were captive-born twins. Two to seven different sequences were identified for each individual, suggesting that some mandrills may have as many as four Mhc-DRB loci on a single haplotype. From these sequences, representatives of at least six Mhc-DRB loci or lineages were identified. As observed in other primates, some new lineages may have arisen through the process of gene conversion. These findings indicate that mandrills have Mhc-DRB diversity not unlike rhesus macaques and humans.

  14. Directed PCR-free engineering of highly repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Preissler Steffen

    2011-09-01

    Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

  15. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

    Directory of Open Access Journals (Sweden)

    Md. Tariqul Islam

    2015-01-01

    Full Text Available MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

  16. Exome Sequence Analysis of 14 Families With High Myopia

    DEFF Research Database (Denmark)

    Kloss, Bethany A.; Tompson, Stuart W.; Whisenhunt, Kristina N.

    2017-01-01

    Purpose: To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. Methods: Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sang...

  17. Draft genome sequence of Streptomyces coelicoflavus ZG0656 reveals the putative biosynthetic gene cluster of acarviostatin family α-amylase inhibitors.

    Science.gov (United States)

    Guo, X; Geng, P; Bai, F; Bai, G; Sun, T; Li, X; Shi, L; Zhong, Q

    2012-08-01

    The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α-amylase inhibitors, and then to reveal the putative acarviostatin-related gene cluster and the biosynthetic pathway. The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct-cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00-7-P, and the extracellular assemblies lead to diverse acarviostatin end products. The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct-cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  18. Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria.

    Science.gov (United States)

    Delamuta, Jakeline Renata Marçon; Ribeiro, Renan Augusto; Menna, Pâmela; Bangel, Eliane Villamil; Hungria, Mariangela

    2012-04-01

    Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

  19. Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum.

    Science.gov (United States)

    Rao, Soumya; Nandineni, Madhusudan R

    2017-01-01

    Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens.

  20. Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

    Science.gov (United States)

    Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

    2017-10-17

    Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

  1. Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations

    KAUST Repository

    Arellano, Shawn M.

    2012-10-11

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ13C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge. © 2012 Springer Science+Business Media New York.

  2. Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

    Science.gov (United States)

    Arellano, Shawn M; Lee, On On; Lafi, Feras F; Yang, Jiangke; Wang, Yong; Young, Craig M; Qian, Pei-Yuan

    2013-02-01

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

  3. Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

    Directory of Open Access Journals (Sweden)

    Chao Shiaoman

    2011-01-01

    Full Text Available Abstract Background Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat. Results Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM analysis. Of these, 52 (54% were polymorphic between parents of the Ogle1040 × TAM O-301 (OT mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry. Conclusions The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide

  4. Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

    Directory of Open Access Journals (Sweden)

    Bruggmann Rémy

    2007-05-01

    Full Text Available Abstract Background Quantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL. To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes. Results To obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5. Conclusion Comparative sequence analysis revealed highly conserved collinear regions

  5. Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

    Science.gov (United States)

    Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

    2011-04-01

    Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

  6. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Directory of Open Access Journals (Sweden)

    Jason D Thompson

    Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  7. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Science.gov (United States)

    Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

    2012-01-01

    Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  8. Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese Rice Wine.

    Science.gov (United States)

    Hong, Xutao; Chen, Jing; Liu, Lin; Wu, Huan; Tan, Haiqin; Xie, Guangfa; Xu, Qian; Zou, Huijun; Yu, Wenjing; Wang, Lan; Qin, Nan

    2016-05-31

    Chinese Rice Wine (CRW) is a common alcoholic beverage in China. To investigate the influence of microbial composition on the quality of CRW, high throughput sequencing was performed for 110 wine samples on bacterial 16S rRNA gene and fungal Internal Transcribed Spacer II (ITS2). Bioinformatic analyses demonstrated that the quality of yeast starter and final wine correlated with microbial taxonomic composition, which was exemplified by our finding that wine spoilage resulted from a high proportion of genus Lactobacillus. Subsequently, based on Lactobacillus abundance of an early stage, a model was constructed to predict final wine quality. In addition, three batches of 20 representative wine samples selected from a pool of 110 samples were further analyzed in metagenomics. The results revealed that wine spoilage was due to rapid growth of Lactobacillus brevis at the early stage of fermentation. Gene functional analysis indicated the importance of some pathways such as synthesis of biotin, malolactic fermentation and production of short-chain fatty acid. These results led to a conclusion that metabolisms of microbes influence the wine quality. Thus, nurturing of beneficial microbes and inhibition of undesired ones are both important for the mechanized brewery.

  9. Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA

    2015-10-24

    Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic diversity

  10. Homozygosity mapping and targeted sanger sequencing reveal genetic defects underlying inherited retinal disease in families from pakistan.

    Directory of Open Access Journals (Sweden)

    Maleeha Maria

    Full Text Available Homozygosity mapping has facilitated the identification of the genetic causes underlying inherited diseases, particularly in consanguineous families with multiple affected individuals. This knowledge has also resulted in a mutation dataset that can be used in a cost and time effective manner to screen frequent population-specific genetic variations associated with diseases such as inherited retinal disease (IRD.We genetically screened 13 families from a cohort of 81 Pakistani IRD families diagnosed with Leber congenital amaurosis (LCA, retinitis pigmentosa (RP, congenital stationary night blindness (CSNB, or cone dystrophy (CD. We employed genome-wide single nucleotide polymorphism (SNP array analysis to identify homozygous regions shared by affected individuals and performed Sanger sequencing of IRD-associated genes located in the sizeable homozygous regions. In addition, based on population specific mutation data we performed targeted Sanger sequencing (TSS of frequent variants in AIPL1, CEP290, CRB1, GUCY2D, LCA5, RPGRIP1 and TULP1, in probands from 28 LCA families.Homozygosity mapping and Sanger sequencing of IRD-associated genes revealed the underlying mutations in 10 families. TSS revealed causative variants in three families. In these 13 families four novel mutations were identified in CNGA1, CNGB1, GUCY2D, and RPGRIP1.Homozygosity mapping and TSS revealed the underlying genetic cause in 13 IRD families, which is useful for genetic counseling as well as therapeutic interventions that are likely to become available in the near future.

  11. Who's for dinner? High-throughput sequencing reveals bat dietary differentiation in a biodiversity hotspot where prey taxonomy is largely undescribed.

    Science.gov (United States)

    Burgar, Joanna M; Murray, Daithi C; Craig, Michael D; Haile, James; Houston, Jayne; Stokes, Vicki; Bunce, Michael

    2014-08-01

    Effective management and conservation of biodiversity requires understanding of predator-prey relationships to ensure the continued existence of both predator and prey populations. Gathering dietary data from predatory species, such as insectivorous bats, often presents logistical challenges, further exacerbated in biodiversity hot spots because prey items are highly speciose, yet their taxonomy is largely undescribed. We used high-throughput sequencing (HTS) and bioinformatic analyses to phylogenetically group DNA sequences into molecular operational taxonomic units (MOTUs) to examine predator-prey dynamics of three sympatric insectivorous bat species in the biodiversity hotspot of south-western Australia. We could only assign between 4% and 20% of MOTUs to known genera or species, depending on the method used, underscoring the importance of examining dietary diversity irrespective of taxonomic knowledge in areas lacking a comprehensive genetic reference database. MOTU analysis confirmed that resource partitioning occurred, with dietary divergence positively related to the ecomorphological divergence of the three bat species. We predicted that bat species' diets would converge during times of high energetic requirements, that is, the maternity season for females and the mating season for males. There was an interactive effect of season on female, but not male, bat species' diets, although small sample sizes may have limited our findings. Contrary to our predictions, females of two ecomorphologically similar species showed dietary convergence during the mating season rather than the maternity season. HTS-based approaches can help elucidate complex predator-prey relationships in highly speciose regions, which should facilitate the conservation of biodiversity in genetically uncharacterized areas, such as biodiversity hotspots. © 2013 John Wiley & Sons Ltd.

  12. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

    Directory of Open Access Journals (Sweden)

    Li Kelvin

    2012-11-01

    Full Text Available Abstract Background In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. Results We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Conclusions Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus

  13. Genomic view of bipolar disorder revealed by whole genome sequencing in a genetic isolate.

    Directory of Open Access Journals (Sweden)

    Benjamin Georgi

    2014-03-01

    Full Text Available Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.

  14. Genomic View of Bipolar Disorder Revealed by Whole Genome Sequencing in a Genetic Isolate

    Science.gov (United States)

    Georgi, Benjamin; Craig, David; Kember, Rachel L.; Liu, Wencheng; Lindquist, Ingrid; Nasser, Sara; Brown, Christopher; Egeland, Janice A.; Paul, Steven M.; Bućan, Maja

    2014-01-01

    Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders. PMID:24625924

  15. Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

    OpenAIRE

    Lanciano, Sophie; Carpentier, M. C.; Llauro, C.; Jobet, E.; Robakowska-Hyzorek, D.; Lasserre, E.; Ghesquière, Alain; Panaud, O.; Mirouze, Marie

    2017-01-01

    Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposon...

  16. Continuous- and Discrete-Time Stimulus Sequences for High Stimulus Rate Paradigm in Evoked Potential Studies

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2013-01-01

    Full Text Available To obtain reliable transient auditory evoked potentials (AEPs from EEGs recorded using high stimulus rate (HSR paradigm, it is critical to design the stimulus sequences of appropriate frequency properties. Traditionally, the individual stimulus events in a stimulus sequence occur only at discrete time points dependent on the sampling frequency of the recording system and the duration of stimulus sequence. This dependency likely causes the implementation of suboptimal stimulus sequences, sacrificing the reliability of resulting AEPs. In this paper, we explicate the use of continuous-time stimulus sequence for HSR paradigm, which is independent of the discrete electroencephalogram (EEG recording system. We employ simulation studies to examine the applicability of the continuous-time stimulus sequences and the impacts of sampling frequency on AEPs in traditional studies using discrete-time design. Results from these studies show that the continuous-time sequences can offer better frequency properties and improve the reliability of recovered AEPs. Furthermore, we find that the errors in the recovered AEPs depend critically on the sampling frequencies of experimental systems, and their relationship can be fitted using a reciprocal function. As such, our study contributes to the literature by demonstrating the applicability and advantages of continuous-time stimulus sequences for HSR paradigm and by revealing the relationship between the reliability of AEPs and sampling frequencies of the experimental systems when discrete-time stimulus sequences are used in traditional manner for the HSR paradigm.

  17. Analysis of complete nucleotide sequences of Angolan hepatitis B virus isolates reveals the existence of a separate lineage within genotype E.

    Directory of Open Access Journals (Sweden)

    Barbara V Lago

    Full Text Available Hepatitis B virus genotype E (HBV/E is highly prevalent in Western Africa. In this work, 30 HBV/E isolates from HBsAg positive Angolans (staff and visitors of a private hospital in Luanda were genetically characterized: 16 of them were completely sequenced and the pre-S/S sequences of the remaining 14 were determined. A high proportion (12/30, 40% of subjects tested positive for both HBsAg and anti-HBs markers. Deduced amino acid sequences revealed the existence of specific substitutions and deletions in the B- and T-cell epitopes of the surface antigen (pre-S1- and pre-S2 regions of the virus isolates derived from 8/12 individuals with concurrent HBsAg/anti-HBs. Phylogenetic analysis performed with 231 HBV/E full-length sequences, including 16 from this study, showed that all isolates from Angola, Namibia and the Democratic Republic of Congo (n = 28 clustered in a separate lineage, divergent from the HBV/E isolates from nine other African countries, namely Cameroon, Central African Republic, Côte d'Ivoire, Ghana, Guinea, Madagascar, Niger, Nigeria and Sudan, with a Bayesian posterior probability of 1. Five specific mutations, namely small S protein T57I, polymerase Q177H, G245W and M612L, and X protein V30L, were observed in 79-96% of the isolates of the separate lineage, compared to a frequency of 0-12% among the other HBV/E African isolates.

  18. 454-sequencing reveals stochastic local reassembly and high disturbance tolerance within arbuscular mycorrhizal fungal communities

    DEFF Research Database (Denmark)

    Lekberg, Karin Ylva Margareta; Schnoor, Tim; Kjøller, Rasmus

    2012-01-01

    unpredictable, with approximately 40%of all sequences within a sample belonging to a single OTU of varying identity. The distribution of two plant species that are often poorly colonized by AMfungi (Dianthus deltoides and Carex arenaria) correlated significantly with the OTU composition, which may indicate...

  19. A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

    Science.gov (United States)

    Ankarklev, Johan; Lebbad, Marianne; Einarsson, Elin; Franzén, Oscar; Ahola, Harri; Troell, Karin; Svärd, Staffan G

    2018-06-01

    Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia. Copyright © 2018. Published by Elsevier B.V.

  20. Evolution of sequence-defined highly functionalized nucleic acid polymers

    Science.gov (United States)

    Chen, Zhen; Lichtor, Phillip A.; Berliner, Adrian P.; Chen, Jonathan C.; Liu, David R.

    2018-03-01

    The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to generate new classes of receptors, catalysts and materials. Here we describe a ligase-mediated DNA-templated polymerization and in vitro selection system to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks that contain eight chemically diverse side chains on a DNA backbone. Through iterated cycles of polymer translation, selection and reverse translation, we discovered HFNAPs that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) and interleukin-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (KD = 3 nM). This evolved polymer potently inhibited the binding between PCSK9 and the low-density lipoprotein receptor. Structure-activity relationship studies revealed that specific side chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires.

  1. Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease.

    Science.gov (United States)

    Lieber, Daniel S; Vafai, Scott B; Horton, Laura C; Slate, Nancy G; Liu, Shangtao; Borowsky, Mark L; Calvo, Sarah E; Schmahmann, Jeremy D; Mootha, Vamsi K

    2012-01-06

    Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.

  2. Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease

    Directory of Open Access Journals (Sweden)

    Lieber Daniel S

    2012-01-01

    Full Text Available Abstract Background Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. Case Presentation Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. Conclusion This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.

  3. Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

    Czech Academy of Sciences Publication Activity Database

    Jakše, J.; Radišek, S.; Pokorn, T.; Matoušek, Jaroslav; Javornik, B.

    2015-01-01

    Roč. 64, č. 4 (2015), s. 831-842 ISSN 0032-0862 R&D Projects: GA MŠk(CZ) LH14255 Institutional support: RVO:60077344 Keywords : Bioinformatic * Citrus bark cracking viroid * Hop * Next-generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.383, year: 2015

  4. Which MRI sequence of the spine best reveals bone-marrow metastases of neuroblastoma?

    International Nuclear Information System (INIS)

    Meyer, James S.; Jaramillo, Diego; Siegel, Marilyn J.; Farooqui, Saleem O.; Fletcher, Barry D.; Hoffer, Fredric A.

    2005-01-01

    MRI is an effective tool in evaluating bone marrow metastases. However, no study has defined which MRI sequences or image characteristics best correlate with bone-marrow metastases in neuroblastoma. To identify and refine MRI criteria and sequence selection for the diagnosis of bone-marrow metastases in children with neuroblastoma. Ninety-one children (mean age: 3.2 years; standard deviation: 2.8 years) enrolled in the RDOG IV study participated in our study. Forty-five children had bone metastases determined by bone-marrow aspiration or biopsy (n=4), radionuclide imaging (n=2), or both (n=39). Spine lesions were characterized using coronal T1-weighted (T1W) sagittal short tau inversion recovery (STIR) and coronal gadolinium-enhanced T1-weighted (GAD) MR sequences. Contingency table analysis was performed to determine which MRI sequences and characteristics were associated with metastases. The MRI criteria for metastatic disease were then developed for each imaging sequence. The sensitivity, specificity, predictive values, and accuracy of these criteria were determined for the whole group, children younger than 12 months old, and children 12 months and older. The MR characteristics that had significant (P ≤ 0.05) associations with metastases were homogeneous low T1-signal intensity, homogeneous high STIR-signal intensity, and heterogeneous pattern on T1, STIR, or GAD. Homogeneous low T1-signal had the highest sensitivity (88%), but a specificity of 62% for detecting metastases. A heterogeneous pattern on GAD was highly specific (97%), but relatively insensitive (65%) for detecting metastases. These MR characteristics were most accurate in children 12 months and older. The combination of non-contrast-enhanced T1W and GAD sequences can be used to determine the presence of spinal metastases in children with neuroblastoma, particularly those children who are 1 year and older. (orig.)

  5. Highly multiplexed targeted DNA sequencing from single nuclei.

    Science.gov (United States)

    Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

    2016-02-01

    Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

  6. Multi-species sequence comparison reveals dynamic evolution of the elastin gene that has involved purifying selection and lineage-specific insertions/deletions

    Directory of Open Access Journals (Sweden)

    Green Eric D

    2004-05-01

    Full Text Available Abstract Background The elastin gene (ELN is implicated as a factor in both supravalvular aortic stenosis (SVAS and Williams Beuren Syndrome (WBS, two diseases involving pronounced complications in mental or physical development. Although the complete spectrum of functional roles of the processed gene product remains to be established, these roles are inferred to be analogous in human and mouse. This view is supported by genomic sequence comparison, in which there are no large-scale differences in the ~1.8 Mb sequence block encompassing the common region deleted in WBS, with the exception of an overall reversed physical orientation between human and mouse. Results Conserved synteny around ELN does not translate to a high level of conservation in the gene itself. In fact, ELN orthologs in mammals show more sequence divergence than expected for a gene with a critical role in development. The pattern of divergence is non-conventional due to an unusually high ratio of gaps to substitutions. Specifically, multi-sequence alignments of eight mammalian sequences reveal numerous non-aligning regions caused by species-specific insertions and deletions, in spite of the fact that the vast majority of aligning sites appear to be conserved and undergoing purifying selection. Conclusions The pattern of lineage-specific, in-frame insertions/deletions in the coding exons of ELN orthologous genes is unusual and has led to unique features of the gene in each lineage. These differences may indicate that the gene has a slightly different functional mechanism in mammalian lineages, or that the corresponding regions are functionally inert. Identified regions that undergo purifying selection reflect a functional importance associated with evolutionary pressure to retain those features.

  7. Nuclear Species-Diagnostic SNP Markers Mined from 454 Amplicon Sequencing Reveal Admixture Genomic Structure of Modern Citrus Varieties

    Science.gov (United States)

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP

  8. Exome sequencing identifies ZNF644 mutations in high myopia.

    Directory of Open Access Journals (Sweden)

    Yi Shi

    2011-06-01

    Full Text Available Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644 was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3'UTR+12 C>G, and 3'UTR+592 G>A in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.

  9. Exome sequencing generates high quality data in non-target regions

    Directory of Open Access Journals (Sweden)

    Guo Yan

    2012-05-01

    Full Text Available Abstract Background Exome sequencing using next-generation sequencing technologies is a cost efficient approach to selectively sequencing coding regions of human genome for detection of disease variants. A significant amount of DNA fragments from the capture process fall outside target regions, and sequence data for positions outside target regions have been mostly ignored after alignment. Result We performed whole exome sequencing on 22 subjects using Agilent SureSelect capture reagent and 6 subjects using Illumina TrueSeq capture reagent. We also downloaded sequencing data for 6 subjects from the 1000 Genomes Project Pilot 3 study. Using these data, we examined the quality of SNPs detected outside target regions by computing consistency rate with genotypes obtained from SNP chips or the Hapmap database, transition-transversion (Ti/Tv ratio, and percentage of SNPs inside dbSNP. For all three platforms, we obtained high-quality SNPs outside target regions, and some far from target regions. In our Agilent SureSelect data, we obtained 84,049 high-quality SNPs outside target regions compared to 65,231 SNPs inside target regions (a 129% increase. For our Illumina TrueSeq data, we obtained 222,171 high-quality SNPs outside target regions compared to 95,818 SNPs inside target regions (a 232% increase. For the data from the 1000 Genomes Project, we obtained 7,139 high-quality SNPs outside target regions compared to 1,548 SNPs inside target regions (a 461% increase. Conclusions These results demonstrate that a significant amount of high quality genotypes outside target regions can be obtained from exome sequencing data. These data should not be ignored in genetic epidemiology studies.

  10. Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns

    Science.gov (United States)

    Coghlan, Megan L.; Haile, James; Houston, Jayne; Murray, Dáithí C.; White, Nicole E.; Moolhuijzen, Paula; Bellgard, Matthew I.; Bunce, Michael

    2012-01-01

    Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when

  11. Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

    Directory of Open Access Journals (Sweden)

    Zhang Xinmin

    2011-05-01

    Full Text Available Abstract Background In highly copy number variable (CNV regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach. Results As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations. Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods. Conclusion Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.

  12. Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

    Directory of Open Access Journals (Sweden)

    Wanli Gao

    2018-01-01

    Full Text Available Drinking water safety is increasingly perceived as one of the top global environmental issues. Plankton has been commonly used as a bioindicator for water quality in lakes and reservoirs. Recently, DNA sequencing technology has been applied to bioassessment. In this study, we compared the effectiveness of the 16S and 18S rRNA high throughput sequencing method (HTS and the traditional optical microscopy method (TOM in the bioassessment of drinking water quality. Five stations reflecting different habitats and hydrological conditions in Danjiangkou Reservoir, one of the largest drinking water reservoirs in Asia, were sampled May 2016. Non-metric multi-dimensional scaling (NMDS analysis showed that plankton assemblages varied among the stations and the spatial patterns revealed by the two methods were consistent. The correlation between TOM and HTS in a symmetric Procrustes analysis was 0.61, revealing overall good concordance between the two methods. Procrustes analysis also showed that site-specific differences between the two methods varied among the stations. Station Heijizui (H, a site heavily influenced by two tributaries, had the largest difference while station Qushou (Q, a confluence site close to the outlet dam, had the smallest difference between the two methods. Our results show that DNA sequencing has the potential to provide consistent identification of taxa, and reliable bioassessment in a long-term biomonitoring and assessment program for drinking water reservoirs.

  13. Cytosolic Glutamine Synthetase is Important for Photosynthetic Efficiency and Water Use Efficiency in Potato as Revealed by High Throughput Sequencing QTL analysis

    DEFF Research Database (Denmark)

    Kaminski, Kacper Piotr; Sørensen, Kirsten Kørup; Andersen, Mathias Neumann

    2015-01-01

    was observed. Two extreme WUE bulks of clones were identified and pools of genomic DNA from them as well as the parents were sequenced and mapped to reference potato genome. Following a novel data analysis approach, two highly resolved QTLs were found on chromosome 1 and 9. Interestingly, three genes encoding...

  14. A priori Considerations When Conducting High-Throughput Amplicon-Based Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Aditi Sengupta

    2016-03-01

    Full Text Available Amplicon-based sequencing strategies that include 16S rRNA and functional genes, alongside “meta-omics” analyses of communities of microorganisms, have allowed researchers to pose questions and find answers to “who” is present in the environment and “what” they are doing. Next-generation sequencing approaches that aid microbial ecology studies of agricultural systems are fast gaining popularity among agronomy, crop, soil, and environmental science researchers. Given the rapid development of these high-throughput sequencing techniques, researchers with no prior experience will desire information about the best practices that can be used before actually starting high-throughput amplicon-based sequence analyses. We have outlined items that need to be carefully considered in experimental design, sampling, basic bioinformatics, sequencing of mock communities and negative controls, acquisition of metadata, and in standardization of reaction conditions as per experimental requirements. Not all considerations mentioned here may pertain to a particular study. The overall goal is to inform researchers about considerations that must be taken into account when conducting high-throughput microbial DNA sequencing and sequences analysis.

  15. Mycobacterium malmesburyense sp. nov., a non-tuberculous species of the genus Mycobacterium revealed by multiple gene sequence characterization

    CSIR Research Space (South Africa)

    Gcebe, N

    2017-04-01

    Full Text Available Journal of Systematic and Evolutionary Microbiology: DOI 10.1099/ijsem.0.001678 Mycobacterium malmesburyense sp. nov., a non-tuberculous species of the genus Mycobacterium revealed by multiple gene sequence characterization Gcebe N Rutten V Gey...

  16. High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

    Directory of Open Access Journals (Sweden)

    Trout-Yakel Keri M

    2010-02-01

    Full Text Available Abstract Background A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

  17. Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

    Science.gov (United States)

    Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

    2018-07-01

    Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    Energy Technology Data Exchange (ETDEWEB)

    Shi, CY; Yang, H; Wei, CL; Yu, O; Zhang, ZZ; Sun, J; Wan, XC

    2011-01-01

    Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real

  19. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    Directory of Open Access Journals (Sweden)

    Chen Qi

    2011-02-01

    Full Text Available Abstract Background Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Results Using high-throughput Illumina RNA-seq, the transcriptome from poly (A+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs. Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010. Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were

  20. Clinical evaluation of further-developed MRCP sequences in comparison with standard MRCP sequences

    International Nuclear Information System (INIS)

    Hundt, W.; Scheidler, J.; Reiser, M.; Petsch, R.

    2002-01-01

    The purpose of this study was the comparison of technically improved single-shot magnetic resonance cholangiopancreatography (MRCP) sequences with standard single-shot rapid acquisition with relaxation enhancement (RARE) and half-Fourier acquired single-shot turbo spin-echo (HASTE) sequences in evaluating the normal and abnormal biliary duct system. The bile duct system of 45 patients was prospectively investigated on a 1.5-T MRI system. The investigation was performed with RARE and HASTE MR cholangiography sequences with standard and high spatial resolutions, and with a delayed-echo half-Fourier RARE (HASTE) sequence. Findings of the improved MRCP sequences were compared with the standard MRCP sequences. The level of confidence in assessing the diagnosis was divided into five groups. The Wilcoxon signed-rank test at a level of p<0.05 was applied. In 15 patients no pathology was found. The MRCP showed stenoses of the bile duct system in 10 patients and choledocholithiasis and cholecystolithiasis in 16 patients. In 12 patients a dilatation of the bile duct system was found. Comparison of the low- and high spatial resolution sequences and the short and long TE times of the half-Fourier RARE (HASTE) sequence revealed no statistically significant differences regarding accuracy of the examination. The diagnostic confidence level in assessing normal or pathological findings for the high-resolution RARE and half-Fourier RARE (HASTE) was significantly better than for the standard sequences. For the delayed-echo half-Fourier RARE (HASTE) sequence no statistically significant difference was seen. The high-resolution RARE and half-Fourier RARE (HASTE) sequences had a higher confidence level, but there was no significant difference in diagnosis in terms of detection and assessment of pathological changes in the biliary duct system compared with standard sequences. (orig.)

  1. Significant strain accumulation between the deformation front and landward out-of-sequence thrusts in accretionary wedge of SW Taiwan revealed by cGPS and SAR interferometry

    Science.gov (United States)

    Tsai, M. C.

    2017-12-01

    High strain accumulation across the fold-and-thrust belt in Southwestern Taiwan are revealed by the Continuous GPS (cGPS) and SAR interferometry. This high strain is generally accommodated by the major active structures in fold-and-thrust belt of western Foothills in SW Taiwan connected to the accretionary wedge in the incipient are-continent collision zone. The active structures across the high strain accumulation include the deformation front around the Tainan Tableland, the Hochiali, Hsiaokangshan, Fangshan and Chishan faults. Among these active structures, the deformation pattern revealed from cGPS and SAR interferometry suggest that the Fangshan transfer fault may be a left-lateral fault zone with thrust component accommodating the westward differential motion of thrust sheets on both side of the fault. In addition, the Chishan fault connected to the splay fault bordering the lower-slope and upper-slope of the accretionary wedge which could be the major seismogenic fault and an out-of-sequence thrust fault in SW Taiwan. The big earthquakes resulted from the reactivation of out-of-sequence thrusts have been observed along the Nankai accretionary wedge, thus the assessment of the major seismogenic structures by strain accumulation between the frontal décollement and out-of-sequence thrusts is a crucial topic. According to the background seismicity, the low seismicity and mid-crust to mantle events are observed inland and the lower- and upper- slope domain offshore SW Taiwan, which rheologically implies the upper crust of the accretionary wedge is more or less aseimic. This result may suggest that the excess fluid pressure from the accretionary wedge not only has significantly weakened the prism materials as well as major fault zone, but also makes the accretionary wedge landward extension, which is why the low seismicity is observed in SW Taiwan area. Key words: Continuous GPS, SAR interferometry, strain rate, out-of-sequence thrust.

  2. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

    Directory of Open Access Journals (Sweden)

    Jonas Binladen

    2007-02-01

    Full Text Available The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources.We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences. Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis.We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%. Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial

  3. Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds.

    Directory of Open Access Journals (Sweden)

    Yao Xu

    Full Text Available Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus and Qinchuan (Bos taurus are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV were identified by aligning Nanyang to Qinchuan genome, 783 of which (27% encompassed the coding regions of 495 functional genes. The gene ontology (GO analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log2 (ratio = -2.34988; P value = 1.53E-102. Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs

  4. Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences

    OpenAIRE

    Bunawan, Hamidun; Yen, Choong Chee; Yaakop, Salmah; Noor, Normah Mohd

    2017-01-01

    Background The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. Results Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consi...

  5. Sequencing of a patient with balanced chromosome abnormalities and neurodevelopmental disease identifies disruption of multiple high risk loci by structural variation.

    Directory of Open Access Journals (Sweden)

    Jonathon Blake

    Full Text Available Balanced chromosome abnormalities (BCAs occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14 that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception.

  6. Sequencing of a Patient with Balanced Chromosome Abnormalities and Neurodevelopmental Disease Identifies Disruption of Multiple High Risk Loci by Structural Variation

    Science.gov (United States)

    Blake, Jonathon; Riddell, Andrew; Theiss, Susanne; Gonzalez, Alexis Perez; Haase, Bettina; Jauch, Anna; Janssen, Johannes W. G.; Ibberson, David; Pavlinic, Dinko; Moog, Ute; Benes, Vladimir; Runz, Heiko

    2014-01-01

    Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception. PMID:24625750

  7. SNP calling using genotype model selection on high-throughput sequencing data

    KAUST Repository

    You, Na

    2012-01-16

    Motivation: A review of the available single nucleotide polymorphism (SNP) calling procedures for Illumina high-throughput sequencing (HTS) platform data reveals that most rely mainly on base-calling and mapping qualities as sources of error when calling SNPs. Thus, errors not involved in base-calling or alignment, such as those in genomic sample preparation, are not accounted for.Results: A novel method of consensus and SNP calling, Genotype Model Selection (GeMS), is given which accounts for the errors that occur during the preparation of the genomic sample. Simulations and real data analyses indicate that GeMS has the best performance balance of sensitivity and positive predictive value among the tested SNP callers. © The Author 2012. Published by Oxford University Press. All rights reserved.

  8. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

    Directory of Open Access Journals (Sweden)

    Zhewei Song

    2017-07-01

    Full Text Available Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces and lactic acid bacteria (genus Lactobacillus classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol to acid (lactic acid and acetic acid in Chinese Maotai-flavor liquor production. Our findings provide

  9. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing.

    Science.gov (United States)

    Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

    2017-01-01

    Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces , and Zygosaccharomyces ) and lactic acid bacteria (genus Lactobacillus ) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into

  10. Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing).

    Science.gov (United States)

    Vengerfeldt, Veiko; Špilka, Katerina; Saag, Mare; Preem, Jens-Konrad; Oopkaup, Kristjan; Truu, Jaak; Mändar, Reet

    2014-11-01

    Chronic apical periodontitis (CAP) is a frequent condition that has a considerable effect on a patient's quality of life. We aimed to reveal root canal microbial communities in antibiotic-naive patients by applying Illumina sequencing (Illumina Inc, San Diego, CA). Samples were collected under strict aseptic conditions from 12 teeth (5 with primary CAP, 3 with secondary CAP, and 4 with a periapical abscess [PA]) and characterized by profiling the microbial community on the basis of the V6 hypervariable region of the 16S ribosomal RNA gene by using Illumina HiSeq2000 sequencing combinatorial sequence-tagged polymerase chain reaction products. Root canal specimens displayed highly polymicrobial communities in all 3 patient groups. One sample contained 5-8 (mean = 6.5) phyla of bacteria. The most numerous were Firmicutes and Bacteroidetes, but Actinobacteria, Fusobacteria, Proteobacteria, Spirochaetes, Tenericutes, and Synergistetes were also present in most of the patients. One sample contained 30-70 different operational taxonomic units; the mean (± standard deviation) was lower in the primary CAP group (36 ± 4) than in the PA (45 ± 4) and secondary CAP (43 ± 13) groups (P < .05). The communities were individually different, but anaerobic bacteria predominated as the rule. Enterococcus faecalis was found only in patients with secondary CAP. One PA sample displayed a significantly high proportion (47%) of Proteobacteria, mainly at the expense of Janthinobacterium lividum. This study provided an in-depth characterization of the microbiota of periapical tissues, revealing highly polymicrobial communities and minor differences between the study groups. A full understanding of the etiology of periodontal disease will only be possible through further in-depth systems-level analyses of the host-microbiome interaction. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  11. Genetic diversity in breonadia salicina based on intra-species sequence variation of chloroplast dna spacer sequence

    International Nuclear Information System (INIS)

    Qurainy, F.A.; Gaafar, A.R.Z.

    2014-01-01

    Assessment and knowledge of the genetic diversity and variation within and between populations of rare and endangered plants is very important for effective conservation. Intergenic spacer sequences variation of psbA-trnH locus of chloroplast genome was assessed within Breonadia salicina (Rubiaceae), a critically endangered and endemic plant species to South western part of Kingdom of Saudi Arabia. The obtained sequence data from 19 individuals in three populations revealed nine haplotypes. The aligned sequences obtained from the overall Saudi accessions extended to 355 bp, revealing nine haplotypes. A high level of haplotype diversity (Hd = 0.842) and low level of nucleotide diversity (Pi = 0.0058) were detected. Consistently, both hierarchical analysis of molecular variance (AMOVA) and constructed neighbor-joining tree indicated null genetic differentiation among populations. This level of differentiation between populations or between regions in psbA-trnH sequences may be due to effects of the abundance of ancestral haplotype sharing and the presence of private haplotypes fixed for each population. Furthermore, the results revealed almost the same level of genetic diversity in comparison with Yemeni accessions, in which Saudi accessions were sharing three haplotypes from the four haplotypes found in Yemeni accessions. (author)

  12. Assembly of the Lactuca sativa, L. cv. Tizian draft genome sequence reveals differences within major resistance complex 1 as compared to the cv. Salinas reference genome.

    Science.gov (United States)

    Verwaaijen, Bart; Wibberg, Daniel; Nelkner, Johanna; Gordin, Miriam; Rupp, Oliver; Winkler, Anika; Bremges, Andreas; Blom, Jochen; Grosch, Rita; Pühler, Alfred; Schlüter, Andreas

    2018-02-10

    Lettuce (Lactuca sativa, L.) is an important annual plant of the family Asteraceae (Compositae). The commercial lettuce cultivar Tizian has been used in various scientific studies investigating the interaction of the plant with phytopathogens or biological control agents. Here, we present the de novo draft genome sequencing and gene prediction for this specific cultivar derived from transcriptome sequence data. The assembled scaffolds amount to a size of 2.22 Gb. Based on RNAseq data, 31,112 transcript isoforms were identified. Functional predictions for these transcripts were determined within the GenDBE annotation platform. Comparison with the cv. Salinas reference genome revealed a high degree of sequence similarity on genome and transcriptome levels, with an average amino acid identity of 99%. Furthermore, it was observed that two large regions are either missing or are highly divergent within the cv. Tizian genome compared to cv. Salinas. One of these regions covers the major resistance complex 1 region of cv. Salinas. The cv. Tizian draft genome sequence provides a valuable resource for future functional and transcriptome analyses focused on this lettuce cultivar. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Genetic variation in the Staphylococcus aureus 8325 strain lineage revealed by whole-genome sequencing.

    Directory of Open Access Journals (Sweden)

    Kristoffer T Bæk

    Full Text Available Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47 required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75% steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3, a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.

  14. Application of high-throughput DNA sequencing in phytopathology.

    Science.gov (United States)

    Studholme, David J; Glover, Rachel H; Boonham, Neil

    2011-01-01

    The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Copyright © 2011 by Annual Reviews. All rights reserved.

  15. Single-nucleotide polymorphism discovery by high-throughput sequencing in sorghum

    Directory of Open Access Journals (Sweden)

    White Frank F

    2011-07-01

    Full Text Available Abstract Background Eight diverse sorghum (Sorghum bicolor L. Moench accessions were subjected to short-read genome sequencing to characterize the distribution of single-nucleotide polymorphisms (SNPs. Two strategies were used for DNA library preparation. Missing SNP genotype data were imputed by local haplotype comparison. The effect of library type and genomic diversity on SNP discovery and imputation are evaluated. Results Alignment of eight genome equivalents (6 Gb to the public reference genome revealed 283,000 SNPs at ≥82% confirmation probability. Sequencing from libraries constructed to limit sequencing to start at defined restriction sites led to genotyping 10-fold more SNPs in all 8 accessions, and correctly imputing 11% more missing data, than from semirandom libraries. The SNP yield advantage of the reduced-representation method was less than expected, since up to one fifth of reads started at noncanonical restriction sites and up to one third of restriction sites predicted in silico to yield unique alignments were not sampled at near-saturation. For imputation accuracy, the availability of a genomically similar accession in the germplasm panel was more important than panel size or sequencing coverage. Conclusions A sequence quantity of 3 million 50-base reads per accession using a BsrFI library would conservatively provide satisfactory genotyping of 96,000 sorghum SNPs. For most reliable SNP-genotype imputation in shallowly sequenced genomes, germplasm panels should consist of pairs or groups of genomically similar entries. These results may help in designing strategies for economical genotyping-by-sequencing of large numbers of plant accessions.

  16. Mitochondrial genome sequences reveal deep divergences among Anopheles punctulatus sibling species in Papua New Guinea

    Directory of Open Access Journals (Sweden)

    Logue Kyle

    2013-02-01

    Full Text Available Abstract Background Members of the Anopheles punctulatus group (AP group are the primary vectors of human malaria in Papua New Guinea. The AP group includes 13 sibling species, most of them morphologically indistinguishable. Understanding why only certain species are able to transmit malaria requires a better comprehension of their evolutionary history. In particular, understanding relationships and divergence times among Anopheles species may enable assessing how malaria-related traits (e.g. blood feeding behaviours, vector competence have evolved. Methods DNA sequences of 14 mitochondrial (mt genomes from five AP sibling species and two species of the Anopheles dirus complex of Southeast Asia were sequenced. DNA sequences from all concatenated protein coding genes (10,770 bp were then analysed using a Bayesian approach to reconstruct phylogenetic relationships and date the divergence of the AP sibling species. Results Phylogenetic reconstruction using the concatenated DNA sequence of all mitochondrial protein coding genes indicates that the ancestors of the AP group arrived in Papua New Guinea 25 to 54 million years ago and rapidly diverged to form the current sibling species. Conclusion Through evaluation of newly described mt genome sequences, this study has revealed a divergence among members of the AP group in Papua New Guinea that would significantly predate the arrival of humans in this region, 50 thousand years ago. The divergence observed among the mtDNA sequences studied here may have resulted from reproductive isolation during historical changes in sea-level through glacial minima and maxima. This leads to a hypothesis that the AP sibling species have evolved independently for potentially thousands of generations. This suggests that the evolution of many phenotypes, such as insecticide resistance will arise independently in each of the AP sibling species studied here.

  17. High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

    Science.gov (United States)

    Feng, Juanjuan; Wang, Jinhui; Fan, Pengxiang; Jia, Weitao; Nie, Lingling; Jiang, Ping; Chen, Xianyang; Lv, Sulian; Wan, Lichuan; Chang, Sandra; Li, Shizhong; Li, Yinxin

    2015-02-26

    microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far. Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE. Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable

  18. WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

    Science.gov (United States)

    Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

    2010-07-01

    High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

  19. Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

    Science.gov (United States)

    Yu, Xin-Yi; Du, Bei-Bei; Gao, Zhi-Hong; Zhang, Shi-Jie; Tu, Xu-Tong; Chen, Xiao-Yun; Zhang, Zhen; Qu, Shen-Chun

    2014-08-01

    MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.

  20. Probabilistic Methods for Processing High-Throughput Sequencing Signals

    DEFF Research Database (Denmark)

    Sørensen, Lasse Maretty

    High-throughput sequencing has the potential to answer many of the big questions in biology and medicine. It can be used to determine the ancestry of species, to chart complex ecosystems and to understand and diagnose disease. However, going from raw sequencing data to biological or medical insig....... By estimating the genotypes on a set of candidate variants obtained from both a standard mapping-based approach as well as de novo assemblies, we are able to find considerably more structural variation than previous studies...... for reconstructing transcript sequences from RNA sequencing data. The method is based on a novel sparse prior distribution over transcript abundances and is markedly more accurate than existing approaches. The second chapter describes a new method for calling genotypes from a fixed set of candidate variants....... The method queries the reads using a graph representation of the variants and hereby mitigates the reference-bias that characterise standard genotyping methods. In the last chapter, we apply this method to call the genotypes of 50 deeply sequencing parent-offspring trios from the GenomeDenmark project...

  1. Sources of PCR-induced distortions in high-throughput sequencing data sets

    Science.gov (United States)

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  2. Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

    Directory of Open Access Journals (Sweden)

    Takeru Nakazato

    Full Text Available High-throughput sequencing technology, also called next-generation sequencing (NGS, has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA. As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/. This service will improve accessibility to high-quality data from SRA.

  3. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    Directory of Open Access Journals (Sweden)

    Aurélien Chateigner

    2015-07-01

    Full Text Available Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%. K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs. Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  4. Tracking TCRβ sequence clonotype expansions during antiviral therapy using high-throughput sequencing of the hypervariable region

    Directory of Open Access Journals (Sweden)

    Mark W Robinson

    2016-04-01

    Full Text Available To maintain a persistent infection viruses such as hepatitis C virus (HCV employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilise either antigen stimulation (e.g. ELISpot, proliferation assays, cytokine production or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently high-throughput sequencing (HTS technologies have been developed for the analysis of T cell repertoires. In the present study we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing anti-viral therapy we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study however the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high joining region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101–restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of

  5. Communicating the Benefits of a Full Sequence of High School Science Courses

    Science.gov (United States)

    Nicholas, Catherine Marie

    2014-01-01

    High school students are generally uninformed about the benefits of enrolling in a full sequence of science courses, therefore only about a third of our nation's high school graduates have completed the science sequence of Biology, Chemistry and Physics. The lack of students completing a full sequence of science courses contributes to the deficit…

  6. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing

    DEFF Research Database (Denmark)

    Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P

    2007-01-01

    BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine...... primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution...

  7. Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts.

    Science.gov (United States)

    Webster, Nicole S; Taylor, Michael W; Behnam, Faris; Lücker, Sebastian; Rattei, Thomas; Whalan, Stephen; Horn, Matthias; Wagner, Michael

    2010-08-01

    Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250,000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described 'sponge-specific' clusters that were detected in this study, 48% were found exclusively in adults and larvae - implying vertical transmission of these groups. The remaining taxa, including 'Poribacteria', were also found at very low abundance among the 135,000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

  8. Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging.

    Science.gov (United States)

    Logan, Grace; Newman, Joseph; Wright, Caroline F; Lasecka-Dykes, Lidia; Haydon, Daniel T; Cottam, Eleanor M; Tuthill, Tobias J

    2017-10-18

    Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the

  9. Scrutinizing virus genome termini by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Shasha Li

    Full Text Available Analysis of genomic terminal sequences has been a major step in studies on viral DNA replication and packaging mechanisms. However, traditional methods to study genome termini are challenging due to the time-consuming protocols and their inefficiency where critical details are lost easily. Recent advances in next generation sequencing (NGS have enabled it to be a powerful tool to study genome termini. In this study, using NGS we sequenced one iridovirus genome and twenty phage genomes and confirmed for the first time that the high frequency sequences (HFSs found in the NGS reads are indeed the terminal sequences of viral genomes. Further, we established a criterion to distinguish the type of termini and the viral packaging mode. We also obtained additional terminal details such as terminal repeats, multi-termini, asymmetric termini. With this approach, we were able to simultaneously detect details of the genome termini as well as obtain the complete sequence of bacteriophage genomes. Theoretically, this application can be further extended to analyze larger and more complicated genomes of plant and animal viruses. This study proposed a novel and efficient method for research on viral replication, packaging, terminase activity, transcription regulation, and metabolism of the host cell.

  10. SUGAR: graphical user interface-based data refiner for high-throughput DNA sequencing.

    Science.gov (United States)

    Sato, Yukuto; Kojima, Kaname; Nariai, Naoki; Yamaguchi-Kabata, Yumi; Kawai, Yosuke; Takahashi, Mamoru; Mimori, Takahiro; Nagasaki, Masao

    2014-08-08

    Next-generation sequencers (NGSs) have become one of the main tools for current biology. To obtain useful insights from the NGS data, it is essential to control low-quality portions of the data affected by technical errors such as air bubbles in sequencing fluidics. We develop a software SUGAR (subtile-based GUI-assisted refiner) which can handle ultra-high-throughput data with user-friendly graphical user interface (GUI) and interactive analysis capability. The SUGAR generates high-resolution quality heatmaps of the flowcell, enabling users to find possible signals of technical errors during the sequencing. The sequencing data generated from the error-affected regions of a flowcell can be selectively removed by automated analysis or GUI-assisted operations implemented in the SUGAR. The automated data-cleaning function based on sequence read quality (Phred) scores was applied to a public whole human genome sequencing data and we proved the overall mapping quality was improved. The detailed data evaluation and cleaning enabled by SUGAR would reduce technical problems in sequence read mapping, improving subsequent variant analysis that require high-quality sequence data and mapping results. Therefore, the software will be especially useful to control the quality of variant calls to the low population cells, e.g., cancers, in a sample with technical errors of sequencing procedures.

  11. Targeted exome sequencing reveals novel USH2A mutations in Chinese patients with simplex Usher syndrome.

    Science.gov (United States)

    Shu, Hai-Rong; Bi, Huai; Pan, Yang-Chun; Xu, Hang-Yu; Song, Jian-Xin; Hu, Jie

    2015-09-16

    Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing. In this study, we applied a next-generation sequencing approach to detect genetic abnormalities in patients with USH. Two unrelated Chinese families were recruited, consisting of two USH afflicted patients and four unaffected relatives. We selected 199 genes related to inherited retinal diseases as targets for deep exome sequencing. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis. A homozygous frameshift mutation (c.4382delA, p.T1462Lfs*2) was revealed in exon20 of gene USH2A in the F1 family. Two compound heterozygous mutations, IVS47 + 1G > A and c.13156A > T (p.I4386F), located in intron 48 and exon 63 respectively, of USH2A, were identified as causative mutations for the F2 family. Of note, the missense mutation c.13156A > T has not been reported so far. In conclusion, targeted exome sequencing precisely and rapidly identified the genetic defects in two Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.

  12. RNA-Sequencing Reveals Unique Transcriptional Signatures of Running and Running-Independent Environmental Enrichment in the Adult Mouse Dentate Gyrus.

    Science.gov (United States)

    Grégoire, Catherine-Alexandra; Tobin, Stephanie; Goldenstein, Brianna L; Samarut, Éric; Leclerc, Andréanne; Aumont, Anne; Drapeau, Pierre; Fulton, Stephanie; Fernandes, Karl J L

    2018-01-01

    Environmental enrichment (EE) is a powerful stimulus of brain plasticity and is among the most accessible treatment options for brain disease. In rodents, EE is modeled using multi-factorial environments that include running, social interactions, and/or complex surroundings. Here, we show that running and running-independent EE differentially affect the hippocampal dentate gyrus (DG), a brain region critical for learning and memory. Outbred male CD1 mice housed individually with a voluntary running disk showed improved spatial memory in the radial arm maze compared to individually- or socially-housed mice with a locked disk. We therefore used RNA sequencing to perform an unbiased interrogation of DG gene expression in mice exposed to either a voluntary running disk (RUN), a locked disk (LD), or a locked disk plus social enrichment and tunnels [i.e., a running-independent complex environment (CE)]. RNA sequencing revealed that RUN and CE mice showed distinct, non-overlapping patterns of transcriptomic changes versus the LD control. Bio-informatics uncovered that the RUN and CE environments modulate separate transcriptional networks, biological processes, cellular compartments and molecular pathways, with RUN preferentially regulating synaptic and growth-related pathways and CE altering extracellular matrix-related functions. Within the RUN group, high-distance runners also showed selective stress pathway alterations that correlated with a drastic decline in overall transcriptional changes, suggesting that excess running causes a stress-induced suppression of running's genetic effects. Our findings reveal stimulus-dependent transcriptional signatures of EE on the DG, and provide a resource for generating unbiased, data-driven hypotheses for novel mediators of EE-induced cognitive changes.

  13. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  14. 3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2013-09-21

    Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

  15. Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

    Science.gov (United States)

    Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Okuno, Yusuke; Yamato, Genki; Hara, Yusuke; Nagata, Yasunobu; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Kato, Motohiro; Park, Myoung-Ja; Ohki, Kentaro; Shimada, Akira; Takita, Junko; Tomizawa, Daisuke; Kudo, Kazuko; Arakawa, Hirokazu; Adachi, Souichi; Taga, Takashi; Tawa, Akio; Ito, Etsuro; Horibe, Keizo; Sanada, Masashi; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

    2016-11-01

    Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence. © 2016 John Wiley & Sons Ltd.

  16. Whale phylogeny and rapid radiation events revealed using novel retroposed elements and their flanking sequences.

    Science.gov (United States)

    Chen, Zhuo; Xu, Shixia; Zhou, Kaiya; Yang, Guang

    2011-10-27

    A diversity of hypotheses have been proposed based on both morphological and molecular data to reveal phylogenetic relationships within the order Cetacea (dolphins, porpoises, and whales), and great progress has been made in the past two decades. However, there is still some controversy concerning relationships among certain cetacean taxa such as river dolphins and delphinoid species, which needs to be further addressed with more markers in an effort to address unresolved portions of the phylogeny. An analysis of additional SINE insertions and SINE-flanking sequences supported the monophyly of the order Cetacea as well as Odontocete, Delphinoidea (Delphinidae + Phocoenidae + Mondontidae), and Delphinidae. A sister relationship between Delphinidae and Phocoenidae + Mondontidae was supported, and members of classical river dolphins and the genera Tursiops and Stenella were found to be paraphyletic. Estimates of divergence times revealed rapid divergences of basal Odontocete lineages in the Oligocene and Early Miocene, and a recent rapid diversification of Delphinidae in the Middle-Late Miocene and Pliocene within a narrow time frame. Several novel SINEs were found to differentiate Delphinidae from the other two families (Monodontidae and Phocoenidae), whereas the sister grouping of the latter two families with exclusion of Delphinidae was further revealed using the SINE-flanking sequences. Interestingly, some anomalous PCR amplification patterns of SINE insertions were detected, which can be explained as the result of potential ancestral SINE polymorphisms and incomplete lineage sorting. Although a few loci were potentially anomalous, this study demonstrated that the SINE-based approach is a powerful tool in phylogenetic studies. Identifying additional SINE elements that resolve the relationships in the superfamily Delphinoidea and family Delphinidae will be important steps forward in completely resolving cetacean phylogenetic relationships in the future.

  17. Whale phylogeny and rapid radiation events revealed using novel retroposed elements and their flanking sequences

    Directory of Open Access Journals (Sweden)

    Zhou Kaiya

    2011-10-01

    Full Text Available Abstract Background A diversity of hypotheses have been proposed based on both morphological and molecular data to reveal phylogenetic relationships within the order Cetacea (dolphins, porpoises, and whales, and great progress has been made in the past two decades. However, there is still some controversy concerning relationships among certain cetacean taxa such as river dolphins and delphinoid species, which needs to be further addressed with more markers in an effort to address unresolved portions of the phylogeny. Results An analysis of additional SINE insertions and SINE-flanking sequences supported the monophyly of the order Cetacea as well as Odontocete, Delphinoidea (Delphinidae + Phocoenidae + Mondontidae, and Delphinidae. A sister relationship between Delphinidae and Phocoenidae + Mondontidae was supported, and members of classical river dolphins and the genera Tursiops and Stenella were found to be paraphyletic. Estimates of divergence times revealed rapid divergences of basal Odontocete lineages in the Oligocene and Early Miocene, and a recent rapid diversification of Delphinidae in the Middle-Late Miocene and Pliocene within a narrow time frame. Conclusions Several novel SINEs were found to differentiate Delphinidae from the other two families (Monodontidae and Phocoenidae, whereas the sister grouping of the latter two families with exclusion of Delphinidae was further revealed using the SINE-flanking sequences. Interestingly, some anomalous PCR amplification patterns of SINE insertions were detected, which can be explained as the result of potential ancestral SINE polymorphisms and incomplete lineage sorting. Although a few loci were potentially anomalous, this study demonstrated that the SINE-based approach is a powerful tool in phylogenetic studies. Identifying additional SINE elements that resolve the relationships in the superfamily Delphinoidea and family Delphinidae will be important steps forward in completely resolving

  18. Deep sequencing analysis of the developing mouse brain reveals a novel microRNA

    Directory of Open Access Journals (Sweden)

    Piltz Sandra

    2011-04-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small non-coding RNAs that can exert multilevel inhibition/repression at a post-transcriptional or protein synthesis level during disease or development. Characterisation of miRNAs in adult mammalian brains by deep sequencing has been reported previously. However, to date, no small RNA profiling of the developing brain has been undertaken using this method. We have performed deep sequencing and small RNA analysis of a developing (E15.5 mouse brain. Results We identified the expression of 294 known miRNAs in the E15.5 developing mouse brain, which were mostly represented by let-7 family and other brain-specific miRNAs such as miR-9 and miR-124. We also discovered 4 putative 22-23 nt miRNAs: mm_br_e15_1181, mm_br_e15_279920, mm_br_e15_96719 and mm_br_e15_294354 each with a 70-76 nt predicted pre-miRNA. We validated the 4 putative miRNAs and further characterised one of them, mm_br_e15_1181, throughout embryogenesis. Mm_br_e15_1181 biogenesis was Dicer1-dependent and was expressed in E3.5 blastocysts and E7 whole embryos. Embryo-wide expression patterns were observed at E9.5 and E11.5 followed by a near complete loss of expression by E13.5, with expression restricted to a specialised layer of cells within the developing and early postnatal brain. Mm_br_e15_1181 was upregulated during neurodifferentiation of P19 teratocarcinoma cells. This novel miRNA has been identified as miR-3099. Conclusions We have generated and analysed the first deep sequencing dataset of small RNA sequences of the developing mouse brain. The analysis revealed a novel miRNA, miR-3099, with potential regulatory effects on early embryogenesis, and involvement in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development.

  19. Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants.

    Science.gov (United States)

    Guo, Shengye; Li, Xingyu; He, Pengfei; Ho, Honhing; Wu, Yixin; He, Yueqiu

    2015-06-01

    Bacillus subtilis XF-1 is a gram-positive, plant-associated bacterium that stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. In particular, it is especially highly efficient at controlling the clubroot disease of cruciferous crops. Its 4,061,186-bp genome contains an estimated 3853 protein-coding sequences and the 1155 genes of XF-1 are present in most genome-sequenced Bacillus strains: 3757 genes in B. subtilis 168, and 1164 in B. amyloliquefaciens FZB42. Analysis using the Cluster of Orthologous Groups database of proteins shows that 60 genes control bacterial mobility, 221 genes are related to cell wall and membrane biosynthesis, and more than 112 are genes associated with secondary metabolites. In addition, the genes contributed to the strain's plant colonization, bio-control and stimulation of plant growth. Sequencing of the genome is a fundamental step for developing a desired strain to serve as an efficient biological control agent and plant growth stimulator. Similar to other members of the taxon, XF-1 has a genome that contains giant gene clusters for the non-ribosomal synthesis of antifungal lipopeptides (surfactin and fengycin), the polyketides (macrolactin and bacillaene), the siderophore bacillibactin, and the dipeptide bacilysin. There are two synthesis pathways for volatile growth-promoting compounds. The expression of biosynthesized antibiotic peptides in XF-1 was revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

  20. A search for pre-main-sequence stars in high-latitude molecular clouds. 3: A survey of the Einstein database

    Science.gov (United States)

    Caillault, Jean-Pierre; Magnani, Loris; Fryer, Chris

    1995-01-01

    In order to discern whether the high-latitude molecular clouds are regions of ongoing star formation, we have used X-ray emission as a tracer of youthful stars. The entire Einstein database yields 18 images which overlap 10 of the clouds mapped partially or completely in the CO (1-0) transition, providing a total of approximately 6 deg squared of overlap. Five previously unidentified X-ray sources were detected: one has an optical counterpart which is a pre-main-sequence (PMS) star, and two have normal main-sequence stellar counterparts, while the other two are probably extragalactic sources. The PMS star is located in a high Galactic latitude Lynds dark cloud, so this result is not too suprising. The translucent clouds, though, have yet to reveal any evidence of star formation.

  1. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

    Science.gov (United States)

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-07-15

    In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

  2. The application of the high throughput sequencing technology in the transposable elements.

    Science.gov (United States)

    Liu, Zhen; Xu, Jian-hong

    2015-09-01

    High throughput sequencing technology has dramatically improved the efficiency of DNA sequencing, and decreased the costs to a great extent. Meanwhile, this technology usually has advantages of better specificity, higher sensitivity and accuracy. Therefore, it has been applied to the research on genetic variations, transcriptomics and epigenomics. Recently, this technology has been widely employed in the studies of transposable elements and has achieved fruitful results. In this review, we summarize the application of high throughput sequencing technology in the fields of transposable elements, including the estimation of transposon content, preference of target sites and distribution, insertion polymorphism and population frequency, identification of rare copies, transposon horizontal transfers as well as transposon tagging. We also briefly introduce the major common sequencing strategies and algorithms, their advantages and disadvantages, and the corresponding solutions. Finally, we envision the developing trends of high throughput sequencing technology, especially the third generation sequencing technology, and its application in transposon studies in the future, hopefully providing a comprehensive understanding and reference for related scientific researchers.

  3. Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing

    Science.gov (United States)

    Alana Alexander; Debbie Steel; Beth Slikas; Kendra Hoekzema; Colm Carraher; Matthew Parks; Richard Cronn; C. Scott Baker

    2012-01-01

    Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20...

  4. Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants.

    Directory of Open Access Journals (Sweden)

    Sophie Lanciano

    2017-02-01

    Full Text Available Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

  5. Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants.

    Science.gov (United States)

    Lanciano, Sophie; Carpentier, Marie-Christine; Llauro, Christel; Jobet, Edouard; Robakowska-Hyzorek, Dagmara; Lasserre, Eric; Ghesquière, Alain; Panaud, Olivier; Mirouze, Marie

    2017-02-01

    Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

  6. Evolution of MHC class I genes in the endangered loggerhead sea turtle (Caretta caretta) revealed by 454 amplicon sequencing.

    Science.gov (United States)

    Stiebens, Victor A; Merino, Sonia E; Chain, Frédéric J J; Eizaguirre, Christophe

    2013-04-30

    In evolutionary and conservation biology, parasitism is often highlighted as a major selective pressure. To fight against parasites and pathogens, genetic diversity of the immune genes of the major histocompatibility complex (MHC) are particularly important. However, the extensive degree of polymorphism observed in these genes makes it difficult to conduct thorough population screenings. We utilized a genotyping protocol that uses 454 amplicon sequencing to characterize the MHC class I in the endangered loggerhead sea turtle (Caretta caretta) and to investigate their evolution at multiple relevant levels of organization. MHC class I genes revealed signatures of trans-species polymorphism across several reptile species. In the studied loggerhead turtle individuals, it results in the maintenance of two ancient allelic lineages. We also found that individuals carrying an intermediate number of MHC class I alleles are larger than those with either a low or high number of alleles. Multiple modes of evolution seem to maintain MHC diversity in the loggerhead turtles, with relatively high polymorphism for an endangered species.

  7. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  8. Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing

    KAUST Repository

    Rashid, Jonaira; Kobiyama, Atsushi; Reza, Md. Shaheed; Yamada, Yuichiro; Ikeda, Yuri; Ikeda, Daisuke; Mizusawa, Nanami; Ikeo, Kazuho; Sato, Shigeru; Ogata, Takehiko; Kudo, Toshiaki; Kaga, Shinnosuke; Watanabe, Shiho; Naiki, Kimiaki; Kaga, Yoshimasa; Mineta, Katsuhiko; Bajic, Vladimir B.; Gojobori, Takashi; Watabe, Shugo

    2018-01-01

    Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42–49%, 35–49% and 13–17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and late autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

  9. Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing

    KAUST Repository

    Rashid, Jonaira

    2018-04-30

    Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42–49%, 35–49% and 13–17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and late autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

  10. Exome sequencing in Jewish and Arab patients with rhabdomyolysis reveals single-gene etiology in 43% of cases.

    Science.gov (United States)

    Vivante, Asaf; Ityel, Hadas; Pode-Shakked, Ben; Chen, Jing; Shril, Shirlee; van der Ven, Amelie T; Mann, Nina; Schmidt, Johanna Magdalena; Segel, Reeval; Aran, Adi; Zeharia, Avraham; Staretz-Chacham, Orna; Bar-Yosef, Omer; Raas-Rothschild, Annick; Landau, Yuval E; Lifton, Richard P; Anikster, Yair; Hildebrandt, Friedhelm

    2017-12-01

    Rhabdomyolysis is a clinical emergency that may cause acute kidney injury (AKI). It can be acquired or due to monogenic mutations. Around 60 different rare monogenic forms of rhabdomyolysis have been reported to date. In the clinical setting, identifying the underlying molecular diagnosis is challenging due to nonspecific presentation, the high number of causative genes, and current lack of data on the prevalence of monogenic forms. We employed whole exome sequencing (WES) to reveal the percentage of rhabdomyolysis cases explained by single-gene (monogenic) mutations in one of 58 candidate genes. We investigated a cohort of 21 unrelated families with rhabdomyolysis, in whom no underlying etiology had been previously established. Using WES, we identified causative mutations in candidate genes in nine of the 21 families (43%). We detected disease-causing mutations in eight of 58 candidate genes, grouped into the following categories: (1) disorders of fatty acid metabolism (CPT2), (2) disorders of glycogen metabolism (PFKM and PGAM2), (3) disorders of abnormal skeletal muscle relaxation and contraction (CACNA1S, MYH3, RYR1 and SCN4A), and (4) disorders of purine metabolism (AHCY). Our findings demonstrate a very high detection rate for monogenic etiologies using WES and reveal broad genetic heterogeneity for rhabdomyolysis. These results highlight the importance of molecular genetic diagnostics for establishing an etiologic diagnosis. Because these patients are at risk for recurrent episodes of rhabdomyolysis and subsequent risk for AKI, WES allows adequate prophylaxis and treatment for these patients and their family members and enables a personalized medicine approach.

  11. Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context.

    Science.gov (United States)

    Candresse, Thierry; Filloux, Denis; Muhire, Brejnev; Julian, Charlotte; Galzi, Serge; Fort, Guillaume; Bernardo, Pauline; Daugrois, Jean-Heindrich; Fernandez, Emmanuel; Martin, Darren P; Varsani, Arvind; Roumagnac, Philippe

    2014-01-01

    Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non

  12. Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context.

    Directory of Open Access Journals (Sweden)

    Thierry Candresse

    Full Text Available Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS of both virus-derived small interfering RNAs (siRNAs and virion-associated nucleic acids (VANA for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae, but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV. This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non

  13. Highly accurate sequence imputation enables precise QTL mapping in Brown Swiss cattle.

    Science.gov (United States)

    Frischknecht, Mirjam; Pausch, Hubert; Bapst, Beat; Signer-Hasler, Heidi; Flury, Christine; Garrick, Dorian; Stricker, Christian; Fries, Ruedi; Gredler-Grandl, Birgit

    2017-12-29

    Within the last few years a large amount of genomic information has become available in cattle. Densities of genomic information vary from a few thousand variants up to whole genome sequence information. In order to combine genomic information from different sources and infer genotypes for a common set of variants, genotype imputation is required. In this study we evaluated the accuracy of imputation from high density chips to whole genome sequence data in Brown Swiss cattle. Using four popular imputation programs (Beagle, FImpute, Impute2, Minimac) and various compositions of reference panels, the accuracy of the imputed sequence variant genotypes was high and differences between the programs and scenarios were small. We imputed sequence variant genotypes for more than 1600 Brown Swiss bulls and performed genome-wide association studies for milk fat percentage at two stages of lactation. We found one and three quantitative trait loci for early and late lactation fat content, respectively. Known causal variants that were imputed from the sequenced reference panel were among the most significantly associated variants of the genome-wide association study. Our study demonstrates that whole-genome sequence information can be imputed at high accuracy in cattle populations. Using imputed sequence variant genotypes in genome-wide association studies may facilitate causal variant detection.

  14. Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

    Science.gov (United States)

    Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

    2017-11-13

    A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

  15. RNA Sequencing and Coexpression Analysis Reveal Key Genes Involved in α-Linolenic Acid Biosynthesis in Perilla frutescens Seed

    Directory of Open Access Journals (Sweden)

    Tianyuan Zhang

    2017-11-01

    Full Text Available Perilla frutescen is used as traditional food and medicine in East Asia. Its seeds contain high levels of α-linolenic acid (ALA, which is important for health, but is scarce in our daily meals. Previous reports on RNA-seq of perilla seed had identified fatty acid (FA and triacylglycerol (TAG synthesis genes, but the underlying mechanism of ALA biosynthesis and its regulation still need to be further explored. So we conducted Illumina RNA-sequencing in seven temporal developmental stages of perilla seeds. Sequencing generated a total of 127 million clean reads, containing 15.88 Gb of valid data. The de novo assembly of sequence reads yielded 64,156 unigenes with an average length of 777 bp. A total of 39,760 unigenes were annotated and 11,693 unigenes were found to be differentially expressed in all samples. According to Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis, 486 unigenes were annotated in the “lipid metabolism” pathway. Of these, 150 unigenes were found to be involved in fatty acid (FA biosynthesis and triacylglycerol (TAG assembly in perilla seeds. A coexpression analysis showed that a total of 104 genes were highly coexpressed (r > 0.95. The coexpression network could be divided into two main subnetworks showing over expression in the medium or earlier and late phases, respectively. In order to identify the putative regulatory genes, a transcription factor (TF analysis was performed. This led to the identification of 45 gene families, mainly including the AP2-EREBP, bHLH, MYB, and NAC families, etc. After coexpression analysis of TFs with highly expression of FAD2 and FAD3 genes, 162 TFs were found to be significantly associated with two FAD genes (r > 0.95. Those TFs were predicted to be the key regulatory factors in ALA biosynthesis in perilla seed. The qRT-PCR analysis also verified the relevance of expression pattern between two FAD genes and partial candidate TFs. Although it has been reported that some TFs

  16. Sedimentology and High Resolution Sequence Stratigraphy of the Middle Jurassic Dhruma Formation Carbonates Outcrops in the Central Saudi Arabia

    Science.gov (United States)

    Yousif, Ibrahim; Abdullatif, Osman; Makkawi, Mohammed; Abdulghani, Waleed

    2017-04-01

    This study investigates the microfacies and sequence stratigraphic frame work of the Middle Jurassic Dhruma Formation in outcrops in central Saudi Arabia. The study contributes to the efforts to understand and enhance local and regional stratigraphic relationship and correlation of the Jurassic carbonate sequences and their significance to reservoir description and prediction in the subsurcae. The study describes and characterizes the sedimentology, microfacies and the stratigraphy of Dhruma Formation from outcrop sections having a total thickness of 70 m. Detailed microfacies and high-resolution stratigraphical analysis were carried out to determine microfacies, cyclicity, sequences and staking pattern. The study revealed ten lithofacies namely: oolitic grainstone,bioclastic oolitic grainstone, oolitic grapestone, bioclastic grainstone,foraminiferal packstone, echinoderm packstone, peloidal packstone to grainstone,skeletal wackestone to packstone, mudstone, and marlstone.These lithofacies were grouped into five lithofacies associations that deposited on a carbonate ramp setting. The depositional environment ranging from low energy lagoonal setting to high-energy shoals and banks to low energy outer ramp setting. Five high-resolution composite sequences have been defined and each sequence is composed at the bottom of intercalated mudstone/wackestone that passing up into grainstone lithofacies.The composite sequences range in thickness from 7 to 15 m, while the parasequences range from 0.5 to 1.5 m. The composite sequences extend laterally for a distance of more than 350 m. The overall composite section shows a shallowing upward succession of the 4th to the 5th order high-resolution sequences.The dominant lithofacies are the grainy ones, which constitute 30%, 50% and 80% of the studied sections. Furthermore, the parasequences thickness and their bio-components are increasing towards the top. The muddy lithofacies intensively affected the vertical continuity of the

  17. Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

    Science.gov (United States)

    Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

    2017-01-01

    Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

  18. Deep sequencing of the viral phoH gene reveals temporal variation, depth-specific composition, and persistent dominance of the same viral phoH genes in the Sargasso Sea

    Directory of Open Access Journals (Sweden)

    Dawn B. Goldsmith

    2015-06-01

    Full Text Available Deep sequencing of the viral phoH gene, a host-derived auxiliary metabolic gene, was used to track viral diversity throughout the water column at the Bermuda Atlantic Time-series Study (BATS site in the summer (September and winter (March of three years. Viral phoH sequences reveal differences in the viral communities throughout a depth profile and between seasons in the same year. Variation was also detected between the same seasons in subsequent years, though these differences were not as great as the summer/winter distinctions. Over 3,600 phoH operational taxonomic units (OTUs; 97% sequence identity were identified. Despite high richness, most phoH sequences belong to a few large, common OTUs whereas the majority of the OTUs are small and rare. While many OTUs make sporadic appearances at just a few times or depths, a small number of OTUs dominate the community throughout the seasons, depths, and years.

  19. High resolution sea-level curve for the latest Frasnian and earliest Famennian derived for high frequency sequences in the Appalachian Basin

    Energy Technology Data Exchange (ETDEWEB)

    Filer, J.K. (Washington and Lee Univ., Lexington, VA (United States). Dept. of Geology)

    1992-01-01

    Siliciclastic sequences have been mapped in the subsurface and outcrop of much of the Appalachian basin in facies ranging from shale in the basin plain to shelf sandstone. Eleven transgressive/regressive cycles have been defined in an estimated 1.5 to 2.0 Ma period in the latest Frasnian and earliest Famennian, and range in duration from about 75,000 to 400,000 years. Lithofacies maps, covering most of the basin, were prepared for each sequence. These maps show both the area of basinal black shale deposition, which defines the base of each cycle, and the areal extent of subsequent clinoform siltstone and shelf sandstone deposition in the upper portion of each cycle. The stratigraphic patterns show two stacked sets of progradational basinwide sequences. Geographic scale of the study precludes autocyclic controls of cycles. Sea-level/climate cycles, probably superimposed on longer term tectonic cycles, are the proposed cause of these observed depositional patterns. Removal of the long-term progradational trend of Upper Devonian basin filling results in a proposed eustatic sea-level curve (Johnson and others (1985)) reveals correspondence of three regressive maxima in both models. The curve presented here reveals that an ongoing process of higher frequency sea-level modification was active at this time. Higher frequency sea-level events, nested within previously interpreted lower frequency global events, are inferred to also be eustatic. Models of a biotic crises which occurs at this time should consider the implications of these high frequency sea-level cycles. The patterns observed are consistent with latest Frasnian initiation of glaciation in South America. This would be somewhat earlier than has generally been accepted.

  20. RNA-Sequencing Reveals Unique Transcriptional Signatures of Running and Running-Independent Environmental Enrichment in the Adult Mouse Dentate Gyrus

    Directory of Open Access Journals (Sweden)

    Catherine-Alexandra Grégoire

    2018-04-01

    Full Text Available Environmental enrichment (EE is a powerful stimulus of brain plasticity and is among the most accessible treatment options for brain disease. In rodents, EE is modeled using multi-factorial environments that include running, social interactions, and/or complex surroundings. Here, we show that running and running-independent EE differentially affect the hippocampal dentate gyrus (DG, a brain region critical for learning and memory. Outbred male CD1 mice housed individually with a voluntary running disk showed improved spatial memory in the radial arm maze compared to individually- or socially-housed mice with a locked disk. We therefore used RNA sequencing to perform an unbiased interrogation of DG gene expression in mice exposed to either a voluntary running disk (RUN, a locked disk (LD, or a locked disk plus social enrichment and tunnels [i.e., a running-independent complex environment (CE]. RNA sequencing revealed that RUN and CE mice showed distinct, non-overlapping patterns of transcriptomic changes versus the LD control. Bio-informatics uncovered that the RUN and CE environments modulate separate transcriptional networks, biological processes, cellular compartments and molecular pathways, with RUN preferentially regulating synaptic and growth-related pathways and CE altering extracellular matrix-related functions. Within the RUN group, high-distance runners also showed selective stress pathway alterations that correlated with a drastic decline in overall transcriptional changes, suggesting that excess running causes a stress-induced suppression of running’s genetic effects. Our findings reveal stimulus-dependent transcriptional signatures of EE on the DG, and provide a resource for generating unbiased, data-driven hypotheses for novel mediators of EE-induced cognitive changes.

  1. Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    Full Text Available The Nkrp1 (Klrb1-Clr (Clec2 genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b, Nkrp1c (Klrb1c, and Clr-c (Clec2f genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d and Clr-g (Clec2i showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells, as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

  2. Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

    Science.gov (United States)

    Yamamoto, F; Höglund, B; Fernandez-Vina, M; Tyan, D; Rastrou, M; Williams, T; Moonsamy, P; Goodridge, D; Anderson, M; Erlich, H A; Holcomb, C L

    2015-12-01

    Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. Copyright © 2015. Published by Elsevier Inc.

  3. High-Throughput Sequencing of Microbial Community Diversity and Dynamics during Douchi Fermentation

    Science.gov (United States)

    Tu, Zong-cai; Wang, Xiao-lan

    2016-01-01

    Douchi is a type of Chinese traditional fermented food that is an important source of protein and is used in flavouring ingredients. The end product is affected by the microbial community present during fermentation, but exactly how microbes influence the fermentation process remains poorly understood. We used an Illumina MiSeq approach to investigate bacterial and fungal community diversity during both douchi-koji making and fermentation. A total of 181,443 high quality bacterial 16S rRNA sequences and 221,059 high quality fungal internal transcribed spacer reads were used for taxonomic classification, revealing eight bacterial and three fungal phyla. Firmicutes, Actinobacteria and Proteobacteria were the dominant bacterial phyla, while Ascomycota and Zygomycota were the dominant fungal phyla. At the genus level, Staphylococcus and Weissella were the dominant bacteria, while Aspergillus and Lichtheimia were the dominant fungi. Principal coordinate analysis showed structural separation between the composition of bacteria in koji making and fermentation. However, multivariate analysis of variance based on unweighted UniFrac distances did identify distinct differences (p fermentation. This is the first investigation to integrate douchi fermentation and koji making and fermentation processes through this technological approach. The results provide insight into the microbiome of the douchi fermentation process, and reveal a structural separation that may be stratified by the environment during the production of this traditional fermented food. PMID:27992473

  4. On the optimal trimming of high-throughput mRNA sequence data

    Directory of Open Access Journals (Sweden)

    Matthew D MacManes

    2014-01-01

    Full Text Available The widespread and rapid adoption of high-throughput sequencing technologies has afforded researchers the opportunity to gain a deep understanding of genome level processes that underlie evolutionary change, and perhaps more importantly, the links between genotype and phenotype. In particular, researchers interested in functional biology and adaptation have used these technologies to sequence mRNA transcriptomes of specific tissues, which in turn are often compared to other tissues, or other individuals with different phenotypes. While these techniques are extremely powerful, careful attention to data quality is required. In particular, because high-throughput sequencing is more error-prone than traditional Sanger sequencing, quality trimming of sequence reads should be an important step in all data processing pipelines. While several software packages for quality trimming exist, no general guidelines for the specifics of trimming have been developed. Here, using empirically derived sequence data, I provide general recommendations regarding the optimal strength of trimming, specifically in mRNA-Seq studies. Although very aggressive quality trimming is common, this study suggests that a more gentle trimming, specifically of those nucleotides whose Phred score < 2 or < 5, is optimal for most studies across a wide variety of metrics.

  5. Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

    Science.gov (United States)

    Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

    2011-10-01

    The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

  6. Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

    Science.gov (United States)

    Khanna, Namita; Ghosh, Ananta Kumar; Huntemann, Marcel; Deshpande, Shweta; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Pagani, Ioanna; Pati, Amrita; Pitluck, Sam; Nolan, Matt; Woyke, Tanja; Teshima, Hazuki; Chertkov, Olga; Daligault, Hajnalka; Davenport, Karen; Gu, Wei; Munk, Christine; Zhang, Xiaojing; Bruce, David; Detter, Chris; Xu, Yan; Quintana, Beverly; Reitenga, Krista; Kunde, Yulia; Green, Lance; Erkkila, Tracy; Han, Cliff; Brambilla, Evelyne-Marie; Lang, Elke; Klenk, Hans-Peter; Goodwin, Lynne; Chain, Patrick; Das, Debabrata

    2013-12-20

    Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

  7. Supplementary Material for: Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA

    2015-01-01

    Abstract Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic

  8. Analysis of sequences from field samples reveals the presence of the recently described pepper vein yellows virus (genus Polerovirus) in six additional countries.

    Science.gov (United States)

    Knierim, Dennis; Tsai, Wen-Shi; Kenyon, Lawrence

    2013-06-01

    Polerovirus infection was detected by reverse transcription polymerase chain reaction (RT-PCR) in 29 pepper plants (Capsicum spp.) and one black nightshade plant (Solanum nigrum) sample collected from fields in India, Indonesia, Mali, Philippines, Thailand and Taiwan. At least two representative samples for each country were selected to generate a general polerovirus RT-PCR product of 1.4 kb length for sequencing. Sequence analysis of the partial genome sequences revealed the presence of pepper vein yellows virus (PeVYV) in all 13 samples. A 1990 Australian herbarium sample of pepper described by serological means as infected with capsicum yellows virus (CYV) was identified by sequence analysis of a partial CP sequence as probably infected with a potato leaf roll virus (PLRV) isolate.

  9. Using high-throughput barcode sequencing to efficiently map connectomes.

    Science.gov (United States)

    Peikon, Ian D; Kebschull, Justus M; Vagin, Vasily V; Ravens, Diana I; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R; Bressan, Dario; Zador, Anthony M

    2017-07-07

    The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

    Energy Technology Data Exchange (ETDEWEB)

    Zawadzka-Kazimierczuk, Anna; Kozminski, Wiktor, E-mail: kozmin@chem.uw.edu.pl [University of Warsaw, Faculty of Chemistry (Poland); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria, Department of Bacteriology (Czech Republic)

    2012-04-15

    Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using {delta} subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.

  11. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    Directory of Open Access Journals (Sweden)

    De Marzo Angelo M

    2011-06-01

    Full Text Available Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.

  12. Comparative analysis of transcriptomes in aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing

    Directory of Open Access Journals (Sweden)

    Taketo Okada

    2016-12-01

    Full Text Available Ephedra plants are taxonomically classified as gymnosperms, and are medicinally important as the botanical origin of crude drugs and as bioresources that contain pharmacologically active chemicals. Here we show a comparative analysis of the transcriptomes of aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing by RNA-Seq. De novo assembly of short cDNA sequence reads generated 23,358, 13,373, and 28,579 contigs longer than 200 bases from aerial stems, roots, or both aerial stems and roots, respectively. The presumed functions encoded by these contig sequences were annotated by BLAST (blastx. Subsequently, these contigs were classified based on gene ontology slims, Enzyme Commission numbers, and the InterPro database. Furthermore, comparative gene expression analysis was performed between aerial stems and roots. These transcriptome analyses revealed differences and similarities between the transcriptomes of aerial stems and roots in E. sinica. Deep transcriptome sequencing of Ephedra should open the door to molecular biological studies based on the entire transcriptome, tissue- or organ-specific transcriptomes, or targeted genes of interest.

  13. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Directory of Open Access Journals (Sweden)

    Sarah M Hykin

    Full Text Available For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles, attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp. We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens

  14. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Science.gov (United States)

    Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  15. Characteristics of MHC class I genes in house sparrows Passer domesticus as revealed by long cDNA transcripts and amplicon sequencing.

    Science.gov (United States)

    Karlsson, Maria; Westerdahl, Helena

    2013-08-01

    In birds the major histocompatibility complex (MHC) organization differs both among and within orders; chickens Gallus gallus of the order Galliformes have a simple arrangement, while many songbirds of the order Passeriformes have a more complex arrangement with larger numbers of MHC class I and II genes. Chicken MHC genes are found at two independent loci, classical MHC-B and non-classical MHC-Y, whereas non-classical MHC genes are yet to be verified in passerines. Here we characterize MHC class I transcripts (α1 to α3 domain) and perform amplicon sequencing using a next-generation sequencing technique on exon 3 from house sparrow Passer domesticus (a passerine) families. Then we use phylogenetic, selection, and segregation analyses to gain a better understanding of the MHC class I organization. Trees based on the α1 and α2 domain revealed a distinct cluster with short terminal branches for transcripts with a 6-bp deletion. Interestingly, this cluster was not seen in the tree based on the α3 domain. 21 exon 3 sequences were verified in a single individual and the average numbers within an individual were nine and five for sequences with and without a 6-bp deletion, respectively. All individuals had exon 3 sequences with and without a 6-bp deletion. The sequences with a 6-bp deletion have many characteristics in common with non-classical MHC, e.g., highly conserved amino acid positions were substituted compared with the other alleles, low nucleotide diversity and just a single site was subject to positive selection. However, these alleles also have characteristics that suggest they could be classical, e.g., complete linkage and absence of a distinct cluster in a tree based on the α3 domain. Thus, we cannot determine for certain whether or not the alleles with a 6-bp deletion are non-classical based on our present data. Further analyses on segregation patterns of these alleles in combination with dating the 6-bp deletion through MHC characterization across the

  16. Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm

    Directory of Open Access Journals (Sweden)

    Junjie Lu

    2018-04-01

    Full Text Available Differentiation of human pluripotent stem cells towards definitive endoderm (DE is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Keywords: hPSC, Differentiation, Definitive endoderm, Heterogeneity, Single cell, RNA sequencing

  17. Oil palm genome sequence reveals divergence of interfertile species in old and new worlds

    Science.gov (United States)

    Singh, Rajinder; Ong-Abdullah, Meilina; Low, Eng-Ti Leslie; Manaf, Mohamad Arif Abdul; Rosli, Rozana; Nookiah, Rajanaidu; Ooi, Leslie Cheng-Li; Ooi, Siew–Eng; Chan, Kuang-Lim; Halim, Mohd Amin; Azizi, Norazah; Nagappan, Jayanthi; Bacher, Blaire; Lakey, Nathan; Smith, Steven W; He, Dong; Hogan, Michael; Budiman, Muhammad A; Lee, Ernest K; DeSalle, Rob; Kudrna, David; Goicoechea, Jose Louis; Wing, Rod; Wilson, Richard K; Fulton, Robert S; Ordway, Jared M; Martienssen, Robert A; Sambanthamurthi, Ravigadevi

    2013-01-01

    Oil palm is the most productive oil-bearing crop. Planted on only 5% of the total vegetable oil acreage, palm oil accounts for 33% of vegetable oil, and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8 gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators1, which are highly expressed in the kernel. We also report the draft sequence of the S. American oil palm Elaeis oleifera, which has the same number of chromosomes (2n=32) and produces fertile interspecific hybrids with E. guineensis2, but appears to have diverged in the new world. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations which restrict the use of clones in commercial plantings3, and thus helps achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop. PMID:23883927

  18. Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences.

    Science.gov (United States)

    Bunawan, Hamidun; Yen, Choong Chee; Yaakop, Salmah; Noor, Normah Mohd

    2017-01-26

    The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consisting of N. ampullaria, N. mirabilis, N. gracilis and N. rafflesiana, and another containing both intermediately distributed species (N. albomarginata and N. benstonei) and four highland species (N. sanguinea, N. macfarlanei, N. ramispina and N. alba). The trnL intron and ITS sequences proved to provide phylogenetic informative characters for deriving a phylogeny of Nepenthes species in Peninsular Malaysia. To our knowledge, this is the first molecular phylogenetic study of Nepenthes species occurring along an altitudinal gradient in Peninsular Malaysia.

  19. Phylogenetic and ecological analyses of soil and sporocarp DNA sequences reveal high diversity and strong habitat partitioning in the boreal ectomycorrhizal genus Russula (Russulales; Basidiomycota)

    Science.gov (United States)

    József Geml; Gary A. Laursen; Ian C. Herriott; Jack M. McFarland; Michael G. Booth; Niall Lennon; H. Chad Nusbaum; D. Lee Taylor

    2010-01-01

    Although critical for the functioning of ecosystems, fungi are poorly known in high-latitude regions. Here, we provide the first genetic diversity assessment of one of the most diverse and abundant ectomycorrhizal genera in Alaska: Russula. We analyzed internal transcribed spacer rDNA sequences from sporocarps and soil samples using phylogenetic...

  20. High-Throughput Sequencing Reveals Hypothalamic MicroRNAs as Novel Partners Involved in Timing the Rapid Development of Chicken (Gallus gallus) Gonads.

    Science.gov (United States)

    Han, Wei; Zou, Jianmin; Wang, Kehua; Su, Yijun; Zhu, Yunfen; Song, Chi; Li, Guohui; Qu, Liang; Zhang, Huiyong; Liu, Honglin

    2015-01-01

    Onset of the rapid gonad growth is a milestone in sexual development that comprises many genes and regulatory factors. The observations in model organisms and mammals including humans have shown a potential link between miRNAs and development timing. To determine whether miRNAs play roles in this process in the chicken (Gallus gallus), the Solexa deep sequencing was performed to analyze the profiles of miRNA expression in the hypothalamus of hens from two different pubertal stages, before onset of the rapid gonad development (BO) and after onset of the rapid gonad development (AO). 374 conserved and 46 novel miRNAs were identified as hypothalamus-expressed miRNAs in the chicken. 144 conserved miRNAs were showed to be differentially expressed (reads > 10, P time quantitative RT-PCR (qRT-PCR) method. 2013 putative genes were predicted as the targets of the 15 most differentially expressed miRNAs (fold-change > 4.0, P times by the miRNAs. qRT-PCR revealed the basic transcription levels of these clock genes were much higher (P development of chicken gonads. Considering the characteristics of miRNA functional conservation, the results will contribute to the research on puberty onset in humans.

  1. Sequence exploration reveals information bias among molecular markers used in phylogenetic reconstruction for Colletotrichum species.

    Science.gov (United States)

    Rampersad, Sephra N; Hosein, Fazeeda N; Carrington, Christine Vf

    2014-01-01

    The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of isolates of quarantine importance to the intra-specific level is confounded by a number of factors that affect phylogenetic reconstruction. Information bias and quality parameters were investigated to determine whether nucleotide sequence alignments and phylogenetic trees accurately reflect the genetic diversity and phylogenetic relatedness of individuals. Sequence exploration of GAPDH, ACT, TUB2 and ITS markers indicated that the query sequences had different patterns of nucleotide substitution but were without evidence of base substitution saturation. Regions of high entropy were much more dispersed in the ACT and GAPDH marker alignments than for the ITS and TUB2 markers. A discernible bimodal gap in the genetic distance frequency histograms was produced for the ACT and GAPDH markers which indicated successful separation of intra- and inter-specific sequences in the data set. Overall, analyses indicated clear differences in the ability of these markers to phylogenetically separate individuals to the intra-specific level which coincided with information bias.

  2. Whole genome sequencing reveals complex evolution patterns of multidrug-resistant Mycobacterium tuberculosis Beijing strains in patients.

    Directory of Open Access Journals (Sweden)

    Matthias Merker

    Full Text Available Multidrug-resistant (MDR Mycobacterium tuberculosis complex (MTBC strains represent a major threat for tuberculosis (TB control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A and nine (Patient B polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and

  3. 3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

    Science.gov (United States)

    2013-01-01

    Background Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. Results The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). Conclusions 3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. PMID:24053768

  4. Characterization of race 65 of Colletotrichum lindemuthianum by sequencing ITS regions

    Directory of Open Access Journals (Sweden)

    Marcela Coelho

    2016-09-01

    Full Text Available The present work aimed characterize isolates of C. lindemuthianum race 65 from different regions in Brazil by ITS sequencing. A total of 17 isolates of race 65, collected in the states of Mato Grosso, Minas Gerais, Paraná, Santa Catarina and São Paulo, were studied. Analysis of the sequences of isolates 8, 9, 12, 14 and 15 revealed the presence of two single nucleotide polymorphisms (SNPs in the ITS1 region at the same positions. These isolates, when analyzed together with the sequence of isolate 17, revealed a SNP in the ITS2 region. The highest genetic dissimilarity, observed between isolates 11 and  3 and between isolates 11 and 10, was 0.772. In turn, isolates 7 and 2 were the most similar, with a value of 0.002 for genetic distance. The phylogenetic tree obtained based on the sequences of the ITS1 and ITS2 regions revealed the formation of two groups, one with a subgroup. The results reveal high molecular variability among isolates of race 65 of C. lindemuthianum.

  5. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R.; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G.; Ohm, Robin A.; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L.; Bailey, Andrew M.; Billette, Christophe; Coutinho, Pedro M.; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hilden, Kristiina; Kues, Ursula; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Murat, Claude; Riley, Robert W.; Salamov, Asaf A.; Schmutz, Jeremy; Subramanian, Venkataramanan; Wosten, Han A. B.; Xu, Jianping; Eastwood, Daniel C.; Foster, Gary D.; Sonnenberg, Anton S. M.; Cullen, Dan; de Vries, Ronald P.; Lundell, Taina; Hibbett, David S.; Henrissat, Bernard; Burton, Kerry S.; Kerrigan, Richard W.; Challen, Michael P.; Grigoriev, Igor V.; Martin, Francis

    2012-04-27

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the button mushroom forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  6. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

    Science.gov (United States)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G; Ohm, Robin A; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L; Bailey, Andrew M; Billette, Christophe; Coutinho, Pedro M; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hildén, Kristiina; Kües, Ursula; Labutti, Kurt M; Lapidus, Alla; Lindquist, Erika A; Lucas, Susan M; Murat, Claude; Riley, Robert W; Salamov, Asaf A; Schmutz, Jeremy; Subramanian, Venkataramanan; Wösten, Han A B; Xu, Jianping; Eastwood, Daniel C; Foster, Gary D; Sonnenberg, Anton S M; Cullen, Dan; de Vries, Ronald P; Lundell, Taina; Hibbett, David S; Henrissat, Bernard; Burton, Kerry S; Kerrigan, Richard W; Challen, Michael P; Grigoriev, Igor V; Martin, Francis

    2012-10-23

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  7. High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch

    Energy Technology Data Exchange (ETDEWEB)

    Harding, Louisa B. [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Schultz, Irvin R. [Battelle, Marine Sciences Laboratory – Pacific Northwest National Laboratory, 1529 West Sequim Bay Road, Sequim, WA 98382 (United States); Goetz, Giles W. [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Luckenbach, J. Adam [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd E, Seattle, WA 98112 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States); Young, Graham [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States); Goetz, Frederick W. [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Manchester Research Station, P.O. Box 130, Manchester, WA 98353 (United States); Swanson, Penny, E-mail: penny.swanson@noaa.gov [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd E, Seattle, WA 98112 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States)

    2013-10-15

    Highlights: •Studied impacts of ethynylestradiol (EE2) exposure on salmon pituitary transcriptome. •High-throughput sequencing, RNAseq, and pathway analysis were performed. •EE2 altered mRNAs for genes in circadian rhythm, GnRH, and TGFβ signaling pathways. •LH and FSH beta subunit mRNAs were most highly up- and down-regulated by EE2, respectively. •Estrogens may alter processes associated with reproductive timing in salmon. -- Abstract: Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain–pituitary–gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina{sup ®} sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (−3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks

  8. High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch

    International Nuclear Information System (INIS)

    Harding, Louisa B.; Schultz, Irvin R.; Goetz, Giles W.; Luckenbach, J. Adam; Young, Graham; Goetz, Frederick W.; Swanson, Penny

    2013-01-01

    Highlights: •Studied impacts of ethynylestradiol (EE2) exposure on salmon pituitary transcriptome. •High-throughput sequencing, RNAseq, and pathway analysis were performed. •EE2 altered mRNAs for genes in circadian rhythm, GnRH, and TGFβ signaling pathways. •LH and FSH beta subunit mRNAs were most highly up- and down-regulated by EE2, respectively. •Estrogens may alter processes associated with reproductive timing in salmon. -- Abstract: Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain–pituitary–gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina ® sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (−3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks

  9. High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers for Measuring Puberty Onset in Chicken (Gallus gallus).

    Science.gov (United States)

    Han, Wei; Zhu, Yunfen; Su, Yijun; Li, Guohui; Qu, Liang; Zhang, Huiyong; Wang, Kehua; Zou, Jianmin; Liu, Honglin

    2016-01-01

    There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P 1.0, P puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans.

  10. Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes.

    Science.gov (United States)

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan

    2008-01-01

    Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

  11. SNP design from 454 sequencing of Podosphaera plantaginis transcriptome reveals a genetically diverse pathogen metapopulation with high levels of mixed-genotype infection.

    Directory of Open Access Journals (Sweden)

    Charlotte Tollenaere

    Full Text Available Molecular tools may greatly improve our understanding of pathogen evolution and epidemiology but technical constraints have hindered the development of genetic resources for parasites compared to free-living organisms. This study aims at developing molecular tools for Podosphaera plantaginis, an obligate fungal pathogen of Plantago lanceolata. This interaction has been intensively studied in the Åland archipelago of Finland with epidemiological data collected from over 4,000 host populations annually since year 2001.A cDNA library of a pooled sample of fungal conidia was sequenced on the 454 GS-FLX platform. Over 549,411 reads were obtained and annotated into 45,245 contigs. Annotation data was acquired for 65.2% of the assembled sequences. The transcriptome assembly was screened for SNP loci, as well as for functionally important genes (mating-type genes and potential effector proteins. A genotyping assay of 27 SNP loci was designed and tested on 380 infected leaf samples from 80 populations within the Åland archipelago. With this panel we identified 85 multilocus genotypes (MLG with uneven frequencies across the pathogen metapopulation. Approximately half of the sampled populations contain polymorphism. Our genotyping protocol revealed mixed-genotype infection within a single host leaf to be common. Mixed infection has been proposed as one of the main drivers of pathogen evolution, and hence may be an important process in this pathosystem.The developed SNP panel offers exciting research perspectives for future studies in this well-characterized pathosystem. Also, the transcriptome provides an invaluable novel genomic resource for powdery mildews, which cause significant yield losses on commercially important crops annually. Furthermore, the features that render genetic studies in this system a challenge are shared with the majority of obligate parasitic species, and hence our results provide methodological insights from SNP calling to field

  12. Whole Genome Sequencing of Enterovirus species C Isolates by High-throughput Sequencing: Development of Generic Primers

    Directory of Open Access Journals (Sweden)

    Maël Bessaud

    2016-08-01

    Full Text Available Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C consists of more than 20 types, among which the 3 serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions.A simple method was developed to sequence quickly the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to be sequenced by high-throughput technique.The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures.By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.

  13. Quack: A quality assurance tool for high throughput sequence data.

    Science.gov (United States)

    Thrash, Adam; Arick, Mark; Peterson, Daniel G

    2018-05-01

    The quality of data generated by high-throughput DNA sequencing tools must be rapidly assessed in order to determine how useful the data may be in making biological discoveries; higher quality data leads to more confident results and conclusions. Due to the ever-increasing size of data sets and the importance of rapid quality assessment, tools that analyze sequencing data should quickly produce easily interpretable graphics. Quack addresses these issues by generating information-dense visualizations from FASTQ files at a speed far surpassing other publicly available quality assurance tools in a manner independent of sequencing technology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  14. High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

    Science.gov (United States)

    Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

    2016-11-23

    Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

  15. Fast high-resolution MR imaging using the snapshot-FLASH MR sequence

    International Nuclear Information System (INIS)

    Matthaei, D.; Haase, A.; Henrich, D.; Duhmke, E.

    1990-01-01

    Snapshot, fast low-angle short (FLASH) MR imaging using an accelerated FLASH-MR sequence provides MR images with measuring times far below 1 second. The short TE of this sequence prevents susceptibility artifacts in gradient-echo imaging. In this paper variations of the sequence are shown that provide high resolution images with T1-weighted IR, T2-weighted SE, and chemical shift (CHESS) contrast sequences. METHODS AND MATERIALS: A whole-body 2-T system (Bruker-Medizintechnik) were used in combination with a 60-cm gradient system (providing gradient strength of 5 mT/m) to study healthy volunteers. The measuring time for a 256 x 256 image matrix was 800 msec. This sequence has been used in combination with T1-weighted IR, T2-weighted SE, and CHESS variations

  16. High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

    Science.gov (United States)

    Rahman, Abdur; Cailliez-Grimal, Catherine; Bontemps, Cyril; Payot, Sophie; Chaillou, Stéphane; Revol-Junelles, Anne-Marie; Borges, Frédéric

    2014-07-01

    Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  18. High-throughput sequencing of small RNA transcriptome reveals salt stress regulated microRNAs in sugarcane.

    Directory of Open Access Journals (Sweden)

    Mariana Carnavale Bottino

    Full Text Available Salt stress is a primary cause of crop losses worldwide, and it has been the subject of intense investigation to unravel the complex mechanisms responsible for salinity tolerance. MicroRNA is implicated in many developmental processes and in responses to various abiotic stresses, playing pivotal roles in plant adaptation. Deep sequencing technology was chosen to determine the small RNA transcriptome of Saccharum sp cultivars grown on saline conditions. We constructed four small RNAs libraries prepared from plants grown on hydroponic culture submitted to 170 mM NaCl and harvested after 1 h, 6 hs and 24 hs. Each library was sequenced individually and together generated more than 50 million short reads. Ninety-eight conserved miRNAs and 33 miRNAs* were identified by bioinformatics. Several of the microRNA showed considerable differences of expression in the four libraries. To confirm the results of the bioinformatics-based analysis, we studied the expression of the 10 most abundant miRNAs and 1 miRNA* in plants treated with 170 mM NaCl and in plants with a severe treatment of 340 mM NaCl. The results showed that 11 selected miRNAs had higher expression in samples treated with severe salt treatment compared to the mild one. We also investigated the regulation of the same miRNAs in shoots of four cultivars grown on soil treated with 170 mM NaCl. Cultivars could be grouped according to miRNAs expression in response to salt stress. Furthermore, the majority of the predicted target genes had an inverse regulation with their correspondent microRNAs. The targets encode a wide range of proteins, including transcription factors, metabolic enzymes and genes involved in hormone signaling, probably assisting the plants to develop tolerance to salinity. Our work provides insights into the regulatory functions of miRNAs, thereby expanding our knowledge on potential salt-stressed regulated genes.

  19. Comparative genomic sequence analysis of strawberry and other rosids reveals significant microsynteny

    Directory of Open Access Journals (Sweden)

    Abbott Albert

    2010-06-01

    Full Text Available Abstract Background Fragaria belongs to the Rosaceae, an economically important family that includes a number of important fruit producing genera such as Malus and Prunus. Using genomic sequences from 50 Fragaria fosmids, we have examined the microsynteny between Fragaria and other plant models. Results In more than half of the strawberry fosmids, we found syntenic regions that are conserved in Populus, Vitis, Medicago and/or Arabidopsis with Populus containing the greatest number of syntenic regions with Fragaria. The longest syntenic region was between LG VIII of the poplar genome and the strawberry fosmid 72E18, where seven out of twelve predicted genes were collinear. We also observed an unexpectedly high level of conserved synteny between Fragaria (rosid I and Vitis (basal rosid. One of the strawberry fosmids, 34E24, contained a cluster of R gene analogs (RGAs with NBS and LRR domains. We detected clusters of RGAs with high sequence similarity to those in 34E24 in all the genomes compared. In the phylogenetic tree we have generated, all the NBS-LRR genes grouped together with Arabidopsis CNL-A type NBS-LRR genes. The Fragaria RGA grouped together with those of Vitis and Populus in the phylogenetic tree. Conclusions Our analysis shows considerable microsynteny between Fragaria and other plant genomes such as Populus, Medicago, Vitis, and Arabidopsis to a lesser degree. We also detected a cluster of NBS-LRR type genes that are conserved in all the genomes compared.

  20. Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii.

    Science.gov (United States)

    Tanigawa, Kana; Watanabe, Koichi

    2011-03-01

    Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

  1. A robust, simple genotyping-by-sequencing (GBS approach for high diversity species.

    Directory of Open Access Journals (Sweden)

    Robert J Elshire

    Full Text Available Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs. This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM and barley (Oregon Wolfe Barley recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

  2. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation.

    Directory of Open Access Journals (Sweden)

    Si-Yang Liu

    Full Text Available Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.

  3. Alignment of high-throughput sequencing data inside in-memory databases.

    Science.gov (United States)

    Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

    2014-01-01

    In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

  4. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

    Directory of Open Access Journals (Sweden)

    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

  5. Genome sequencing and analysis reveals possible determinants of Staphylococcus aureus nasal carriage

    Directory of Open Access Journals (Sweden)

    Cole Alexander M

    2008-09-01

    Full Text Available Abstract Background Nasal carriage of Staphylococcus aureus is a major risk factor in clinical and community settings due to the range of etiologies caused by the organism. We have identified unique immunological and ultrastructural properties associated with nasal carriage isolates denoting a role for bacterial factors in nasal carriage. However, despite extensive molecular level characterizations by several groups suggesting factors necessary for colonization on nasal epithelium, genetic determinants of nasal carriage are unknown. Herein, we have set a genomic foundation for unraveling the bacterial determinants of nasal carriage in S. aureus. Results MLST analysis revealed no lineage specific differences between carrier and non-carrier strains suggesting a role for mobile genetic elements. We completely sequenced a model carrier isolate (D30 and a model non-carrier strain (930918-3 to identify differential gene content. Comparison revealed the presence of 84 genes unique to the carrier strain and strongly suggests a role for Type VII secretion systems in nasal carriage. These genes, along with a putative pathogenicity island (SaPIBov present uniquely in the carrier strains are likely important in affecting carriage. Further, PCR-based genotyping of other clinical isolates for a specific subset of these 84 genes raise the possibility of nasal carriage being caused by multiple gene sets. Conclusion Our data suggest that carriage is likely a heterogeneic phenotypic trait and implies a role for nucleotide level polymorphism in carriage. Complete genome level analyses of multiple carriage strains of S. aureus will be important in clarifying molecular determinants of S. aureus nasal carriage.

  6. Molecular characterization, sequence analysis and tissue expression of a porcine gene – MOSPD2

    Directory of Open Access Journals (Sweden)

    Yang Jie

    2017-01-01

    Full Text Available The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2 of five species: horse (89%, human (90%, chimpanzee (89%, rhesus monkey (89% and mouse (85%; thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.

  7. Expression Profiling during Arabidopsis/Downy Mildew Interaction Reveals a Highly-Expressed Effector That Attenuates Responses to Salicylic Acid

    Science.gov (United States)

    Asai, Shuta; Caillaud, Marie-Cécile; Furzer, Oliver J.; Ishaque, Naveed; Wirthmueller, Lennart; Fabro, Georgina; Shirasu, Ken; Jones, Jonathan D. G.

    2014-01-01

    Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome. PMID:25329884

  8. Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

    Directory of Open Access Journals (Sweden)

    Shuta Asai

    2014-10-01

    Full Text Available Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

  9. Deep amplicon sequencing reveals mixed phytoplasma infection within single grapevine plants

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Contaldo, Nicoletta; Makarova, Olga

    2011-01-01

    The diversity of phytoplasmas within single plants has not yet been fully investigated. In this project, deep amplicon sequencing was used to generate 50,926 phytoplasma sequences from 11 phytoplasma-infected grapevine samples from a PCR amplicon in the 5' end of the 16S region. After clustering ...

  10. Molecular analyses reveal high species diversity of trematodes in a sub-Arctic lake

    Science.gov (United States)

    Soldánová, Miroslava; Georgieva, Simona; Roháčováa, Jana; Knudsen, Rune; Kuhn, Jesper A.; Henriksen, Eirik H.; Siwertsson, Anna; Shaw, Jenny C.; Kuris, Armand M.; Amundsen, Per-Arne; Scholz, Tomáš; Lafferty, Kevin D.; Kostadinova, Aneta

    2017-01-01

    To identify trematode diversity and life-cycles in the sub-Arctic Lake Takvatn, Norway, we characterised 120 trematode isolates from mollusc first intermediate hosts, metacercariae from second intermediate host fishes and invertebrates, and adults from fish and invertebrate definitive hosts, using molecular techniques. Phylogenies based on nuclear and/or mtDNA revealed high species richness (24 species or species-level genetic lineages), and uncovered trematode diversity (16 putative new species) from five families typical in lake ecosystems (Allocreadiidae, Diplostomidae, Plagiorchiidae, Schistosomatidae and Strigeidae). Sampling potential invertebrate hosts allowed matching of sequence data for different stages, thus achieving molecular elucidation of trematode life-cycles and exploration of host-parasite interactions. Phylogenetic analyses also helped identify three major mollusc intermediate hosts (Radix balthica, Pisidium casertanum and Sphaerium sp.) in the lake. Our findings increase the known trematode diversity at the sub-Arctic Lake Takvatn, showing that digenean diversity is high in this otherwise depauperate sub-Arctic freshwater ecosystem, and indicating that sub-Arctic and Arctic ecosystems may be characterised by unique trematode assemblages.

  11. Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

    Science.gov (United States)

    Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

    2014-03-04

    Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

  12. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Science.gov (United States)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R.; Foulongne-Oriol, Marie; Lombard, Vincent; Nagye, Laszlo G.; Ohm, Robin A.; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L.; Bailey, Andrew M.; Billette, Christophe; Coutinho, Pedro M.; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hildén, Kristiina; Kües, Ursula; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Murat, Claude; Riley, Robert W.; Salamov, Asaf A.; Schmutz, Jeremy; Subramanian, Venkataramanan; Wösten, Han A. B.; Xu, Jianping; Eastwood, Daniel C.; Foster, Gary D.; Sonnenberg, Anton S. M.; Cullen, Dan; de Vries, Ronald P.; Lundell, Taina; Hibbett, David S.; Henrissat, Bernard; Burton, Kerry S.; Kerrigan, Richard W.; Challen, Michael P.; Grigoriev, Igor V.; Martin, Francis

    2012-01-01

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics. PMID:23045686

  13. Complete sequence analysis reveals two distinct poleroviruses infecting cucurbits in China.

    Science.gov (United States)

    Xiang, Hai-ying; Shang, Qiao-xia; Han, Cheng-gui; Li, Da-wei; Yu, Jia-lin

    2008-01-01

    The complete RNA genomes of a Chinese isolate of cucurbit aphid-borne yellows virus (CABYV-CHN) and a new polerovirus tentatively referred to as melon aphid-borne yellows virus (MABYV) were determined. The entire genome of CABYV-CHN shared 89.0% nucleotide sequence identity with the French CABYV isolate. In contrast, nucleotide sequence identities between MABYV and CABYV and other poleroviruses were in the range of 50.7-74.2%, with amino acid sequence identities ranging from 24.8 to 82.9% for individual gene products. We propose that CABYV-CHN is a strain of CABYV and that MABYV is a member of a tentative distinct species within the genus Polerovirus.

  14. Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.

    Science.gov (United States)

    Hanson, E; Ingold, S; Haas, C; Ballantyne, J

    2018-05-01

    The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and

  15. ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

    Science.gov (United States)

    Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

    2014-02-01

    Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

  16. The subclonal structure and genomic evolution of oral squamous cell carcinoma revealed by ultra-deep sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin J

    2017-01-01

    Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolutethe clonal structure and describe the genomic cancer evolution......, we applied whole-exome sequencing combined with ultra-deep targeted sequencing on oral squamous cell carcinomas (OSCC). From each patient, a set of biopsies was sampled from distinct geographical sites in primary tumor and lymph node metastasis.We demonstrate that the included OSCCs show a high...

  17. High-throughput Sequencing Based Immune Repertoire Study during Infectious Disease

    Directory of Open Access Journals (Sweden)

    Dongni Hou

    2016-08-01

    Full Text Available The selectivity of the adaptive immune response is based on the enormous diversity of T and B cell antigen-specific receptors. The immune repertoire, the collection of T and B cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. In this article, we review the recent advances in immune repertoire study of infectious diseases that achieved by traditional techniques and high-throughput sequencing techniques. High-throughput sequencing techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. This progress in methodology enhances the understanding of immunologic changes during pathogen challenge, and also provides a basis for further development of novel diagnostic markers, immunotherapies and vaccines.

  18. Comparative Genomics Reveals High Genomic Diversity in the Genus Photobacterium.

    Science.gov (United States)

    Machado, Henrique; Gram, Lone

    2017-01-01

    Vibrionaceae is a large marine bacterial family, which can constitute up to 50% of the prokaryotic population in marine waters. Photobacterium is the second largest genus in the family and we used comparative genomics on 35 strains representing 16 of the 28 species described so far, to understand the genomic diversity present in the Photobacterium genus. Such understanding is important for ecophysiology studies of the genus. We used whole genome sequences to evaluate phylogenetic relationships using several analyses (16S rRNA, MLSA, fur , amino-acid usage, ANI), which allowed us to identify two misidentified strains. Genome analyses also revealed occurrence of higher and lower GC content clades, correlating with phylogenetic clusters. Pan- and core-genome analysis revealed the conservation of 25% of the genome throughout the genus, with a large and open pan-genome. The major source of genomic diversity could be traced to the smaller chromosome and plasmids. Several of the physiological traits studied in the genus did not correlate with phylogenetic data. Since horizontal gene transfer (HGT) is often suggested as a source of genetic diversity and a potential driver of genomic evolution in bacterial species, we looked into evidence of such in Photobacterium genomes. Genomic islands were the source of genomic differences between strains of the same species. Also, we found transposase genes and CRISPR arrays that suggest multiple encounters with foreign DNA. Presence of genomic exchange traits was widespread and abundant in the genus, suggesting a role in genomic evolution. The high genetic variability and indications of genetic exchange make it difficult to elucidate genome evolutionary paths and raise the awareness of the roles of foreign DNA in the genomic evolution of environmental organisms.

  19. The determination of high-resolution spatio-temporal glacier motion fields from time-lapse sequences

    Science.gov (United States)

    Schwalbe, Ellen; Maas, Hans-Gerd

    2017-12-01

    This paper presents a comprehensive method for the determination of glacier surface motion vector fields at high spatial and temporal resolution. These vector fields can be derived from monocular terrestrial camera image sequences and are a valuable data source for glaciological analysis of the motion behaviour of glaciers. The measurement concepts for the acquisition of image sequences are presented, and an automated monoscopic image sequence processing chain is developed. Motion vector fields can be derived with high precision by applying automatic subpixel-accuracy image matching techniques on grey value patterns in the image sequences. Well-established matching techniques have been adapted to the special characteristics of the glacier data in order to achieve high reliability in automatic image sequence processing, including the handling of moving shadows as well as motion effects induced by small instabilities in the camera set-up. Suitable geo-referencing techniques were developed to transform image measurements into a reference coordinate system.The result of monoscopic image sequence analysis is a dense raster of glacier surface point trajectories for each image sequence. Each translation vector component in these trajectories can be determined with an accuracy of a few centimetres for points at a distance of several kilometres from the camera. Extensive practical validation experiments have shown that motion vector and trajectory fields derived from monocular image sequences can be used for the determination of high-resolution velocity fields of glaciers, including the analysis of tidal effects on glacier movement, the investigation of a glacier's motion behaviour during calving events, the determination of the position and migration of the grounding line and the detection of subglacial channels during glacier lake outburst floods.

  20. Multilocus Sequence Typing Reveals Relevant Genetic Variation and Different Evolutionary Dynamics among Strains of Xanthomonas arboricola pv. juglandis

    Directory of Open Access Journals (Sweden)

    Marco Scortichini

    2010-11-01

    Full Text Available Forty-five Xanthomonas arboricola pv. juglandis (Xaj strains originating from Juglans regia cultivation in different countries were molecularly typed by means of MultiLocus Sequence Typing (MLST, using acnB, gapA, gyrB and rpoD gene fragments. A total of 2.5 kilobases was used to infer the phylogenetic relationship among the strains and possible recombination events. Haplotype diversity, linkage disequilibrium analysis, selection tests, gene flow estimates and codon adaptation index were also assessed. The dendrograms built by maximum likelihood with concatenated nucleotide and amino acid sequences revealed two major and two minor phylotypes. The same haplotype was found in strains originating from different continents, and different haplotypes were found in strains isolated in the same year from the same location. A recombination breakpoint was detected within the rpoD gene fragment. At the pathovar level, the Xaj populations studied here are clonal and under neutral selection. However, four Xaj strains isolated from walnut fruits with apical necrosis are under diversifying selection, suggesting a possible new adaptation. Gene flow estimates do not support the hypothesis of geographic isolation of the strains, even though the genetic diversity between the strains increases as the geographic distance between them increases. A triplet deletion, causing the absence of valine, was found in the rpoD fragment of all 45 Xaj strains when compared with X. axonopodis pv. citri strain 306. The codon adaptation index was high in all four genes studied, indicating a relevant metabolic activity.

  1. Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa.

    Science.gov (United States)

    Geisen, S; Laros, I; Vizcaíno, A; Bonkowski, M; de Groot, G A

    2015-09-01

    Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over-represented, while those of most amoebae and flagellates were under-represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities. © 2015 John Wiley & Sons Ltd.

  2. The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

    Science.gov (United States)

    Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

    2017-11-17

    MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

  3. Profiling of the metabolically active community from a production-scale biogas plant by means of high-throughput metatranscriptome sequencing

    DEFF Research Database (Denmark)

    Zakrzewski, Martha; Goesmann, Alexander; Jaenicke, Sebastian

    2012-01-01

    of the community by classification of 16S ribosomal sequence tags revealed that members of the Euryarchaeota and Firmicutes account for the dominant phyla. Only smaller fractions of the 16S ribosomal sequence tags were assigned to the phyla Bacteroidetes, Actinobacteria and Synergistetes. Among the m...

  4. Mitochondrial DNA markers reveal high genetic diversity but low genetic differentiation in the black fly Simulium tani Takaoka & Davies along an elevational gradient in Malaysia.

    Directory of Open Access Journals (Sweden)

    Van Lun Low

    Full Text Available The population genetic structure of Simulium tani was inferred from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI and II (COII along an elevational gradient in Cameron Highlands, Malaysia. A statistical parsimony network of 71 individuals revealed 71 haplotypes in the COI gene and 43 haplotypes in the COII gene; the concatenated sequences of the COI and COII genes revealed 71 haplotypes. High levels of genetic diversity but low levels of genetic differentiation were observed among populations of S. tani at five elevations. The degree of genetic diversity, however, was not in accordance with an altitudinal gradient, and a Mantel test indicated that elevation did not have a limiting effect on gene flow. No ancestral haplotype of S. tani was found among the populations. Pupae with unique structural characters at the highest elevation showed a tendency to form their own haplotype cluster, as revealed by the COII gene. Tajima's D, Fu's Fs, and mismatch distribution tests revealed population expansion of S. tani in Cameron Highlands. A strong correlation was found between nucleotide diversity and the levels of dissolved oxygen in the streams where S. tani was collected.

  5. Metagenome Sequence Analysis of Filamentous Microbial Communities Obtained from Geochemically Distinct Geothermal Channels Reveals Specialization of Three Aquificales Lineages

    Directory of Open Access Journals (Sweden)

    Cristina eTakacs-vesbach

    2013-05-01

    Full Text Available The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal ‘filamentous streamer’ communities (~40 Mbp per site, which targeted three different groups of Aquificales found in Yellowstone National Park (YNP. Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae populations, whereas the circumneutral pH (6.5 - 7.8 sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae. Thermocrinis (Aquificaceae populations were found primarily in the circumneutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl. The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl CoA synthetase (Ccs and citryl CoA lyase (Ccl. All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2 have resulted in niche specialization among members of the Aquificales.

  6. Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

    Science.gov (United States)

    Starcevic, Antonio; Dunlap, Walter C; Cullum, John; Shick, J Malcolm; Hranueli, Daslav; Long, Paul F

    2010-11-12

    The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+)-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

  7. Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

    Directory of Open Access Journals (Sweden)

    Antonio Starcevic

    2010-11-01

    Full Text Available The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues.A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs, which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis.Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

  8. Comparative Genomics in Switchgrass Using 61,585 High-Quality Expressed Sequence Tags

    Directory of Open Access Journals (Sweden)

    Christian M. Tobias

    2008-11-01

    Full Text Available The development of genomic resources for switchgrass ( L., a perennial NAD-malic enzyme type C grass, is required to enable molecular breeding and biotechnological approaches for improving its value as a forage and bioenergy crop. Expressed sequence tag (EST sequencing is one method that can quickly sample gene inventories and produce data suitable for marker development or analysis of tissue-specific patterns of expression. Toward this goal, three cDNA libraries from callus, crown, and seedling tissues of ‘Kanlow’ switchgrass were end-sequenced to generate a total of 61,585 high-quality ESTs from 36,565 separate clones. Seventy-three percent of the assembled consensus sequences could be aligned with the sorghum [ (L. Moench] genome at a -value of <1 × 10, indicating a high degree of similarity. Sixty-five percent of the ESTs matched with gene ontology molecular terms, and 3.3% of the sequences were matched with genes that play potential roles in cell-wall biogenesis. The representation in the three libraries of gene families known to be associated with C photosynthesis, cellulose and β-glucan synthesis, phenylpropanoid biosynthesis, and peroxidase activity indicated likely roles for individual family members. Pairwise comparisons of synonymous codon substitutions were used to assess genome sequence diversity and indicated an overall similarity between the two genome copies present in the tetraploid. Identification of EST–simple sequence repeat markers and amplification on two individual parents of a mapping population yielded an average of 2.18 amplicons per individual, and 35% of the markers produced fragment length polymorphisms.

  9. Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding.

    Directory of Open Access Journals (Sweden)

    Hai Li

    2010-01-01

    Full Text Available Chitinases are prevalent in life and are found in species including archaea, bacteria, fungi, plants, and animals. They break down chitin, which is the second most abundant carbohydrate in nature after cellulose. Hence, they are important for maintaining a balance between carbon and nitrogen trapped as insoluble chitin in biomass. Chitinases are classified into two families, 18 and 19 glycoside hydrolases. In addition to a catalytic domain, which is a triosephosphate isomerase barrel, many family 18 chitinases contain another module, i.e., chitinase insertion domain. While numerous studies focus on the biological role of the catalytic domain in chitinase activity, the function of the chitinase insertion domain is not completely understood. Bioinformatics offers an important avenue in which to facilitate understanding the role of residues within the chitinase insertion domain in chitinase function.Twenty-seven chitinase insertion domain sequences, which include four experimentally determined structures and span five kingdoms, were aligned and analyzed using a modified sequence entropy parameter. Thirty-two positions with conserved residues were identified. The role of these conserved residues was explored by conducting a structural analysis of a number of holo-enzymes. Hydrogen bonding and van der Waals calculations revealed a distinct subset of four conserved residues constituting two sequence motifs that interact with oligosaccharides. The other conserved residues may be key to the structure, folding, and stability of this domain.Sequence and structural studies of the chitinase insertion domains conducted within the framework of evolution identified four conserved residues which clearly interact with the substrates. Furthermore, evolutionary studies propose a link between the appearance of the chitinase insertion domain and the function of family 18 chitinases in the subfamily A.

  10. The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081.

    Directory of Open Access Journals (Sweden)

    Nicholas R Thomson

    2006-12-01

    Full Text Available The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common

  11. Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    Directory of Open Access Journals (Sweden)

    Janine Maimanakos

    2016-08-01

    Full Text Available Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76 are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99% of the (R-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

  12. High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China.

    Science.gov (United States)

    Wei, Yu-Jie; Wu, Yun; Yan, Yin-Zhuo; Zou, Wan; Xue, Jie; Ma, Wen-Rui; Wang, Wei; Tian, Ge; Wang, Li-Ye

    2018-01-01

    In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine.

  13. High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China

    Science.gov (United States)

    Yan, Yin-zhuo; Zou, Wan; Ma, Wen-rui; Wang, Wei; Tian, Ge; Wang, Li-ye

    2018-01-01

    In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine. PMID:29565999

  14. Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes.

    Science.gov (United States)

    Bolsheva, Nadezhda L; Melnikova, Nataliya V; Kirov, Ilya V; Speranskaya, Anna S; Krinitsina, Anastasia A; Dmitriev, Alexey A; Belenikin, Maxim S; Krasnov, George S; Lakunina, Valentina A; Snezhkina, Anastasiya V; Rozhmina, Tatiana A; Samatadze, Tatiana E; Yurkevich, Olga Yu; Zoshchuk, Svyatoslav A; Amosova, Аlexandra V; Kudryavtseva, Anna V; Muravenko, Olga V

    2017-12-28

    The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n = 30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes. High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH. Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n = 30) could originate either as the result of hybridization of two diploid species (2n = 16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n = 16) and a diploid ancestor of modern L. narbonense (2n = 14). High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the

  15. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries

    Science.gov (United States)

    Talkowski, Michael E.; Rosenfeld, Jill A.; Blumenthal, Ian; Pillalamarri, Vamsee; Chiang, Colby; Heilbut, Adrian; Ernst, Carl; Hanscom, Carrie; Rossin, Elizabeth; Lindgren, Amelia; Pereira, Shahrin; Ruderfer, Douglas; Kirby, Andrew; Ripke, Stephan; Harris, David; Lee, Ji-Hyun; Ha, Kyungsoo; Kim, Hyung-Goo; Solomon, Benjamin D.; Gropman, Andrea L.; Lucente, Diane; Sims, Katherine; Ohsumi, Toshiro K.; Borowsky, Mark L.; Loranger, Stephanie; Quade, Bradley; Lage, Kasper; Miles, Judith; Wu, Bai-Lin; Shen, Yiping; Neale, Benjamin; Shaffer, Lisa G.; Daly, Mark J.; Morton, Cynthia C.; Gusella, James F.

    2012-01-01

    SUMMARY Balanced chromosomal abnormalities (BCAs) represent a reservoir of single gene disruptions in neurodevelopmental disorders (NDD). We sequenced BCAs in autism and related NDDs, revealing disruption of 33 loci in four general categories: 1) genes associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, CDKL5), 2) single gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, SNURF-SNRPN), 3) novel risk loci (e.g., CHD8, KIRREL3, ZNF507), and 4) genes associated with later onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, ANK3). We also discovered profoundly increased burden of copy number variants among 19,556 neurodevelopmental cases compared to 13,991 controls (p = 2.07×10−47) and enrichment of polygenic risk alleles from autism and schizophrenia genome-wide association studies (p = 0.0018 and 0.0009, respectively). Our findings suggest a polygenic risk model of autism incorporating loci of strong effect and indicate that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages. PMID:22521361

  16. miRBase: annotating high confidence microRNAs using deep sequencing data.

    Science.gov (United States)

    Kozomara, Ana; Griffiths-Jones, Sam

    2014-01-01

    We describe an update of the miRBase database (http://www.mirbase.org/), the primary microRNA sequence repository. The latest miRBase release (v20, June 2013) contains 24 521 microRNA loci from 206 species, processed to produce 30 424 mature microRNA products. The rate of deposition of novel microRNAs and the number of researchers involved in their discovery continue to increase, driven largely by small RNA deep sequencing experiments. In the face of these increases, and a range of microRNA annotation methods and criteria, maintaining the quality of the microRNA sequence data set is a significant challenge. Here, we describe recent developments of the miRBase database to address this issue. In particular, we describe the collation and use of deep sequencing data sets to assign levels of confidence to miRBase entries. We now provide a high confidence subset of miRBase entries, based on the pattern of mapped reads. The high confidence microRNA data set is available alongside the complete microRNA collection at http://www.mirbase.org/. We also describe embedding microRNA-specific Wikipedia pages on the miRBase website to encourage the microRNA community to contribute and share textual and functional information.

  17. Frequency-locked pulse sequencer for high-frame-rate monochromatic tissue motion imaging.

    Science.gov (United States)

    Azar, Reza Zahiri; Baghani, Ali; Salcudean, Septimiu E; Rohling, Robert

    2011-04-01

    To overcome the inherent low frame rate of conventional ultrasound, we have previously presented a system that can be implemented on conventional ultrasound scanners for high-frame-rate imaging of monochromatic tissue motion. The system employs a sector subdivision technique in the sequencer to increase the acquisition rate. To eliminate the delays introduced during data acquisition, a motion phase correction algorithm has also been introduced to create in-phase displacement images. Previous experimental results from tissue- mimicking phantoms showed that the system can achieve effective frame rates of up to a few kilohertz on conventional ultrasound systems. In this short communication, we present a new pulse sequencing strategy that facilitates high-frame-rate imaging of monochromatic motion such that the acquired echo signals are inherently in-phase. The sequencer uses the knowledge of the excitation frequency to synchronize the acquisition of the entire imaging plane to that of an external exciter. This sequencing approach eliminates any need for synchronization or phase correction and has applications in tissue elastography, which we demonstrate with tissue-mimicking phantoms. © 2011 IEEE

  18. Complete genome sequence of Fer-de-Lance Virus reveals a novel gene in reptilian Paramyxoviruses

    Science.gov (United States)

    Kurath, G.; Batts, W.N.; Ahne, W.; Winton, J.R.

    2004-01-01

    The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3??? N-U-P-M-F-HN-L 5???, coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae. The FDLV P gene expression strategy is like that of rubulaviruses, which express the accessory V protein from the primary transcript and edit a portion of the mRNA to encode P and I proteins. There is also an overlapping open reading frame potentially encoding a small basic protein in the P gene. The gene designated U (unknown), encodes a deduced protein of 19.4 kDa that has no counterpart in other paramyxoviruses and has no similarity with sequences in the National Center for Biotechnology Information database. Active transcription of the U gene in infected cells was demonstrated by Northern blot analysis, and bicistronic N-U mRNA was also evident. The genomes of two other snake paramyxovirus genotypes were also found to have U genes, with 11 to 16% nucleotide divergence from the FDLV U gene. Pairwise comparisons of amino acid identities and phylogenetic analyses of all deduced FDLV protein sequences with homologous sequences from other Paramyxoviridae indicate that FDLV represents a new genus within the subfamily Paramyxovirinae. We suggest the name Ferlavirus for the new genus, with FDLV as the type species.

  19. High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

    Directory of Open Access Journals (Sweden)

    Gomes Paula

    2010-10-01

    Full Text Available Abstract Background Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. Results A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR and their RNA transcription level by quantitative PCR (q

  20. CoLIde: a bioinformatics tool for CO-expression-based small RNA Loci Identification using high-throughput sequencing data.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-07-01

    Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

  1. Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

    Science.gov (United States)

    Romanchuk, Artur; Chang, Jeff H.; Mukhtar, M. Shahid; Cherkis, Karen; Roach, Jeff; Grant, Sarah R.; Jones, Corbin D.; Dangl, Jeffery L.

    2011-01-01

    Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. PMID:21799664

  2. Metatranscriptome Sequencing Reveals Insights into the Gene Expression and Functional Potential of Rumen Wall Bacteria

    Directory of Open Access Journals (Sweden)

    Evelyne Mann

    2018-01-01

    Full Text Available Microbiota of the rumen wall constitute an important niche of rumen microbial ecology and their composition has been elucidated in different ruminants during the last years. However, the knowledge about the function of rumen wall microbes is still limited. Rumen wall biopsies were taken from three fistulated dairy cows under a standard forage-based diet and after 4 weeks of high concentrate feeding inducing a subacute rumen acidosis (SARA. Extracted RNA was used for metatranscriptome sequencing using Illumina HiSeq sequencing technology. The gene expression of the rumen wall microbial community was analyzed by mapping 35 million sequences against the Kyoto Encyclopedia for Genes and Genomes (KEGG database and determining differentially expressed genes. A total of 1,607 functional features were assigned with high expression of genes involved in central metabolism, galactose, starch and sucrose metabolism. The glycogen phosphorylase (EC:2.4.1.1 which degrades (1->4-alpha-D-glucans was among the highest expressed genes being transcribed by 115 bacterial genera. Energy metabolism genes were also highly expressed, including the pyruvate orthophosphate dikinase (EC:2.7.9.1 involved in pyruvate metabolism, which was covered by 177 genera. Nitrogen metabolism genes, in particular glutamate dehydrogenase (EC:1.4.1.4, glutamine synthetase (EC:6.3.1.2 and glutamate synthase (EC:1.4.1.13, EC:1.4.1.14 were also found to be highly expressed and prove rumen wall microbiota to be actively involved in providing host-relevant metabolites for exchange across the rumen wall. In addition, we found all four urease subunits (EC:3.5.1.5 transcribed by members of the genera Flavobacterium, Corynebacterium, Helicobacter, Clostridium, and Bacillus, and the dissimilatory sulfate reductase (EC 1.8.99.5 dsrABC, which is responsible for the reduction of sulfite to sulfide. We also provide in situ evidence for cellulose and cellobiose degradation, a key step in fiber-rich feed

  3. Internal Transcribed Spacer 1 (ITS1 based sequence typing reveals phylogenetically distinct Ascaris population

    Directory of Open Access Journals (Sweden)

    Koushik Das

    2015-01-01

    Full Text Available Taxonomic differentiation among morphologically identical Ascaris species is a debatable scientific issue in the context of Ascariasis epidemiology. To explain the disease epidemiology and also the taxonomic position of different Ascaris species, genome information of infecting strains from endemic areas throughout the world is certainly crucial. Ascaris population from human has been genetically characterized based on the widely used genetic marker, internal transcribed spacer1 (ITS1. Along with previously reported and prevalent genotype G1, 8 new sequence variants of ITS1 have been identified. Genotype G1 was significantly present among female patients aged between 10 to 15 years. Intragenic linkage disequilibrium (LD analysis at target locus within our study population has identified an incomplete LD value with potential recombination events. A separate cluster of Indian isolates with high bootstrap value indicate their distinct phylogenetic position in comparison to the global Ascaris population. Genetic shuffling through recombination could be a possible reason for high population diversity and frequent emergence of new sequence variants, identified in present and other previous studies. This study explores the genetic organization of Indian Ascaris population for the first time which certainly includes some fundamental information on the molecular epidemiology of Ascariasis.

  4. Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China.

    Science.gov (United States)

    Shu, Fan-Fan; Lv, Rui-Qing; Zhang, Yi-Fang; Duan, Gang; Wu, Ding-Yu; Li, Bi-Feng; Yang, Jian-Fa; Zou, Feng-Cai

    2012-08-01

    On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.

  5. Centroid based clustering of high throughput sequencing reads based on n-mer counts.

    Science.gov (United States)

    Solovyov, Alexander; Lipkin, W Ian

    2013-09-08

    Many problems in computational biology require alignment-free sequence comparisons. One of the common tasks involving sequence comparison is sequence clustering. Here we apply methods of alignment-free comparison (in particular, comparison using sequence composition) to the challenge of sequence clustering. We study several centroid based algorithms for clustering sequences based on word counts. Study of their performance shows that using k-means algorithm with or without the data whitening is efficient from the computational point of view. A higher clustering accuracy can be achieved using the soft expectation maximization method, whereby each sequence is attributed to each cluster with a specific probability. We implement an open source tool for alignment-free clustering. It is publicly available from github: https://github.com/luscinius/afcluster. We show the utility of alignment-free sequence clustering for high throughput sequencing analysis despite its limitations. In particular, it allows one to perform assembly with reduced resources and a minimal loss of quality. The major factor affecting performance of alignment-free read clustering is the length of the read.

  6. Highly divergent 16S rRNA sequences in ribosomal operons of Scytonema hyalinum (Cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Jeffrey R Johansen

    Full Text Available A highly divergent 16S rRNA gene was found in one of the five ribosomal operons present in a species complex currently circumscribed as Scytonema hyalinum (Nostocales, Cyanobacteria using clone libraries. If 16S rRNA sequence macroheterogeneity among ribosomal operons due to insertions, deletions or truncation is excluded, the sequence heterogeneity observed in S. hyalinum was the highest observed in any prokaryotic species thus far (7.3-9.0%. The secondary structure of the 16S rRNA molecules encoded by the two divergent operons was nearly identical, indicating possible functionality. The 23S rRNA gene was examined for a few strains in this complex, and it was also found to be highly divergent from the gene in Type 2 operons (8.7%, and likewise had nearly identical secondary structure between the Type 1 and Type 2 operons. Furthermore, the 16S-23S ITS showed marked differences consistent between operons among numerous strains. Both operons have promoter sequences that satisfy consensus requirements for functional prokaryotic transcription initiation. Horizontal gene transfer from another unknown heterocytous cyanobacterium is considered the most likely explanation for the origin of this molecule, but does not explain the ultimate origin of this sequence, which is very divergent from all 16S rRNA sequences found thus far in cyanobacteria. The divergent sequence is highly conserved among numerous strains of S. hyalinum, suggesting adaptive advantage and selective constraint of the divergent sequence.

  7. The Douglas-fir genome sequence reveals specialization of the photosynthetic apparatus in Pinaceae

    Science.gov (United States)

    David B. Neale; Patrick E. McGuire; Nicholas C. Wheeler; Kristian A. Stevens; Marc W. Crepeau; Charis Cardeno; Aleksey V. Zimin; Daniela Puiu; Geo M. Pertea; U. Uzay Sezen; Claudio Casola; Tomasz E. Koralewski; Robin Paul; Daniel Gonzalez-Ibeas; Sumaira Zaman; Richard Cronn; Mark Yandell; Carson Holt; Charles H. Langley; James A. Yorke; Steven L. Salzberg; Jill L. Wegrzyn

    2017-01-01

    A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50...

  8. Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence.

    Science.gov (United States)

    Iquebal, M A; Tomar, Rukam S; Parakhia, M V; Singla, Deepak; Jaiswal, Sarika; Rathod, V M; Padhiyar, S M; Kumar, Neeraj; Rai, Anil; Kumar, Dinesh

    2017-07-13

    Groundnut (Arachis hypogaea L.) is an important oil seed crop having major biotic constraint in production due to stem rot disease caused by fungus, Athelia rolfsii causing 25-80% loss in productivity. As chemical and biological combating strategies of this fungus are not very effective, thus genome sequencing can reveal virulence and pathogenicity related genes for better understanding of the host-parasite interaction. We report draft assembly of Athelia rolfsii genome of ~73 Mb having 8919 contigs. Annotation analysis revealed 16830 genes which are involved in fungicide resistance, virulence and pathogenicity along with putative effector and lethal genes. Secretome analysis revealed CAZY genes representing 1085 enzymatic genes, glycoside hydrolases, carbohydrate esterases, carbohydrate-binding modules, auxillary activities, glycosyl transferases and polysaccharide lyases. Repeat analysis revealed 11171 SSRs, LTR, GYPSY and COPIA elements. Comparative analysis with other existing ascomycotina genome predicted conserved domain family of WD40, CYP450, Pkinase and ABC transporter revealing insight of evolution of pathogenicity and virulence. This study would help in understanding pathogenicity and virulence at molecular level and development of new combating strategies. Such approach is imperative in endeavour of genome based solution in stem rot disease management leading to better productivity of groundnut crop in tropical region of world.

  9. Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes.

    Science.gov (United States)

    Jensen, Peter D; Zhang, Yuanji; Wiggins, B Elizabeth; Petrick, Jay S; Zhu, Jin; Kerstetter, Randall A; Heck, Gregory R; Ivashuta, Sergey I

    2013-01-01

    Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

  10. Ontogeny of hepatic energy metabolism genes in mice as revealed by RNA-sequencing.

    Directory of Open Access Journals (Sweden)

    Helen J Renaud

    Full Text Available The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age. The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5-Day 5 (perinatal-enriched, Day 10-Day 20 (pre-weaning-enriched, and Day 25-Day 60 (adolescence/adulthood-enriched. Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty

  11. Genetic variation in Rhodomyrtus tomentosa (Kemunting) populations from Malaysia as revealed by inter-simple sequence repeat markers.

    Science.gov (United States)

    Hue, T S; Abdullah, T L; Abdullah, N A P; Sinniah, U R

    2015-12-14

    Kemunting (Rhodomyrtus tomentosa) from the Myrtaceae family, is native to Malaysia. It is widely used in traditional medicine to treat various illnesses and possesses significant antibacterial properties. In addition, it has great potential as ornamental in landscape design. Genetic variability studies are important for the rational management and conservation of genetic material. In the present study, inter-simple sequence repeat markers were used to assess the genetic diversity of 18 R. tomentosa populations collected from ten states of Peninsular Malaysia. The 11 primers selected generated 173 bands that ranged in size from 1.6 kb to 130 bp, which corresponded to an average of 15.73 bands per primer. Of these bands, 97.69% (169 in total) were polymorphic. High genetic diversity was documented at the species level (H(T) = 0.2705; I = 0.3973; PPB = 97.69%) but there was a low diversity at population level (H(S) = 0.0073; I = 0 .1085; PPB = 20.14%). The high level of genetic differentiation revealed by G(ST) (73%) and analysis of molecular variance (63%), together with the limited gene flow among population (N(m) = 0.1851), suggests that the populations examined are isolated. Results from an unweighted pair group method with arithmetic mean dendrogram and principal coordinate analysis clearly grouped the populations into two geographic groups. This clear grouping can also be demonstrated by the significant Mantel test (r = 0.581, P = 0.001). We recommend that all the R. tomentosa populations be preserved in conservation program.

  12. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    DEFF Research Database (Denmark)

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie

    2014-01-01

    The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sec...

  13. Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

    Science.gov (United States)

    Chang, Lixian; Yuan, Weiping; Zeng, Huimin; Zhou, Quanquan; Wei, Wei; Zhou, Jianfeng; Li, Miaomiao; Wang, Xiaomin; Xu, Mingjiang; Yang, Fengchun; Yang, Yungui; Cheng, Tao; Zhu, Xiaofan

    2014-05-15

    Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients' clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.

  14. Multiple Teaching Approaches, Teaching Sequence and Concept Retention in High School Physics Education

    Science.gov (United States)

    Fogarty, Ian; Geelan, David

    2013-01-01

    Students in 4 Canadian high school physics classes completed instructional sequences in two key physics topics related to motion--Straight Line Motion and Newton's First Law. Different sequences of laboratory investigation, teacher explanation (lecture) and the use of computer-based scientific visualizations (animations and simulations) were…

  15. Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication

    Directory of Open Access Journals (Sweden)

    Lippold Sebastian

    2011-11-01

    Full Text Available Abstract Background DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region were investigated for larger sample sets. Results In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds and a single Przewalski horse (Equus przewalski using a recently described multiplex micro-array capture approach. We found 473 variable positions within the domestic horses, 292 of which are parsimony-informative, providing a well resolved phylogenetic tree. Our divergence time estimate suggests that the mitochondrial genomes of modern horse breeds shared a common ancestor around 93,000 years ago and no later than 38,000 years ago. A Bayesian skyline plot (BSP reveals a significant population expansion beginning 6,000-8,000 years ago with an ongoing exponential growth until the present, similar to other domestic animal species. Our data further suggest that a large sample of wild horse diversity was incorporated into the domestic population; specifically, at least 46 of the mtDNA lineages observed in domestic horses (73% already existed before the beginning of domestication about 5,000 years ago. Conclusions Our study provides a window into the maternal origins of extant domestic horses and confirms that modern domestic breeds present a wide sample of the mtDNA diversity found in ancestral, now extinct, wild horse populations. The data obtained allow us to detect a population expansion event coinciding with the beginning of domestication and to estimate both the minimum number of female horses incorporated into the domestic gene pool and the

  16. Whole exome sequencing in 342 congenital cardiac left sided lesion cases reveals extensive genetic heterogeneity and complex inheritance patterns

    Directory of Open Access Journals (Sweden)

    Alexander H. Li

    2017-10-01

    Full Text Available Abstract Background Left-sided lesions (LSLs account for an important fraction of severe congenital cardiovascular malformations (CVMs. The genetic contributions to LSLs are complex, and the mutations that cause these malformations span several diverse biological signaling pathways: TGFB, NOTCH, SHH, and more. Here, we use whole exome sequence data generated in 342 LSL cases to identify likely damaging variants in putative candidate CVM genes. Methods Using a series of bioinformatics filters, we focused on genes harboring population-rare, putative loss-of-function (LOF, and predicted damaging variants in 1760 CVM candidate genes constructed a priori from the literature and model organism databases. Gene variants that were not observed in a comparably sequenced control dataset of 5492 samples without severe CVM were then subjected to targeted validation in cases and parents. Whole exome sequencing data from 4593 individuals referred for clinical sequencing were used to bolster evidence for the role of candidate genes in CVMs and LSLs. Results Our analyses revealed 28 candidate variants in 27 genes, including 17 genes not previously associated with a human CVM disorder, and revealed diverse patterns of inheritance among LOF carriers, including 9 confirmed de novo variants in both novel and newly described human CVM candidate genes (ACVR1, JARID2, NR2F2, PLRG1, SMURF1 as well as established syndromic CVM genes (KMT2D, NF1, TBX20, ZEB2. We also identified two genes (DNAH5, OFD1 with evidence of recessive and hemizygous inheritance patterns, respectively. Within our clinical cohort, we also observed heterozygous LOF variants in JARID2 and SMAD1 in individuals with cardiac phenotypes, and collectively, carriers of LOF variants in our candidate genes had a four times higher odds of having CVM (odds ratio = 4.0, 95% confidence interval 2.5–6.5. Conclusions Our analytical strategy highlights the utility of bioinformatic resources, including human

  17. High Throughput Sequencing for Detection of Foodborne Pathogens

    Directory of Open Access Journals (Sweden)

    Camilla Sekse

    2017-10-01

    Full Text Available High-throughput sequencing (HTS is becoming the state-of-the-art technology for typing of microbial isolates, especially in clinical samples. Yet, its application is still in its infancy for monitoring and outbreak investigations of foods. Here we review the published literature, covering not only bacterial but also viral and Eukaryote food pathogens, to assess the status and potential of HTS implementation to inform stakeholders, improve food safety and reduce outbreak impacts. The developments in sequencing technology and bioinformatics have outpaced the capacity to analyze and interpret the sequence data. The influence of sample processing, nucleic acid extraction and purification, harmonized protocols for generation and interpretation of data, and properly annotated and curated reference databases including non-pathogenic “natural” strains are other major obstacles to the realization of the full potential of HTS in analytical food surveillance, epidemiological and outbreak investigations, and in complementing preventive approaches for the control and management of foodborne pathogens. Despite significant obstacles, the achieved progress in capacity and broadening of the application range over the last decade is impressive and unprecedented, as illustrated with the chosen examples from the literature. Large consortia, often with broad international participation, are making coordinated efforts to cope with many of the mentioned obstacles. Further rapid progress can therefore be prospected for the next decade.

  18. High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

    Directory of Open Access Journals (Sweden)

    Wenqin Wang

    Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

  19. Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

    Directory of Open Access Journals (Sweden)

    Yuichi Shiraishi

    Full Text Available Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV-related hepatocellular carcinomas (HCCs and their matched controls. Comparison of whole genome sequence (WGS and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3, and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

  20. High signals in the uterine cervix on T2-weighted MRI sequences

    International Nuclear Information System (INIS)

    Graef, De M.; Karam, R.; Daclin, P.Y.; Rouanet, J.P.; Juhan, V.; Maubon, A.J.

    2003-01-01

    The aim of this pictorial review was to illustrate the normal cervix appearance on T2-weighted images, and give a review of common or less common disorders of the uterine cervix that appear as high signal intensity lesions on T2-weighted sequences. Numerous aetiologies dominated by cervical cancer are reviewed and discussed. This gamut is obviously incomplete; however, radiologists who perform MR women's imaging should perform T2-weighted sequences in the sagittal plane regardless of the indication for pelvic MR. Those sequences will diagnose some previously unknown cervical cancers as well as many other unknown cervical or uterine lesions. (orig.)

  1. Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

    KAUST Repository

    Lee, Onon

    2010-11-18

    Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored. © 2011 International Society for Microbial Ecology All rights reserved.

  2. Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents.

    Science.gov (United States)

    Johnston, Christine; Magaret, Amalia; Roychoudhury, Pavitra; Greninger, Alexander L; Cheng, Anqi; Diem, Kurt; Fitzgibbon, Matthew P; Huang, Meei-Li; Selke, Stacy; Lingappa, Jairam R; Celum, Connie; Jerome, Keith R; Wald, Anna; Koelle, David M

    2017-10-01

    Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. Genital lesion swabs containing ≥ 10 7 log 10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (U L _U S ) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated U L_ U S segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids. Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

    Science.gov (United States)

    Kebschull, Justus M; Garcia da Silva, Pedro; Reid, Ashlan P; Peikon, Ian D; Albeanu, Dinu F; Zador, Anthony M

    2016-09-07

    Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

    Directory of Open Access Journals (Sweden)

    Chang Su

    2014-12-01

    Full Text Available DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees.

  5. Novel viral genomes identified from six metagenomes reveal wide distribution of archaeal viruses and high viral diversity in terrestrial hot springs

    DEFF Research Database (Denmark)

    Islin, Sóley Ruth; Menzel, Peter; Krogh, Anders

    2016-01-01

    Limited by culture-dependent methods the number of viruses identified from thermophilic Archaea and Bacteria is still very small. In this study we retrieved viral sequences from six hot spring metagenomes isolated worldwide, revealing a wide distribution of four archaeal viral families....... Among the novel genomes, one belongs to a putative thermophilic virus infecting the bacterium Hydrogenobaculum, for which no virus has been reported in the literature. Moreover, a high viral diversity was observed in the metagenomes, especially among the Lipothrixviridae, as indicated by the large...

  6. Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

    Science.gov (United States)

    Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

    2016-04-01

    Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

  7. High-throughput sequencing of Campylobacter jejuni insertion mutant libraries reveals mapA as a fitness factor for chicken colonization.

    Science.gov (United States)

    Johnson, Jeremiah G; Livny, Jonathan; Dirita, Victor J

    2014-06-01

    Campylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture.

  8. Subfamily logos: visualization of sequence deviations at alignment positions with high information content

    Directory of Open Access Journals (Sweden)

    Beitz Eric

    2006-06-01

    Full Text Available Abstract Background Recognition of relevant sequence deviations can be valuable for elucidating functional differences between protein subfamilies. Interesting residues at highly conserved positions can then be mutated and experimentally analyzed. However, identification of such sites is tedious because automated approaches are scarce. Results Subfamily logos visualize subfamily-specific sequence deviations. The display is similar to classical sequence logos but extends into the negative range. Positive, upright characters correspond to residues which are characteristic for the subfamily, negative, upside-down characters to residues typical for the remaining sequences. The symbol height is adjusted to the information content of the alignment position. Residues which are conserved throughout do not appear. Conclusion Subfamily logos provide an intuitive display of relevant sequence deviations. The method has proven to be valid using a set of 135 aligned aquaporin sequences in which established subfamily-specific positions were readily identified by the algorithm.

  9. High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae.

    Science.gov (United States)

    Chang, Perng-Kuang; Scharfenstein, Leslie L; Solorzano, Cesar D; Abbas, Hamed K; Hua, Sui-Sheng T; Jones, Walker A; Zablotowicz, Robert M

    2015-05-04

    Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus morphotype that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5 kb deletion in the norB-cypA region of the aflatoxin gene cluster (i.e. the S genotype). Phylogenetic studies have indicated that an isolate of the nonaflatoxigenic A. flavus with the S genotype is the ancestor of A. oryzae. Genome sequence comparison between A. flavus NRRL3357, which produces large sclerotia (L strain), and S-strain A. flavus 70S identified a region (samA-rosA) that was highly variable in the two morphotypes. A third type of samA-rosA region was found in A. oryzae RIB40. The three samA-rosA types were later revealed to be commonly present in A. flavus L-strain populations. Of the 182 L-strain A. flavus field isolates examined, 46%, 15% and 39% had the samA-rosA type of NRRL3357, 70S and RIB40, respectively. The three types also were found in 18 S-strain A. flavus isolates with different proportions. For A. oryzae, however, the majority (80%) of the 16 strains examined had the RIB40 type and none had the NRRL3357 type. The results suggested that A. oryzae strains in the current culture collections were mostly derived from the samA-rosA/RIB40 lineage of the nonaflatoxigenic A. flavus with the S genotype. Published by Elsevier B.V.

  10. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    Science.gov (United States)

    Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

    2015-01-01

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  11. Sequencing of emerging canine distemper virus strain reveals new distinct genetic lineage in the United States associated with disease in wildlife and domestic canine populations.

    Science.gov (United States)

    Riley, Matthew C; Wilkes, Rebecca P

    2015-12-18

    Recent outbreaks of canine distemper have prompted examination of strains from clinical samples submitted to the University of Tennessee College of Veterinary Medicine (UTCVM) Clinical Virology Lab. We previously described a new strain of CDV that significantly diverged from all genotypes reported to date including America 2, the genotype proposed to be the main lineage currently circulating in the US. The aim of this study was to determine when this new strain appeared and how widespread it is in animal populations, given that it has also been detected in fully vaccinated adult dogs. Additionally, we sequenced complete viral genomes to characterize the strain and determine if variation is confined to known variable regions of the genome or if the changes are also present in more conserved regions. Archived clinical samples were genotyped using real-time RT-PCR amplification and sequencing. The genomes of two unrelated viruses from a dog and fox each from a different state were sequenced and aligned with previously published genomes. Phylogenetic analysis was performed using coding, non-coding and genome-length sequences. Virus neutralization assays were used to evaluate potential antigenic differences between this strain and a vaccine strain and mixed ANOVA test was used to compare the titers. Genotyping revealed this strain first appeared in 2011 and was detected in dogs from multiple states in the Southeast region of the United States. It was the main strain detected among the clinical samples that were typed from 2011-2013, including wildlife submissions. Genome sequencing demonstrated that it is highly conserved within a new lineage and preliminary serologic testing showed significant differences in neutralizing antibody titers between this strain and the strain commonly used in vaccines. This new strain represents an emerging CDV in domestic dogs in the US, may be associated with a stable reservoir in the wildlife population, and could facilitate vaccine

  12. The last frontier? High-resolution, near-bottom measurements of the Hawaiian Jurassic magnetic anomaly sequence

    Science.gov (United States)

    Tivey, M.; Tominaga, M.; Sager, W. W.

    2012-12-01

    The Jurassic sequence of marine magnetic anomalies i.e. older than M29 remain the last part of the marine magnetic anomaly sequence of the geomagnetic polarity timescale (GPTS) that can be gleaned from the ocean crustal record. While Jurassic crust is present in several areas of the world's ocean basins, the oldest and arguably best preserved sequence is in the western Pacific where three lineations sets (Japanese, Hawaiian and Phoenix) converge on the oldest remaining ocean crust on the planet (i.e. crust that has not been subducted). The magnetic anomalies in these 3 lineation sets are marked by low amplitude, relatively indistinct anomalies (tiny wiggles) that collectively have been called the Jurassic quiet Zone (JQZ). Over the past 20 years we have been working on resolving the character and origin of these anomalies with various technologies to improve our resolution of this period. Following an aeromagnetic survey that revealed the possible presence of lineated anomalies older than M29 in the Japanese lineations, we conducted a deeptow magnetometer survey of the Japanese sequence in 1992. In 2002/03 we extended and confirmed this deeptow record with a deeptowed sidescan and magnetometer survey of the Japanese lineation sequence by tying in ODP Hole 801C and extending the anomaly sequence between M29 and M44. These surveys reveal remarkably fast reversals that are lineated and decrease in intensity back in time until M38, prior to which the sequence becomes somewhat confused (the LAZ or low amplitude zone) before recovering in both amplitude and lineated character around Hole 801C (M42). These results are partially supported by recently reported terrestrial magnetostratigraphy records that show the existence of reversals back to M38. A Jurassic GPTS was constructed from this Japanese anomaly sequence, but the overall global significance of the reversal sequence and systematic field intensity changes require confirmation from crustal records created at

  13. High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

    Directory of Open Access Journals (Sweden)

    Chiao-Ling Lo

    2016-08-01

    Full Text Available Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP. This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross resulted in small haplotype blocks (HB with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS, were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50% of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284 and intronic regions (169 with the least in exon's (4, suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a, excitatory receptors (Grin2a, Gria3, Grip1, neurotransmitters (Pomc, and synapses (Snap29. This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

  14. High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

    Science.gov (United States)

    Lo, Chiao-Ling; Lossie, Amy C; Liang, Tiebing; Liu, Yunlong; Xuei, Xiaoling; Lumeng, Lawrence; Zhou, Feng C; Muir, William M

    2016-08-01

    Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

  15. The gut microbiotassay – a high-throughput real-time PCR chip combined with next generation sequencing

    DEFF Research Database (Denmark)

    Hermann-Bank, Marie Louise; Skovgaard, Kerstin; Mølbak, Lars

    it demonstrated distinct quantities of bacteria in the different gut sections, with the highest number found in colon as expected. From the sequence data it was evident that primer systems targeting lower taxonomical levels, contributed with a higher resolution, revealing species that primer system targeting...

  16. A High Resolution Genetic Map Anchoring Scaffolds of the Sequenced Watermelon Genome

    Science.gov (United States)

    Kou, Qinghe; Jiang, Jiao; Guo, Shaogui; Zhang, Haiying; Hou, Wenju; Zou, Xiaohua; Sun, Honghe; Gong, Guoyi; Levi, Amnon; Xu, Yong

    2012-01-01

    As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F8 population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ∼800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits. PMID:22247776

  17. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M

    2005-12-01

    Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

  18. Whole-exome sequencing of a patient with severe and complex hemostatic abnormalities reveals a possible contributing frameshift mutation in C3AR1

    DEFF Research Database (Denmark)

    Leinøe, Eva; Nielsen, Ove Juul; Jønson, Lars

    2016-01-01

    -threatening coagulation disorder causing recurrent venous thromboembolic events, severe thrombocytopenia, and subdural hematomas. Whole-exome sequencing revealed a frameshift mutation (C3AR1 c.355-356dup, p.Asp119Alafs*19) resulting in a premature stop codon in C3AR1 (Complement Component 3a Receptor 1). Based...

  19. Self-Exciting Point Process Modeling of Conversation Event Sequences

    Science.gov (United States)

    Masuda, Naoki; Takaguchi, Taro; Sato, Nobuo; Yano, Kazuo

    Self-exciting processes of Hawkes type have been used to model various phenomena including earthquakes, neural activities, and views of online videos. Studies of temporal networks have revealed that sequences of social interevent times for individuals are highly bursty. We examine some basic properties of event sequences generated by the Hawkes self-exciting process to show that it generates bursty interevent times for a wide parameter range. Then, we fit the model to the data of conversation sequences recorded in company offices in Japan. In this way, we can estimate relative magnitudes of the self excitement, its temporal decay, and the base event rate independent of the self excitation. These variables highly depend on individuals. We also point out that the Hawkes model has an important limitation that the correlation in the interevent times and the burstiness cannot be independently modulated.

  20. Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish

    Directory of Open Access Journals (Sweden)

    Xiang Li-xin

    2010-08-01

    Full Text Available Abstract Background Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish. Results RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution. Conclusion This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host

  1. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    Science.gov (United States)

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  2. Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution

    NARCIS (Netherlands)

    Chena, X.G.; Jiang, X.; Gu, J.; Xu, M.; Wu, Y.; Deng, Y.; Zhang, C.; Bonizzoni, M.; Dermauw, W.; Vontas, J.; Armbruster, P.; Huang, X.; Yang, Y.; Zhang, H.; He, W.; Peng, H.; Liu, Y.; Wu, K.; Chen, J.; Lirakis, M.; Topalis, P.; Van Leeuwen, T.; Hall, B.A.; Thorpe, C.; Mueller, R.L.; Sun, C.; Waterhouse, R.M.; Yan, G.; Tu, Z.J.; Fang, X.; James, A.A.

    2015-01-01

    The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined

  3. SSR_pipeline--computer software for the identification of microsatellite sequences from paired-end Illumina high-throughput DNA sequence data

    Science.gov (United States)

    Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

    2013-01-01

    SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (SSRs; for example, microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains three analysis modules along with a fourth control module that can be used to automate analyses of large volumes of data. The modules are used to (1) identify the subset of paired-end sequences that pass quality standards, (2) align paired-end reads into a single composite DNA sequence, and (3) identify sequences that possess microsatellites conforming to user specified parameters. Each of the three separate analysis modules also can be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc). All modules are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, Windows). The program suite relies on a compiled Python extension module to perform paired-end alignments. Instructions for compiling the extension from source code are provided in the documentation. Users who do not have Python installed on their computers or who do not have the ability to compile software also may choose to download packaged executable files. These files include all Python scripts, a copy of the compiled extension module, and a minimal installation of Python in a single binary executable. See program documentation for more information.

  4. Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

    Directory of Open Access Journals (Sweden)

    Randi Holm Jensen

    Full Text Available Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

  5. Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges.

    Science.gov (United States)

    Vora, Neeta L; Powell, Bradford; Brandt, Alicia; Strande, Natasha; Hardisty, Emily; Gilmore, Kelly; Foreman, Ann Katherine M; Wilhelmsen, Kirk; Bizon, Chris; Reilly, Jason; Owen, Phil; Powell, Cynthia M; Skinner, Debra; Rini, Christine; Lyerly, Anne D; Boggess, Kim A; Weck, Karen; Berg, Jonathan S; Evans, James P

    2017-11-01

    PurposeWe investigated the diagnostic and clinical performance of exome sequencing in fetuses with sonographic abnormalities with normal karyotype and microarray and, in some cases, normal gene-specific sequencing.MethodsExome sequencing was performed on DNA from 15 anomalous fetuses and from the peripheral blood of their parents. Parents provided consent to be informed of diagnostic results in the fetus, medically actionable findings in the parents, and their identification as carrier couples for significant autosomal recessive conditions. We assessed the perceptions and understanding of exome sequencing using mixed methods in 15 mother-father dyads.ResultsIn seven (47%) of 15 fetuses, exome sequencing provided a diagnosis or possible diagnosis with identification of variants in the following genes: COL1A1, MUSK, KCTD1, RTTN, TMEM67, PIEZO1 and DYNC2H1. One additional case revealed a de novo nonsense mutation in a novel candidate gene (MAP4K4). The perceived likelihood that exome sequencing would explain the results (5.2 on a 10-point scale) was higher than the approximately 30% diagnostic yield discussed in pretest counseling.ConclusionExome sequencing had diagnostic utility in a highly select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy and variant interpretation must be addressed by highly tailored pre- and posttest genetic counseling.

  6. Chemistry, the Central Science? The History of the High School Science Sequence

    Science.gov (United States)

    Sheppard, Keith; Robbins, Dennis M.

    2005-01-01

    Chemistry became the ''central science'' not by design but by accident in the US high schools. The three important factors, which had their influence on the high school science, are sequenced and their impact on the development of US science education, are mentioned.

  7. Fetal anatomy revealed with fast MR sequences.

    Science.gov (United States)

    Levine, D; Hatabu, H; Gaa, J; Atkinson, M W; Edelman, R R

    1996-10-01

    Although all the imaging studies in this pictorial essay were done for maternal rather than fetal indications, fetal anatomy was well visualized. However, when scans are undertaken for fetal indications, fetal motion in between scout views and imaging sequences may make specific image planes difficult to obtain. Of the different techniques described in this review, we preferred the HASTE technique and use it almost exclusively for scanning pregnant patients. The T2-weighting is ideal for delineating fetal organs. Also, the HASTE technique allows images to be obtained in 430 msec, limiting artifacts arising from maternal and fetal motion. MR imaging should play a more important role in evaluating equivocal sonographic cases as fast scanning techniques are more widely used. Obstetric MR imaging no longer will be limited by fetal motion artifacts. When complex anatomy requires definition in a complicated pregnant patient, MR imaging should be considered as a useful adjunct to sonography.

  8. Get your high-quality low-cost genome sequence

    NARCIS (Netherlands)

    Faino, L.; Thomma, B.P.H.J.

    2014-01-01

    The study of whole-genome sequences has become essential for almost all branches of biological research. Next-generation sequencing (NGS) has revolutionized the scalability, speed, and resolution of sequencing and brought genomic science within reach of academic laboratories that study non-model

  9. Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing.

    Science.gov (United States)

    Monteiro, Carolina Cunha; Villegas, Luis Eduardo Martinez; Campolina, Thais Bonifácio; Pires, Ana Clara Machado Araújo; Miranda, Jose Carlos; Pimenta, Paulo Filemon Paolucci; Secundino, Nagila Francinete Costa

    2016-08-31

    Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing

  10. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

  11. Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

    Directory of Open Access Journals (Sweden)

    Perry Evans

    Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

  12. High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

    Science.gov (United States)

    Preston, Jessica L; Royall, Ariel E; Randel, Melissa A; Sikkink, Kristin L; Phillips, Patrick C; Johnson, Eric A

    2016-06-14

    Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

  13. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    Science.gov (United States)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  14. A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

    Science.gov (United States)

    Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike; Khan, Arifa S

    2018-01-01

    Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have

  15. Biphasic Study to Characterize Agricultural Biogas Plants by High-Throughput 16S rRNA Gene Amplicon Sequencing and Microscopic Analysis.

    Science.gov (United States)

    Maus, Irena; Kim, Yong Sung; Wibberg, Daniel; Stolze, Yvonne; Off, Sandra; Antonczyk, Sebastian; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

    2017-02-28

    Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus , were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

  16. Genome Sequencing Reveals the Potential of Achromobacter sp. HZ01 for Bioremediation

    Directory of Open Access Journals (Sweden)

    Yue-Hui Hong

    2017-08-01

    Full Text Available Petroleum pollution is a severe environmental issue. Comprehensively revealing the genetic backgrounds of hydrocarbon-degrading microorganisms contributes to developing effective methods for bioremediation of crude oil-polluted environments. Marine bacterium Achromobacter sp. HZ01 is capable of degrading hydrocarbons and producing biosurfactants. In this study, the draft genome (5.5 Mbp of strain HZ01 has been obtained by Illumina sequencing, containing 5,162 predicted genes. Genome annotation shows that “amino acid metabolism” is the most abundant metabolic pathway. Strain HZ01 is not capable of using some common carbohydrates as the sole carbon sources, which is due to that it contains few genes associated with carbohydrate transport and lacks some important enzymes related to glycometabolism. It contains abundant proteins directly related to petroleum hydrocarbon degradation. AlkB hydroxylase and its homologs were not identified. It harbors a complete enzyme system of terminal oxidation pathway for n-alkane degradation, which may be initiated by cytochrome P450. The enzymes involved in the catechol pathway are relatively complete for the degradation of aromatic compounds. This bacterium lacks several essential enzymes for methane oxidation, and Baeyer-Villiger monooxygenase involved in the subterminal oxidation pathway and cycloalkane degradation was not identified. These results suggest that strain HZ01 degrades n-alkanes via the terminal oxidation pathway, degrades aromatic compounds primarily via the catechol pathway and cannot perform methane oxidation or cycloalkane degradation. Additionally, strain HZ01 possesses abundant genes related to the metabolism of secondary metabolites, including some genes involved in biosurfactant (such as glycolipids and lipopeptides synthesis. The genome analysis also reveals its genetic basis for nitrogen metabolism, antibiotic resistance, regulatory responses to environmental changes, cell motility

  17. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    Directory of Open Access Journals (Sweden)

    Soichi Inagaki

    Full Text Available Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  18. Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes.

    Science.gov (United States)

    Dürig, N; Jude, R; Holl, H; Brooks, S A; Lafayette, C; Jagannathan, V; Leeb, T

    2017-08-01

    White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re-investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes' individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9-kb deletion spanning exons 10-13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses. © 2017 Stichting International Foundation for Animal Genetics.

  19. Essential Genes for In Vitro Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing.

    Science.gov (United States)

    Rosconi, Federico; de Vries, Stefan P W; Baig, Abiyad; Fabiano, Elena; Grant, Andrew J

    2016-11-15

    The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle. A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we

  20. BAC and RNA sequencing reveal the brown planthopper resistance gene BPH15 in a recombination cold spot that mediates a unique defense mechanism.

    Science.gov (United States)

    Lv, Wentang; Du, Ba; Shangguan, Xinxin; Zhao, Yan; Pan, Yufang; Zhu, Lili; He, Yuqing; He, Guangcun

    2014-08-11

    Brown planthopper (BPH, Nilaparvata lugens Stål), is the most destructive phloem-feeding insect pest of rice (Oryza sativa). The BPH-resistance gene BPH15 has been proved to be effective in controlling the pest and widely applied in rice breeding programs. Nevertheless, molecular mechanism of the resistance remain unclear. In this study, we narrowed down the position of BPH15 on chromosome 4 and investigated the transcriptome of BPH15 rice after BPH attacked. We analyzed 13,000 BC2F2 plants of cross between susceptible rice TN1 and the recombinant inbred line RI93 that carrying the BPH15 gene from original resistant donor B5. BPH15 was mapped to a 0.0269 cM region on chromosome 4, which is 210-kb in the reference genome of Nipponbare. Sequencing bacterial artificial chromosome (BAC) clones that span the BPH15 region revealed that the physical size of BPH15 region in resistant rice B5 is 580-kb, much bigger than the corresponding region in the reference genome of Nipponbare. There were 87 predicted genes in the BPH15 region in resistant rice. The expression profiles of predicted genes were analyzed. Four jacalin-related lectin proteins genes and one LRR protein gene were found constitutively expressed in resistant parent and considered the candidate genes of BPH15. The transcriptomes of resistant BPH15 introgression line and the susceptible recipient line were analyzed using high-throughput RNA sequencing. In total, 2,914 differentially expressed genes (DEGs) were identified. BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6 h after infestation, HAI) and late stage (48 HAI). The key defense mechanism was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca(2+) signaling, PR genes, transcription factors, and protein posttranslational modifications. Our work combined BAC and RNA sequencing to identify candidate genes of BPH15 and revealed the resistance mechanism

  1. Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method

    Science.gov (United States)

    Geens, Tom; Desplanques, Ann; Van Loock, Marnix; Bönner, Brigitte M.; Kaleta, Erhard F.; Magnino, Simone; Andersen, Arthur A.; Everett, Karin D. E.; Vanrompay, Daisy

    2005-01-01

    Twenty-one avian Chlamydophila psittaci isolates from different European countries were characterized using ompA restriction fragment length polymorphism, ompA sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method. PMID:15872282

  2. The need for high-quality whole-genome sequence databases in microbial forensics.

    Science.gov (United States)

    Sjödin, Andreas; Broman, Tina; Melefors, Öjar; Andersson, Gunnar; Rasmusson, Birgitta; Knutsson, Rickard; Forsman, Mats

    2013-09-01

    Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

  3. Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

    Science.gov (United States)

    Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

    2017-10-01

    Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. SMRT Sequencing Revealed Mitogenome Characteristics and Mitogenome-Wide DNA Modification Pattern in Ophiocordyceps sinensis.

    Science.gov (United States)

    Kang, Xincong; Hu, Liqin; Shen, Pengyuan; Li, Rui; Liu, Dongbo

    2017-01-01

    Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis , with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3 , 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis , and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO 2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

  5. Genomic sequencing of Pleistocene cave bears

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, James P.; Hofreiter, Michael; Smith, Doug; Priest, JamesR.; Rohland, Nadin; Rabeder, Gernot; Krause, Johannes; Detter, J. Chris; Paabo, Svante; Rubin, Edward M.

    2005-04-01

    Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome, the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.

  6. Working memory for sequences of temporal durations reveals a volatile single-item store

    Directory of Open Access Journals (Sweden)

    Sanjay G Manohar

    2016-10-01

    Full Text Available When a sequence is held in working memory, different items are retained with differing fidelity. Here we ask whether a sequence of brief time intervals that must be remembered show recency effects, similar to those observed in verbal and visuospatial working memory. It has been suggested that prioritising some items over others can be accounted for by a focus of attention, maintaining some items in a privileged state. We therefore also investigated whether such benefits are vulnerable to disruption by attention or expectation. Participants listened to sequences of one to five tones, of varying durations (200ms to 2s. Subsequently, the length of one of the tones in the sequence had to be reproduced by holding a key. The discrepancy between the reproduced and actual durations quantified the fidelity of memory for auditory durations. Recall precision decreased with the number of items that had to be remembered, and was better for the first and last items of sequences, in line with set-size and serial position effects seen in other modalities. To test whether attentional filtering demands might impair performance, an irrelevant variation in pitch was introduced in some blocks of trials. In those blocks, memory precision was worse for sequences that consisted of only one item, i.e. the smallest memory set size. Thus, when irrelevant information was present, the benefit of having only one item in memory is attenuated. Finally we examined whether expectation could interfere with memory. On half the trials, the number of items in the upcoming sequence was cued. When the number of items was known in advance, performance was paradoxically worse when the sequence consisted of only one item. Thus the benefit of having only one item to remember is stronger when it is unexpectedly the only item. Our results suggest that similar mechanisms are used to hold auditory time durations in working memory, as for visual or verbal stimuli. Further, solitary items were

  7. The double main sequence of Omega Centauri

    Science.gov (United States)

    Bedin, L. R.; Piotto, G.; Anderson, J.; King, I. R.; Cassisi, S.; Momany, Y.

    Recent, high precision photometry of Omega Centauri, the biggest Galactic globular cluster, has been obtained with Hubble Space Telescope (HST). The color magnitude diagram reveals an unexpected bifurcation of colors in the main sequence (MS). The newly found double MS, the multiple turnoffs and subgiant branches, and other sequences discovered in the past along the red giant branch of this cluster add up to a fascinating but frustrating puzzle. Among the possible explanations for the blue main sequence an anomalous overabundance of helium is suggested. The hypothesis will be tested with a set of FLAMES@VLT data we have recently obtained (ESO DDT program), and with forthcoming ACS@HST images. Based on observations with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by AURA, Inc., under NASA contract NAS 5-26555.

  8. Combining Amplification Typing of L1 Active Subfamilies (ATLAS) with High-Throughput Sequencing.

    Science.gov (United States)

    Rahbari, Raheleh; Badge, Richard M

    2016-01-01

    With the advent of new generations of high-throughput sequencing technologies, the catalog of human genome variants created by retrotransposon activity is expanding rapidly. However, despite these advances in describing L1 diversity and the fact that L1 must retrotranspose in the germline or prior to germline partitioning to be evolutionarily successful, direct assessment of de novo L1 retrotransposition in the germline or early embryogenesis has not been achieved for endogenous L1 elements. A direct study of de novo L1 retrotransposition into susceptible loci within sperm DNA (Freeman et al., Hum Mutat 32(8):978-988, 2011) suggested that the rate of L1 retrotransposition in the germline is much lower than previously estimated (ATLAS L1 display technique (Badge et al., Am J Hum Genet 72(4):823-838, 2003) to investigate de novo L1 retrotransposition in human genomes. In this chapter, we describe how we combined a high-coverage ATLAS variant with high-throughput sequencing, achieving 11-25× sequence depth per single amplicon, to study L1 retrotransposition in whole genome amplified (WGA) DNAs.

  9. NeSSM: a Next-generation Sequencing Simulator for Metagenomics.

    Directory of Open Access Journals (Sweden)

    Ben Jia

    Full Text Available BACKGROUND: Metagenomics can reveal the vast majority of microbes that have been missed by traditional cultivation-based methods. Due to its extremely wide range of application areas, fast metagenome sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of metagenomics analysis tools. RESULTS: We present here a customizable metagenome simulation system: NeSSM (Next-generation Sequencing Simulator for Metagenomics. Combining complete genomes currently available, a community composition table, and sequencing parameters, it can simulate metagenome sequencing better than existing systems. Sequencing error models based on the explicit distribution of errors at each base and sequencing coverage bias are incorporated in the simulation. In order to improve the fidelity of simulation, tools are provided by NeSSM to estimate the sequencing error models, sequencing coverage bias and the community composition directly from existing metagenome sequencing data. Currently, NeSSM supports single-end and pair-end sequencing for both 454 and Illumina platforms. In addition, a GPU (graphics processing units version of NeSSM is also developed to accelerate the simulation. By comparing the simulated sequencing data from NeSSM with experimental metagenome sequencing data, we have demonstrated that NeSSM performs better in many aspects than existing popular metagenome simulators, such as MetaSim, GemSIM and Grinder. The GPU version of NeSSM is more than one-order of magnitude faster than MetaSim. CONCLUSIONS: NeSSM is a fast simulation system for high-throughput metagenome sequencing. It can be helpful to develop tools and evaluate strategies for metagenomics analysis and it's freely available for academic users at http://cbb.sjtu.edu.cn/~ccwei/pub/software/NeSSM.php.

  10. 18S rDNA Sequences from Microeukaryotes Reveal Oil Indicators in Mangrove Sediment

    Science.gov (United States)

    Santos, Henrique F.; Cury, Juliano C.; Carmo, Flavia L.; Rosado, Alexandre S.; Peixoto, Raquel S.

    2010-01-01

    Background Microeukaryotes are an effective indicator of the presence of environmental contaminants. However, the characterisation of these organisms by conventional tools is often inefficient, and recent molecular studies have revealed a great diversity of microeukaryotes. The full extent of this diversity is unknown, and therefore, the distribution, ecological role and responses to anthropogenic effects of microeukaryotes are rather obscure. The majority of oil from oceanic oil spills (e.g., the May 2010 accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves, which are threatened with worldwide disappearance, highlighting the need for efficient tools to indicate the presence of oil in these environments. However, no studies have used molecular methods to assess the effects of oil contamination in mangrove sediment on microeukaryotes as a group. Methodology/Principal Findings We evaluated the population dynamics and the prevailing 18S rDNA phylotypes of microeukaryotes in mangrove sediment microcosms with and without oil contamination, using PCR/DGGE and clone libraries. We found that microeukaryotes are useful for monitoring oil contamination in mangroves. Our clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the Nematoda. After contamination, a large increase in the abundance of the groups Bacillariophyta (diatoms) and Biosoecida was detected. The oil-contaminated samples were almost entirely dominated by organisms related to Bacillariophyta sp. and Cafeteria minima, which indicates that these groups are possible targets for biomonitoring oil in mangroves. The DGGE fingerprints also indicated shifts in microeukaryote profiles; specific band sequencing indicated the appearance of Bacillariophyta sp. only in contaminated samples and Nematoda only in non-contaminated sediment. Conclusions/Significance We believe that

  11. Improvements and impacts of GRCh38 human reference on high throughput sequencing data analysis.

    Science.gov (United States)

    Guo, Yan; Dai, Yulin; Yu, Hui; Zhao, Shilin; Samuels, David C; Shyr, Yu

    2017-03-01

    Analyses of high throughput sequencing data starts with alignment against a reference genome, which is the foundation for all re-sequencing data analyses. Each new release of the human reference genome has been augmented with improved accuracy and completeness. It is presumed that the latest release of human reference genome, GRCh38 will contribute more to high throughput sequencing data analysis by providing more accuracy. But the amount of improvement has not yet been quantified. We conducted a study to compare the genomic analysis results between the GRCh38 reference and its predecessor GRCh37. Through analyses of alignment, single nucleotide polymorphisms, small insertion/deletions, copy number and structural variants, we show that GRCh38 offers overall more accurate analysis of human sequencing data. More importantly, GRCh38 produced fewer false positive structural variants. In conclusion, GRCh38 is an improvement over GRCh37 not only from the genome assembly aspect, but also yields more reliable genomic analysis results. Copyright © 2017. Published by Elsevier Inc.

  12. Genome Sequencing and Mapping Reveal Loss of Heterozygosity as a Mechanism for Rapid Adaptation in the Vegetable Pathogen Phytophthora capsici

    Energy Technology Data Exchange (ETDEWEB)

    Lamour, Kurt H.; Mudge, Joann; Gobena, Daniel; Hurtado-Gonzales, Oscar P.; Schmutz, Jeremy; Kuo, Alan; Miller, Neil A.; Rice, Brandon J.; Raffaele, Sylvain; Cano, Liliana M.; Bharti, Arvind K.; Donahoo, Ryan S.; Finely, Sabra; Huitema, Edgar; Hulvey, Jon; Platt, Darren; Salamov, Asaf; Savidor, Alon; Sharma, Rahul; Stam, Remco; Sotrey, Dylan; Thines, Marco; Win, Joe; Haas, Brian J.; Dinwiddie, Darrell L.; Jenkins, Jerry; Knight, James R.; Affourtit, Jason P.; Han, Cliff S.; Chertkov, Olga; Lindquist, Erika A.; Detter, Chris; Grigoriev, Igor V.; Kamoun, Sophien; Kingsmore, Stephen F.

    2012-02-07

    The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30percent of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

  13. Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

    Science.gov (United States)

    Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

    2015-12-15

    MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

    Directory of Open Access Journals (Sweden)

    Matthew W. Blair

    2011-11-01

    Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

  15. Implication of the cause of differences in 3D structures of proteins with high sequence identity based on analyses of amino acid sequences and 3D structures.

    Science.gov (United States)

    Matsuoka, Masanari; Sugita, Masatake; Kikuchi, Takeshi

    2014-09-18

    Proteins that share a high sequence homology while exhibiting drastically different 3D structures are investigated in this study. Recently, artificial proteins related to the sequences of the GA and IgG binding GB domains of human serum albumin have been designed. These artificial proteins, referred to as GA and GB, share 98% amino acid sequence identity but exhibit different 3D structures, namely, a 3α bundle versus a 4β + α structure. Discriminating between their 3D structures based on their amino acid sequences is a very difficult problem. In the present work, in addition to using bioinformatics techniques, an analysis based on inter-residue average distance statistics is used to address this problem. It was hard to distinguish which structure a given sequence would take only with the results of ordinary analyses like BLAST and conservation analyses. However, in addition to these analyses, with the analysis based on the inter-residue average distance statistics and our sequence tendency analysis, we could infer which part would play an important role in its structural formation. The results suggest possible determinants of the different 3D structures for sequences with high sequence identity. The possibility of discriminating between the 3D structures based on the given sequences is also discussed.

  16. Genetic signatures of adaptation revealed from transcriptome sequencing of Arctic and red foxes.

    Science.gov (United States)

    Kumar, Vikas; Kutschera, Verena E; Nilsson, Maria A; Janke, Axel

    2015-08-07

    The genus Vulpes (true foxes) comprises numerous species that inhabit a wide range of habitats and climatic conditions, including one species, the Arctic fox (Vulpes lagopus) which is adapted to the arctic region. A close relative to the Arctic fox, the red fox (Vulpes vulpes), occurs in subarctic to subtropical habitats. To study the genetic basis of their adaptations to different environments, transcriptome sequences from two Arctic foxes and one red fox individual were generated and analyzed for signatures of positive selection. In addition, the data allowed for a phylogenetic analysis and divergence time estimate between the two fox species. The de novo assembly of reads resulted in more than 160,000 contigs/transcripts per individual. Approximately 17,000 homologous genes were identified using human and the non-redundant databases. Positive selection analyses revealed several genes involved in various metabolic and molecular processes such as energy metabolism, cardiac gene regulation, apoptosis and blood coagulation to be under positive selection in foxes. Branch site tests identified four genes to be under positive selection in the Arctic fox transcriptome, two of which are fat metabolism genes. In the red fox transcriptome eight genes are under positive selection, including molecular process genes, notably genes involved in ATP metabolism. Analysis of the three transcriptomes and five Sanger re-sequenced genes in additional individuals identified a lower genetic variability within Arctic foxes compared to red foxes, which is consistent with distribution range differences and demographic responses to past climatic fluctuations. A phylogenomic analysis estimated that the Arctic and red fox lineages diverged about three million years ago. Transcriptome data are an economic way to generate genomic resources for evolutionary studies. Despite not representing an entire genome, this transcriptome analysis identified numerous genes that are relevant to arctic

  17. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth.

    Science.gov (United States)

    Peng, Yu; Leung, Henry C M; Yiu, S M; Chin, Francis Y L

    2012-06-01

    Next-generation sequencing allows us to sequence reads from a microbial environment using single-cell sequencing or metagenomic sequencing technologies. However, both technologies suffer from the problem that sequencing depth of different regions of a genome or genomes from different species are highly uneven. Most existing genome assemblers usually have an assumption that sequencing depths are even. These assemblers fail to construct correct long contigs. We introduce the IDBA-UD algorithm that is based on the de Bruijn graph approach for assembling reads from single-cell sequencing or metagenomic sequencing technologies with uneven sequencing depths. Several non-trivial techniques have been employed to tackle the problems. Instead of using a simple threshold, we use multiple depthrelative thresholds to remove erroneous k-mers in both low-depth and high-depth regions. The technique of local assembly with paired-end information is used to solve the branch problem of low-depth short repeat regions. To speed up the process, an error correction step is conducted to correct reads of high-depth regions that can be aligned to highconfident contigs. Comparison of the performances of IDBA-UD and existing assemblers (Velvet, Velvet-SC, SOAPdenovo and Meta-IDBA) for different datasets, shows that IDBA-UD can reconstruct longer contigs with higher accuracy. The IDBA-UD toolkit is available at our website http://www.cs.hku.hk/~alse/idba_ud

  18. Spatio-temporal alignment of pedobarographic image sequences.

    Science.gov (United States)

    Oliveira, Francisco P M; Sousa, Andreia; Santos, Rubim; Tavares, João Manuel R S

    2011-07-01

    This article presents a methodology to align plantar pressure image sequences simultaneously in time and space. The spatial position and orientation of a foot in a sequence are changed to match the foot represented in a second sequence. Simultaneously with the spatial alignment, the temporal scale of the first sequence is transformed with the aim of synchronizing the two input footsteps. Consequently, the spatial correspondence of the foot regions along the sequences as well as the temporal synchronizing is automatically attained, making the study easier and more straightforward. In terms of spatial alignment, the methodology can use one of four possible geometric transformation models: rigid, similarity, affine, or projective. In the temporal alignment, a polynomial transformation up to the 4th degree can be adopted in order to model linear and curved time behaviors. Suitable geometric and temporal transformations are found by minimizing the mean squared error (MSE) between the input sequences. The methodology was tested on a set of real image sequences acquired from a common pedobarographic device. When used in experimental cases generated by applying geometric and temporal control transformations, the methodology revealed high accuracy. In addition, the intra-subject alignment tests from real plantar pressure image sequences showed that the curved temporal models produced better MSE results (P alignment of pedobarographic image data, since previous methods can only be applied on static images.

  19. Genome Microscale Heterogeneity among Wild Potatoes Revealed by Diversity Arrays Technology Marker Sequences

    Directory of Open Access Journals (Sweden)

    Alessandra Traini

    2013-01-01

    Full Text Available Tuber-bearing potato species possess several genes that can be exploited to improve the genetic background of the cultivated potato Solanum tuberosum. Among them, S. bulbocastanum and S. commersonii are well known for their strong resistance to environmental stresses. However, scant information is available for these species in terms of genome organization, gene function, and regulatory networks. Consequently, genomic tools to assist breeding are meager, and efficient exploitation of these species has been limited so far. In this paper, we employed the reference genome sequences from cultivated potato and tomato and a collection of sequences of 1,423 potato Diversity Arrays Technology (DArT markers that show polymorphic representation across the genomes of S. bulbocastanum and/or S. commersonii genotypes. Our results highlighted microscale genome sequence heterogeneity that may play a significant role in functional and structural divergence between related species. Our analytical approach provides knowledge of genome structural and sequence variability that could not be detected by transcriptome and proteome approaches.

  20. Genome Microscale Heterogeneity among Wild Potatoes Revealed by Diversity Arrays Technology Marker Sequences.

    Science.gov (United States)

    Traini, Alessandra; Iorizzo, Massimo; Mann, Harpartap; Bradeen, James M; Carputo, Domenico; Frusciante, Luigi; Chiusano, Maria Luisa

    2013-01-01

    Tuber-bearing potato species possess several genes that can be exploited to improve the genetic background of the cultivated potato Solanum tuberosum. Among them, S. bulbocastanum and S. commersonii are well known for their strong resistance to environmental stresses. However, scant information is available for these species in terms of genome organization, gene function, and regulatory networks. Consequently, genomic tools to assist breeding are meager, and efficient exploitation of these species has been limited so far. In this paper, we employed the reference genome sequences from cultivated potato and tomato and a collection of sequences of 1,423 potato Diversity Arrays Technology (DArT) markers that show polymorphic representation across the genomes of S. bulbocastanum and/or S. commersonii genotypes. Our results highlighted microscale genome sequence heterogeneity that may play a significant role in functional and structural divergence between related species. Our analytical approach provides knowledge of genome structural and sequence variability that could not be detected by transcriptome and proteome approaches.

  1. Applications of High Throughput Nucleotide Sequencing

    DEFF Research Database (Denmark)

    Waage, Johannes Eichler

    equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......-sequencing, a study of the effects on alternative RNA splicing of KO of the nonsense mediated RNA decay system in Mus, using digital gene expression and a custom-built exon-exon junction mapping pipeline is presented (article I). Evolved from this work, a Bioconductor package, spliceR, for classifying alternative...

  2. Transcriptome sequence analysis of an ornamental plant, Ananas comosus var. bracteatus, revealed the potential unigenes involved in terpenoid and phenylpropanoid biosynthesis.

    Science.gov (United States)

    Ma, Jun; Kanakala, S; He, Yehua; Zhang, Junli; Zhong, Xiaolan

    2015-01-01

    Ananas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies. The Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis. The sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus.

  3. Transcriptome sequence analysis of an ornamental plant, Ananas comosus var. bracteatus, revealed the potential unigenes involved in terpenoid and phenylpropanoid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Jun Ma

    Full Text Available Ananas comosus var. bracteatus (Red Pineapple is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies.The Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis.The sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus.

  4. ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences.

    Science.gov (United States)

    Mas, Philippe J; Hart, Darren J

    2017-01-01

    Production of soluble, purifiable domains or multi-domain fragments of proteins is a prerequisite for structural biology and other applications. When target sequences are poorly annotated, or when there are few similar sequences available for alignments, identification of domains can be problematic. A method called expression of soluble proteins by random incremental truncation (ESPRIT) addresses this problem by high-throughput automated screening of tens of thousands of enzymatically truncated gene fragments. Rare soluble constructs are identified by experimental screening, and the boundaries revealed by DNA sequencing.

  5. High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L..

    Directory of Open Access Journals (Sweden)

    Livia Donaire

    Full Text Available Small RNAs (sRNAs of 20 to 25 nucleotides (nt in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.. sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

  6. SEED 2: a user-friendly platform for amplicon high-throughput sequencing data analyses.

    Science.gov (United States)

    Vetrovský, Tomáš; Baldrian, Petr; Morais, Daniel; Berger, Bonnie

    2018-02-14

    Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4GB of data. SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. daniel.morais@biomed.cas.cz. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press.

  7. Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis

    Directory of Open Access Journals (Sweden)

    Zhao Patrick X

    2011-07-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L., a species with high economic value but limited genomic resources. Results The alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1 chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories. Conclusions We describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.

  8. Genetic diversity of Taenia asiatica from Thailand and other geographical locations as revealed by cytochrome c oxidase subunit 1 sequences.

    Science.gov (United States)

    Anantaphruti, Malinee Thairungroj; Thaenkham, Urusa; Watthanakulpanich, Dorn; Phuphisut, Orawan; Maipanich, Wanna; Yoonuan, Tippayarat; Nuamtanong, Supaporn; Pubampen, Somjit; Sanguankiat, Surapol

    2013-02-01

    Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.

  9. Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

    Science.gov (United States)

    Hochgerner, Hannah; Zeisel, Amit; Lönnerberg, Peter; Linnarsson, Sten

    2018-02-01

    The dentate gyrus of the hippocampus is a brain region in which neurogenesis persists into adulthood; however, the relationship between developmental and adult dentate gyrus neurogenesis has not been examined in detail. Here we used single-cell RNA sequencing to reveal the molecular dynamics and diversity of dentate gyrus cell types in perinatal, juvenile, and adult mice. We found distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. We observed shifts in the molecular identity of quiescent and proliferating radial glia and granule cells during the postnatal period that were then maintained through adult stages. In contrast, intermediate progenitor cells, neuroblasts, and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental similarity of postnatal and adult neurogenesis in the hippocampus and pinpoint the early postnatal transformation of radial glia from embryonic progenitors to adult quiescent stem cells.

  10. Ananke: temporal clustering reveals ecological dynamics of microbial communities

    Directory of Open Access Journals (Sweden)

    Michael W. Hall

    2017-09-01

    Full Text Available Taxonomic markers such as the 16S ribosomal RNA gene are widely used in microbial community analysis. A common first step in marker-gene analysis is grouping genes into clusters to reduce data sets to a more manageable size and potentially mitigate the effects of sequencing error. Instead of clustering based on sequence identity, marker-gene data sets collected over time can be clustered based on temporal correlation to reveal ecologically meaningful associations. We present Ananke, a free and open-source algorithm and software package that complements existing sequence-identity-based clustering approaches by clustering marker-gene data based on time-series profiles and provides interactive visualization of clusters, including highlighting of internal OTU inconsistencies. Ananke is able to cluster distinct temporal patterns from simulations of multiple ecological patterns, such as periodic seasonal dynamics and organism appearances/disappearances. We apply our algorithm to two longitudinal marker gene data sets: faecal communities from the human gut of an individual sampled over one year, and communities from a freshwater lake sampled over eleven years. Within the gut, the segregation of the bacterial community around a food-poisoning event was immediately clear. In the freshwater lake, we found that high sequence identity between marker genes does not guarantee similar temporal dynamics, and Ananke time-series clusters revealed patterns obscured by clustering based on sequence identity or taxonomy. Ananke is free and open-source software available at https://github.com/beiko-lab/ananke.

  11. Multilocus sequence data reveal dozens of putative cryptic species in a radiation of endemic Californian mygalomorph spiders (Araneae, Mygalomorphae, Nemesiidae).

    Science.gov (United States)

    Leavitt, Dean H; Starrett, James; Westphal, Michael F; Hedin, Marshal

    2015-10-01

    We use mitochondrial and multi-locus nuclear DNA sequence data to infer both species boundaries and species relationships within California nemesiid spiders. Higher-level phylogenetic data show that the California radiation is monophyletic and distantly related to European members of the genus Brachythele. As such, we consider all California nemesiid taxa to belong to the genus Calisoga Chamberlin, 1937. Rather than find support for one or two taxa as previously hypothesized, genetic data reveal Calisoga to be a species-rich radiation of spiders, including perhaps dozens of species. This conclusion is supported by multiple mitochondrial barcoding analyses, and also independent analyses of nuclear data that reveal general genealogical congruence. We discovered three instances of sympatry, and genetic data indicate reproductive isolation when in sympatry. An examination of female reproductive morphology does not reveal species-specific characters, and observed male morphological differences for a subset of putative species are subtle. Our coalescent species tree analysis of putative species lays the groundwork for future research on the taxonomy and biogeographic history of this remarkable endemic radiation. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. High-sensitivity HLA typing by Saturated Tiling Capture Sequencing (STC-Seq).

    Science.gov (United States)

    Jiao, Yang; Li, Ran; Wu, Chao; Ding, Yibin; Liu, Yanning; Jia, Danmei; Wang, Lifeng; Xu, Xiang; Zhu, Jing; Zheng, Min; Jia, Junling

    2018-01-15

    Highly polymorphic human leukocyte antigen (HLA) genes are responsible for fine-tuning the adaptive immune system. High-resolution HLA typing is important for the treatment of autoimmune and infectious diseases. Additionally, it is routinely performed for identifying matched donors in transplantation medicine. Although many HLA typing approaches have been developed, the complexity, low-efficiency and high-cost of current HLA-typing assays limit their application in population-based high-throughput HLA typing for donors, which is required for creating large-scale databases for transplantation and precision medicine. Here, we present a cost-efficient Saturated Tiling Capture Sequencing (STC-Seq) approach to capturing 14 HLA class I and II genes. The highly efficient capture (an approximately 23,000-fold enrichment) of these genes allows for simplified allele calling. Tests on five genes (HLA-A/B/C/DRB1/DQB1) from 31 human samples and 351 datasets using STC-Seq showed results that were 98% consistent with the known two sets of digitals (field1 and field2) genotypes. Additionally, STC can capture genomic DNA fragments longer than 3 kb from HLA loci, making the library compatible with the third-generation sequencing. STC-Seq is a highly accurate and cost-efficient method for HLA typing which can be used to facilitate the establishment of population-based HLA databases for the precision and transplantation medicine.

  13. Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

    Science.gov (United States)

    Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

    2017-08-16

    High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

  14. Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers

    Directory of Open Access Journals (Sweden)

    Kuzin Alexander

    2008-08-01

    Full Text Available Abstract Background The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers. Results Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS. Conclusion The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.

  15. Whole-exome sequencing reveals genetic variants associated with chronic kidney disease characterized by tubulointerstitial damages in North Central Region, Sri Lanka.

    Science.gov (United States)

    Nanayakkara, Shanika; Senevirathna, S T M L D; Parahitiyawa, Nipuna B; Abeysekera, Tilak; Chandrajith, Rohana; Ratnatunga, Neelakanthi; Hitomi, Toshiaki; Kobayashi, Hatasu; Harada, Kouji H; Koizumi, Akio

    2015-09-01

    The familial clustering observed in chronic kidney disease of uncertain etiology (CKDu) characterized by tubulointerstitial damages in the North Central Region of Sri Lanka strongly suggests the involvement of genetic factors in its pathogenesis. The objective of the present study is to use whole-exome sequencing to identify the genetic variants associated with CKDu. Whole-exome sequencing of eight CKDu cases and eight controls was performed, followed by direct sequencing of candidate loci in 301 CKDu cases and 276 controls. Association study revealed rs34970857 (c.658G > A/p.V220M) located in the KCNA10 gene encoding a voltage-gated K channel as the most promising SNP with the highest odds ratio of 1.74. Four rare variants were identified in gene encoding Laminin beta2 (LAMB2) which is known to cause congenital nephrotic syndrome. Three out of four variants in LAMB2 were novel variants found exclusively in cases. Genetic investigations provide strong evidence on the presence of genetic susceptibility for CKDu. Possibility of presence of several rare variants associated with CKDu in this population is also suggested.

  16. LookSeq: a browser-based viewer for deep sequencing data.

    Science.gov (United States)

    Manske, Heinrich Magnus; Kwiatkowski, Dominic P

    2009-11-01

    Sequencing a genome to great depth can be highly informative about heterogeneity within an individual or a population. Here we address the problem of how to visualize the multiple layers of information contained in deep sequencing data. We propose an interactive AJAX-based web viewer for browsing large data sets of aligned sequence reads. By enabling seamless browsing and fast zooming, the LookSeq program assists the user to assimilate information at different levels of resolution, from an overview of a genomic region to fine details such as heterogeneity within the sample. A specific problem, particularly if the sample is heterogeneous, is how to depict information about structural variation. LookSeq provides a simple graphical representation of paired sequence reads that is more revealing about potential insertions and deletions than are conventional methods.

  17. HTSeq--a Python framework to work with high-throughput sequencing data.

    Science.gov (United States)

    Anders, Simon; Pyl, Paul Theodor; Huber, Wolfgang

    2015-01-15

    A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed. We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes. HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq. © The Author 2014. Published by Oxford University Press.

  18. First applications of a targeted exome sequencing approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with associated gene defects

    Directory of Open Access Journals (Sweden)

    Constantinos Pangalos

    2016-04-01

    Full Text Available Background. Fetal malformations and other structural abnormalities are relatively frequent findings in the course of routine prenatal ultrasonographic examination. Due to their considerable genetic and clinical heterogeneity, the underlying genetic cause is often elusive and the resulting inability to provide a precise diagnosis precludes proper reproductive and fetal risk assessment. We report the development and first applications of an expanded exome sequencing-based test, coupled to a bioinformatics-driven prioritization algorithm, targeting gene disorders presenting with abnormal prenatal ultrasound findings. Methods. We applied the testing strategy to14 euploid fetuses, from 11 on-going pregnancies and three products of abortion, all with various abnormalities or malformations detected through prenatal ultrasound examination. Whole exome sequencing (WES was followed by variant prioritization, utilizing a custom analysis pipeline (Fetalis algorithm, targeting 758 genes associated with genetic disorders which may present with abnormal fetal ultrasound findings. Results. A definitive or highly-likely diagnosis was made in 6 of 14 cases (43%, of which 3 were abortuses (Ellis-van Creveld syndrome, Ehlers-Danlos syndrome and Nemaline myopathy 2 and 3 involved on-going pregnancies (Citrullinemia, Noonan syndrome, PROKR2-related Kallmann syndrome. In the remaining eight on-going pregnancy cases (57%, a ZIC1 variant of unknown clinical significance was detected in one case, while in seven cases testing did not reveal any pathogenic variant(s. Pregnancies were followed-up to birth, resulting in one neonate harboring the PROKR2 mutation, presenting with isolated minor structural cardiac abnormalities, and in seven apparently healthy neonates. Discussion. The expanded targeted exome sequencing-based approach described herein (Fetalis, provides strong evidence suggesting a definite and beneficial increase in our diagnostic capabilities in prenatal

  19. Sequence composition and gene content of the short arm of rye (Secale cereale chromosome 1.

    Directory of Open Access Journals (Sweden)

    Silvia Fluch

    Full Text Available BACKGROUND: The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3% being the most abundant. More than four thousand simple sequence repeat (SSR sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. CONCLUSIONS: The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye.

  20. Exploring evidence of positive selection reveals genetic basis of meat quality traits in Berkshire pigs through whole genome sequencing.

    Science.gov (United States)

    Jeong, Hyeonsoo; Song, Ki-Duk; Seo, Minseok; Caetano-Anollés, Kelsey; Kim, Jaemin; Kwak, Woori; Oh, Jae-Don; Kim, EuiSoo; Jeong, Dong Kee; Cho, Seoae; Kim, Heebal; Lee, Hak-Kyo

    2015-08-20

    Natural and artificial selection following domestication has led to the existence of more than a hundred pig breeds, as well as incredible variation in phenotypic traits. Berkshire pigs are regarded as having superior meat quality compared to other breeds. As the meat production industry seeks selective breeding approaches to improve profitable traits such as meat quality, information about genetic determinants of these traits is in high demand. However, most of the studies have been performed using trained sensory panel analysis without investigating the underlying genetic factors. Here we investigate the relationship between genomic composition and this phenotypic trait by scanning for signatures of positive selection in whole-genome sequencing data. We generated genomes of 10 Berkshire pigs at a total of 100.6 coverage depth, using the Illumina Hiseq2000 platform. Along with the genomes of 11 Landrace and 13 Yorkshire pigs, we identified genomic variants of 18.9 million SNVs and 3.4 million Indels in the mapped regions. We identified several associated genes related to lipid metabolism, intramuscular fatty acid deposition, and muscle fiber type which attribute to pork quality (TG, FABP1, AKIRIN2, GLP2R, TGFBR3, JPH3, ICAM2, and ERN1) by applying between population statistical tests (XP-EHH and XP-CLR). A statistical enrichment test was also conducted to detect breed specific genetic variation. In addition, de novo short sequence read assembly strategy identified several candidate genes (SLC25A14, IGF1, PI4KA, CACNA1A) as also contributing to lipid metabolism. Results revealed several candidate genes involved in Berkshire meat quality; most of these genes are involved in lipid metabolism and intramuscular fat deposition. These results can provide a basis for future research on the genomic characteristics of Berkshire pigs.

  1. Mitochondrial D-loop sequence variation among Italian horse breeds

    Directory of Open Access Journals (Sweden)

    Zanotti Marta

    2004-11-01

    Full Text Available Abstract The genetic variability of the mitochondrial D-loop DNA sequence in seven horse breeds bred in Italy (Giara, Haflinger, Italian trotter, Lipizzan, Maremmano, Thoroughbred and Sarcidano was analysed. Five unrelated horses were chosen in each breed and twenty-two haplotypes were identified. The sequences obtained were aligned and compared with a reference sequence and with 27 mtDNA D-loop sequences selected in the GenBank database, representing Spanish, Portuguese, North African, wild horses and an Equus asinus sequence as the outgroup. Kimura two-parameter distances were calculated and a cluster analysis using the Neighbour-joining method was performed to obtain phylogenetic trees among breeds bred in Italy and among Italian and foreign breeds. The cluster analysis indicates that all the breeds but Giara are divided in the two trees, and no clear relationships were revealed between Italian populations and the other breeds. These results could be interpreted as showing the mixed origin of breeds bred in Italy and probably indicate the presence of many ancient maternal lineages with high diversity in mtDNA sequences.

  2. Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing

    Science.gov (United States)

    Egge, Elianne Sirnæs; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

    2015-01-01

    Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. PMID:25893259

  3. Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

    Science.gov (United States)

    Schwessinger, Benjamin; Rathjen, John P

    2017-01-01

    Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

  4. High Performance Systolic Array Core Architecture Design for DNA Sequencer

    Directory of Open Access Journals (Sweden)

    Saiful Nurdin Dayana

    2018-01-01

    Full Text Available This paper presents a high performance systolic array (SA core architecture design for Deoxyribonucleic Acid (DNA sequencer. The core implements the affine gap penalty score Smith-Waterman (SW algorithm. This time-consuming local alignment algorithm guarantees optimal alignment between DNA sequences, but it requires quadratic computation time when performed on standard desktop computers. The use of linear SA decreases the time complexity from quadratic to linear. In addition, with the exponential growth of DNA databases, the SA architecture is used to overcome the timing issue. In this work, the SW algorithm has been captured using Verilog Hardware Description Language (HDL and simulated using Xilinx ISIM simulator. The proposed design has been implemented in Xilinx Virtex -6 Field Programmable Gate Array (FPGA and improved in the core area by 90% reduction.

  5. Analysis of HIV-1 intersubtype recombination breakpoints suggests region with high pairing probability may be a more fundamental factor than sequence similarity affecting HIV-1 recombination.

    Science.gov (United States)

    Jia, Lei; Li, Lin; Gui, Tao; Liu, Siyang; Li, Hanping; Han, Jingwan; Guo, Wei; Liu, Yongjian; Li, Jingyun

    2016-09-21

    With increasing data on HIV-1, a more relevant molecular model describing mechanism details of HIV-1 genetic recombination usually requires upgrades. Currently an incomplete structural understanding of the copy choice mechanism along with several other issues in the field that lack elucidation led us to perform an analysis of the correlation between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarity to further explore structural mechanisms. Near full length sequences of URFs from Asia, Europe, and Africa (one sequence/patient), and representative sequences of worldwide CRFs were retrieved from the Los Alamos HIV database. Their recombination patterns were analyzed by jpHMM in detail. Then the relationships between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarities were investigated. Pearson correlation test showed that all URF groups and the CRF group exhibit the same breakpoint distribution pattern. Additionally, the Wilcoxon two-sample test indicated a significant and inexplicable limitation of recombination in regions with high pairing probability. These regions have been found to be strongly conserved across distinct biological states (i.e., strong intersubtype similarity), and genetic similarity has been determined to be a very important factor promoting recombination. Thus, the results revealed an unexpected disagreement between intersubtype similarity and breakpoint distribution, which were further confirmed by genetic similarity analysis. Our analysis reveals a critical conflict between results from natural HIV-1 isolates and those from HIV-1-based assay vectors in which genetic similarity has been shown to be a very critical factor promoting recombination. These results indicate the region with high-pairing probabilities may be a more fundamental factor affecting HIV-1 recombination than sequence similarity in natural HIV-1 infections. Our

  6. Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL).

    Science.gov (United States)

    Elouej, Sahar; Beleza-Meireles, Ana; Caswell, Richard; Colclough, Kevin; Ellard, Sian; Desvignes, Jean Pierre; Béroud, Christophe; Lévy, Nicolas; Mohammed, Shehla; De Sandre-Giovannoli, Annachiara

    2017-06-01

    Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing

  7. GEITLERINEMA SPECIES (OSCILLATORIALES, CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY, ULTRASTRUCTURE, AND DNA SEQUENCING(1).

    Science.gov (United States)

    Do Carmo Bittencourt-Oliveira, Maria; Do Nascimento Moura, Ariadne; De Oliveira, Mariana Cabral; Sidnei Massola, Nelson

    2009-06-01

    Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komárek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB-cpcA intergenic spacer (PC-IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC-IGS sequences, in spite of their morphological similarity. PC-IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC-IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium. © 2009 Phycological Society of America.

  8. Genetic variability among Trichuris ovis isolates from different hosts in Guangdong Province, China revealed by sequences of three mitochondrial genes.

    Science.gov (United States)

    Wang, Yan; Liu, Guo-Hua; Li, Jia-Yuan; Xu, Min-Jun; Ye, Yong-Gang; Zhou, Dong-Hui; Song, Hui-Qun; Lin, Rui-Qing; Zhu, Xing-Quan

    2013-02-01

    This study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 5 (nad5) and cytochrome b (cytb), among Trichuris ovis isolates from different hosts in Guangdong Province, China. A portion of the cox1 (pcox1), nad5 (pnad5) and cytb (pcytb) genes was amplified separately from individual whipworms by PCR, and was subjected to sequencing from both directions. The size of the sequences of pcox1, pnad5 and pcytb was 618, 240 and 464 bp, respectively. Although the intra-specific sequence variations within T. ovis were 0-0.8% for pcox1, 0-0.8% for pnad5 and 0-1.9% for pcytb, the inter-specific sequence differences among members of the genus Trichuris were significantly higher, being 24.3-26.5% for pcox1, 33.7-56.4% for pnad5 and 24.8-26.1% for pcytb, respectively. Phylogenetic analyses using combined sequences of pcox1, pnad5 and pcytb, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), indicated that all of the T. ovis isolates grouped together with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA sequences among T. ovis isolates from different hosts, and have implications for studying molecular epidemiology and population genetics of T. ovis.

  9. MUSCLE: multiple sequence alignment with high accuracy and high throughput.

    Science.gov (United States)

    Edgar, Robert C

    2004-01-01

    We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

  10. Microbiota present in cystic fibrosis lungs as revealed by whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Philippe M Hauser

    Full Text Available Determination of the precise composition and variation of microbiota in cystic fibrosis lungs is crucial since chronic inflammation due to microorganisms leads to lung damage and ultimately, death. However, this constitutes a major technical challenge. Culturing of microorganisms does not provide a complete representation of a microbiota, even when using culturomics (high-throughput culture. So far, only PCR-based metagenomics have been investigated. However, these methods are biased towards certain microbial groups, and suffer from uncertain quantification of the different microbial domains. We have explored whole genome sequencing (WGS using the Illumina high-throughput technology applied directly to DNA extracted from sputa obtained from two cystic fibrosis patients. To detect all microorganism groups, we used four procedures for DNA extraction, each with a different lysis protocol. We avoided biases due to whole DNA amplification thanks to the high efficiency of current Illumina technology. Phylogenomic classification of the reads by three different methods produced similar results. Our results suggest that WGS provides, in a single analysis, a better qualitative and quantitative assessment of microbiota compositions than cultures and PCRs. WGS identified a high quantity of Haemophilus spp. (patient 1 or Staphylococcus spp. plus Streptococcus spp. (patient 2 together with low amounts of anaerobic (Veillonella, Prevotella, Fusobacterium and aerobic bacteria (Gemella, Moraxella, Granulicatella. WGS suggested that fungal members represented very low proportions of the microbiota, which were detected by cultures and PCRs because of their selectivity. The future increase of reads' sizes and decrease in cost should ensure the usefulness of WGS for the characterisation of microbiota.

  11. Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

    Science.gov (United States)

    Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

    2015-10-15

    MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system

    Directory of Open Access Journals (Sweden)

    Sandeep Ghatak

    2017-03-01

    Full Text Available Campylobacter is a major cause of foodborne illnesses worldwide. Campylobacter infections, commonly caused by ingestion of undercooked poultry and meat products, can lead to gastroenteritis and chronic reactive arthritis in humans. Whole genome sequencing (WGS is a powerful technology that provides comprehensive genetic information about bacteria and is increasingly being applied to study foodborne pathogens: e.g., evolution, epidemiology/outbreak investigation, and detection. Herein we report the complete genome sequence of Campylobacter coli strain YH502 isolated from retail chicken in the United States. WGS, de novo assembly, and annotation of the genome revealed a chromosome of 1,718,974 bp and a mega-plasmid (pCOS502 of 125,964 bp. GC content of the genome was 31.2% with 1931 coding sequences and 53 non-coding RNAs. Multiple virulence factors including a plasmid-borne type VI secretion system and antimicrobial resistance genes (beta-lactams, fluoroquinolones, and aminoglycoside were found. The presence of T6SS in a mobile genetic element (plasmid suggests plausible horizontal transfer of these virulence genes to other organisms. The C. coli YH502 genome also harbors CRISPR sequences and associated proteins. Phylogenetic analysis based on average nucleotide identity and single nucleotide polymorphisms identified closely related C. coli genomes available in the NCBI database. Taken together, the analyzed genomic data of this potentially virulent strain of C. coli will facilitate further understanding of this important foodborne pathogen most likely leading to better control strategies. The chromosome and plasmid sequences of C. coli YH502 have been deposited in GenBank under the accession numbers CP018900.1 and CP018901.1, respectively.

  13. Application of high-throughput sequencing in understanding human oral microbiome related with health and disease

    OpenAIRE

    Chen, Hui; Jiang, Wen

    2014-01-01

    The oral microbiome is one of most diversity habitat in the human body and they are closely related with oral health and disease. As the technique developing,, high throughput sequencing has become a popular approach applied for oral microbial analysis. Oral bacterial profiles have been studied to explore the relationship between microbial diversity and oral diseases such as caries and periodontal disease. This review describes the application of high-throughput sequencing for characterizati...

  14. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Science.gov (United States)

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  15. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Directory of Open Access Journals (Sweden)

    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  16. Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs

    Science.gov (United States)

    Luo, Jin; Liu, Min-Xuan; Ren, Qiao-Yun; Chen, Ze; Tian, Zhan-Cheng; Hao, Jia-Wei; Wu, Feng; Liu, Xiao-Cui; Luo, Jian-Xun; Yin, Hong; Wang, Hui; Liu, Guang-Yuan

    2017-01-01

    Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species. PMID:28861401

  17. Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

    OpenAIRE

    MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

    2013-01-01

    Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

  18. Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing.

    Science.gov (United States)

    Shaw, Jennifer L A; Monis, Paul; Fabris, Rolando; Ho, Lionel; Braun, Kalan; Drikas, Mary; Cooper, Alan

    2014-12-01

    Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were sampled under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more diverse than those sampled at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest diversity was observed in the MF inlet. However, the MF system revealed the greatest increase in diversity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Comparison of two Next Generation sequencing platforms for full genome sequencing of Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Höper, Dirk

    2013-01-01

    to the consensus sequence. Additionally, we got an average sequence depth for the genome of 4000 for the Iontorrent PGM and 400 for the FLX platform making the mapping suitable for single nucleotide variant (SNV) detection. The analysis revealed a single non-silent SNV A10665G leading to the amino acid change D......Next Generation Sequencing (NGS) is becoming more adopted into viral research and will be the preferred technology in the years to come. We have recently sequenced several strains of Classical Swine Fever Virus (CSFV) by NGS on both Genome Sequencer FLX (GS FLX) and Iontorrent PGM platforms...

  20. Sequence Variation in Toxoplasma gondii rop17 Gene among Strains from Different Hosts and Geographical Locations

    Directory of Open Access Journals (Sweden)

    Nian-Zhang Zhang

    2014-01-01

    Full Text Available Genetic diversity of T. gondii is a concern of many studies, due to the biological and epidemiological diversity of this parasite. The present study examined sequence variation in rhoptry protein 17 (ROP17 gene among T. gondii isolates from different hosts and geographical regions. The rop17 gene was amplified and sequenced from 10 T. gondii strains, and phylogenetic relationship among these T. gondii strains was reconstructed using maximum parsimony (MP, neighbor-joining (NJ, and maximum likelihood (ML analyses. The partial rop17 gene sequences were 1375 bp in length and A+T contents varied from 49.45% to 50.11% among all examined T. gondii strains. Sequence analysis identified 33 variable nucleotide positions (2.1%, 16 of which were identified as transitions. Phylogeny reconstruction based on rop17 gene data revealed two major clusters which could readily distinguish Type I and Type II strains. Analyses of sequence variations in nucleotides and amino acids among these strains revealed high ratio of nonsynonymous to synonymous polymorphisms (>1, indicating that rop17 shows signs of positive selection. This study demonstrated the existence of slightly high sequence variability in the rop17 gene sequences among T. gondii strains from different hosts and geographical regions, suggesting that rop17 gene may represent a new genetic marker for population genetic studies of T. gondii isolates.

  1. Taphonomic expressions of sedimentary hiatuses: field observations on bioclastic concentrations and sequence anatomy in low, moderate and high subsidence settings

    Science.gov (United States)

    Kidwell, S. M.

    1993-07-01

    Field studies of post-Palaeozoic siliciclastic records reveal a strong concordance between different types of bioclastic concentrations and discontinuity surfaces within third-order sequences (≈ 1 My duration), supporting the use of taphonomic criteria in establishing the relative magnitudes of sedimentary hiatuses. Comparison of records across a spectrum of subsidence rates, however (from ≤ 10 m to > 1 km/My), shows that, along with appreciable changes in sequence anatomy, the nature of surface-mantling bioclastic concentrations also changes. The most significant surfaces (second- and third-order sequence boundaries, surfaces or intervals of maximum transgression, transgressive surfaces) tend to be either bare or mantled with taphonomically complex hiatal and lag concentrations. These were more consistently encountered in low subsidence than in moderate subsidence records. In high subsidence records, major surfaces were more often mantled by composite or event concentrations, if they were bioclastic at all. In all subsidence settings, comparatively minor surfaces (parasequence boundaries, bed set boundaries and bedding planes) were bare or mantled with relatively simple event and composite concentrations. Although all fossil assemblages are biased taphonomically to some degree, relative degrees of bias should almost certainly vary among discontinuities as a general rule, suggesting specific adjustments in sampling strategies for evolutionary studies.

  2. Intergenic DNA sequences from the human X chromosome reveal high rates of global gene flow

    Directory of Open Access Journals (Sweden)

    Wall Jeffrey D

    2008-11-01

    Full Text Available Abstract Background Despite intensive efforts devoted to collecting human polymorphism data, little is known about the role of gene flow in the ancestry of human populations. This is partly because most analyses have applied one of two simple models of population structure, the island model or the splitting model, which make unrealistic biological assumptions. Results Here, we analyze 98-kb of DNA sequence from 20 independently evolving intergenic regions on the X chromosome in a sample of 90 humans from six globally diverse populations. We employ an isolation-with-migration (IM model, which assumes that populations split and subsequently exchange migrants, to independently estimate effective population sizes and migration rates. While the maximum effective size of modern humans is estimated at ~10,000, individual populations vary substantially in size, with African populations tending to be larger (2,300–9,000 than non-African populations (300–3,300. We estimate mean rates of bidirectional gene flow at 4.8 × 10-4/generation. Bidirectional migration rates are ~5-fold higher among non-African populations (1.5 × 10-3 than among African populations (2.7 × 10-4. Interestingly, because effective sizes and migration rates are inversely related in African and non-African populations, population migration rates are similar within Africa and Eurasia (e.g., global mean Nm = 2.4. Conclusion We conclude that gene flow has played an important role in structuring global human populations and that migration rates should be incorporated as critical parameters in models of human demography.

  3. Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice.

    Science.gov (United States)

    Brozynska, Marta; Copetti, Dario; Furtado, Agnelo; Wing, Rod A; Crayn, Darren; Fox, Glen; Ishikawa, Ryuji; Henry, Robert J

    2017-06-01

    The related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon-like population, referred to as Taxon A, and O. meridionalis-like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short- and long-read next-generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer.

    Science.gov (United States)

    Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

    2017-06-01

    Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

  5. Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus).

    Science.gov (United States)

    Xinli, Xiao; Lei, Peng

    2015-03-04

    The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

  6. Rapid detection of SMARCB1 sequence variation using high resolution melting

    Directory of Open Access Journals (Sweden)

    Ashley David M

    2009-12-01

    Full Text Available Abstract Background Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM, for detecting sequence variations in SMARCB1. Methods Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4% showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA. A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to

  7. Rapid detection of SMARCB1 sequence variation using high resolution melting

    International Nuclear Information System (INIS)

    Dagar, Vinod; Chow, Chung-Wo; Ashley, David M; Algar, Elizabeth M

    2009-01-01

    Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening

  8. Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway

    Science.gov (United States)

    Murali, Rajmohan; Chandramohan, Raghu; Möller, Inga; Scholz, Simone L.; Berger, Michael; Huberman, Kety; Viale, Agnes; Pirun, Mono; Socci, Nicholas D.; Bouvier, Nancy; Bauer, Sebastian; Artl, Monika; Schilling, Bastian; Schimming, Tobias; Sucker, Antje; Schwindenhammer, Benjamin; Grabellus, Florian; Speicher, Michael R.; Schaller, Jörg; Hillen, Uwe; Schadendorf, Dirk; Mentzel, Thomas; Cheng, Donavan T.; Wiesner, Thomas; Griewank, Klaus G.

    2015-01-01

    Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas. PMID:26440310

  9. RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

    Directory of Open Access Journals (Sweden)

    Sabah Kadri

    Full Text Available microRNAs (miRNAs are small (20-23 nt, non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin and Patiria miniata (sea star are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc. to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads. Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common. We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

  10. RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

    Science.gov (United States)

    Kadri, Sabah; Hinman, Veronica F.; Benos, Panayiotis V.

    2011-01-01

    microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. PMID:22216218

  11. Markovian Model in High Order Sequence Prediction From Log-Motif Patterns in Agbada Paralic Section, Niger Delta, Nigeria

    International Nuclear Information System (INIS)

    Olabode, S. O.; Adekoya, J. A.

    2002-01-01

    Markovian model in the elucidation of high order sequence was applied to repetitive events of regressive and transgressive phases in the Agbada paralic section Niger Delta. The repetitive events are made up of delta front, delta topset and fluvio-deltaic sediments. The sediments consist of sands, sandstones, siltstones and shales in various proportions. Five wells: MN1, AA1, NP2, NP6 and NP8 were studied.Summary of biostratigraphic report and well log-motif patterns was used to delineate the third order depositional sequences in the wells.Various Markovian properties - observed transition frequency matrix, observed transition probability matrix, fixed probability vector, expected random matrix (randomised transition matrix) and difference matrix were determined for stacked high order sequence (high frequency cyclic events) nested within the third-order sequences using the log-motif patterns for the various sand bodies and shales. Flow diagrams were constructed for each of the depositional sequences to know the likely occurrence of number of cycles.Upward transition matrix between the log-motif patterns and flow diagram to elucidate cyclicity show that the overall regressive sequence of the Niger Delta has been modified by deltaic depositional elements and fluctuations in sea level. The predictions of higher order sequence within third order sequences from Markovian Properties provide good basis for correlation within the depositional sequences. The model has also been used to decipher the dominant depositional processes during the formation of the sequences. Discrete reservoir intervals and seal potentials within the sequences were also predicted from the flow diagrams constructed

  12. Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

    Science.gov (United States)

    Scordino, Fabio; Giuffrè, Letterio; Barberi, Giuseppina; Marino Merlo, Francesca; Orlando, Maria Grazia; Giosa, Domenico; Romeo, Orazio

    2018-01-01

    Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6-16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping ( ICL1, MDR1, SAPT2, SAPT4, XYR1 , and ZWF1 ) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

  13. Analysis of the 9p21.3 sequence associated with coronary artery disease reveals a tendency for duplication in a CAD patient

    Science.gov (United States)

    Kouprina, Natalay; Noskov, Vladimir N.; Waterfall, Joshua J.; Walker, Robert L.; Meltzer, Paul S.; Topol, Eric J.; Larionov, Vladimir

    2018-01-01

    Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed ‘duplication blocks’. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ∼50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer. PMID:29632643

  14. Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

    Science.gov (United States)

    Neuer-Nitsche, B; Lu, X N; Werner, D

    1988-09-12

    The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

  15. Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

    Directory of Open Access Journals (Sweden)

    Ritland Carol

    2009-08-01

    Full Text Available Abstract Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs and full-length (FLcDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR and a cytochrome P450 (CYP720B4 from a non-arrayed genomic BAC library of white spruce (Picea glauca. Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR and 94 kbp (CYP720B4 long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs, high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene

  16. Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

    Science.gov (United States)

    Hamberger, Björn; Hall, Dawn; Yuen, Mack; Oddy, Claire; Hamberger, Britta; Keeling, Christopher I; Ritland, Carol; Ritland, Kermit; Bohlmann, Jörg

    2009-08-06

    Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The

  17. Whole-Genome Sequencing of Human Clinical Klebsiella pneumoniae Isolates Reveals Misidentification and Misunderstandings of Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae

    Science.gov (United States)

    Linson, Sarah E.; Ojeda Saavedra, Matthew; Cantu, Concepcion; Davis, James J.; Brettin, Thomas; Olsen, Randall J.

    2017-01-01

    ABSTRACT Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. K. variicola and K. quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K. pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K. variicola and K. quasipneumoniae. Whole-genome sequencing of 95 additional non-ESBL-producing K. pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K. pneumoniae, suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K. variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella. IMPORTANCE Klebsiella

  18. Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology

    Directory of Open Access Journals (Sweden)

    Lesley Joan Collins

    2011-12-01

    Full Text Available ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, snoRNAs and long ncRNAs on a genomic scale making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases.

  19. Characterizing ncRNAs in Human Pathogenic Protists Using High-Throughput Sequencing Technology

    Science.gov (United States)

    Collins, Lesley Joan

    2011-01-01

    ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases. PMID:22303390

  20. Applications of high-throughput sequencing to chromatin structure and function in mammals

    OpenAIRE

    Dunham, Ian

    2009-01-01

    High-throughput DNA sequencing approaches have enabled direct interrogation of chromatin samples from mammalian cells. We are beginning to develop a genome-wide description of nuclear function during development, but further data collection, refinement, and integration are needed.

  1. Phylogenetic and genome-wide deep-sequencing analyses of canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain.

    Directory of Open Access Journals (Sweden)

    Ruben Pérez

    Full Text Available Canine parvovirus (CPV, a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population and a major recombinant strain (86.7%. The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity.

  2. Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain

    Science.gov (United States)

    Pérez, Ruben; Calleros, Lucía; Marandino, Ana; Sarute, Nicolás; Iraola, Gregorio; Grecco, Sofia; Blanc, Hervé; Vignuzzi, Marco; Isakov, Ofer; Shomron, Noam; Carrau, Lucía; Hernández, Martín; Francia, Lourdes; Sosa, Katia; Tomás, Gonzalo; Panzera, Yanina

    2014-01-01

    Canine parvovirus (CPV), a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c) with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population) and a major recombinant strain (86.7%). The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity. PMID:25365348

  3. Transcriptome Sequence Analysis of an Ornamental Plant, Ananas comosus var. bracteatus, Revealed the Potential Unigenes Involved in Terpenoid and Phenylpropanoid Biosynthesis

    OpenAIRE

    Ma, Jun; Kanakala, S.; He, Yehua; Zhang, Junli; Zhong, Xiaolan

    2015-01-01

    Background Ananas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies. Results The Anana...

  4. Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data.

    Science.gov (United States)

    Liu, Yang; Chiaromonte, Francesca; Ross, Howard; Malhotra, Raunaq; Elleder, Daniel; Poss, Mary

    2015-06-30

    Infection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3' half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology. Since rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies - and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase

  5. Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping

    Directory of Open Access Journals (Sweden)

    Walid Mottawea

    2018-05-01

    Full Text Available Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella. In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

  6. High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus

    2016-01-01

    Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards...

  7. Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

    Science.gov (United States)

    Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

    2018-03-09

    High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

  8. Designing a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions

    Directory of Open Access Journals (Sweden)

    Debnath Bhattacharyya

    2013-01-01

    Full Text Available We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant as well as query sequence (virus. Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size. This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length.

  9. Designing a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions

    Science.gov (United States)

    Mandal, Bijoy Kumar; Kim, Tai-hoon

    2013-01-01

    We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant) as well as query sequence (virus). Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size). This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length. PMID:24000321

  10. Exome sequencing and genetic testing for MODY.

    Directory of Open Access Journals (Sweden)

    Stefan Johansson

    Full Text Available Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive.The aim of the study was to examine the performance of exome sequencing for a molecular diagnosis of MODY in patients who have undergone conventional diagnostic sequencing of candidate genes with negative results.We performed exome enrichment followed by high-throughput sequencing in nine patients with suspected MODY. They were Sanger sequencing-negative for mutations in the HNF1A, HNF4A, GCK, HNF1B and INS genes. We excluded common, non-coding and synonymous gene variants, and performed in-depth analysis on filtered sequence variants in a pre-defined set of 111 genes implicated in glucose metabolism.On average, we obtained 45 X median coverage of the entire targeted exome and found 199 rare coding variants per individual. We identified 0-4 rare non-synonymous and nonsense variants per individual in our a priori list of 111 candidate genes. Three of the variants were considered pathogenic (in ABCC8, HNF4A and PPARG, respectively, thus exome sequencing led to a genetic diagnosis in at least three of the nine patients. Approximately 91% of known heterozygous SNPs in the target exomes were detected, but we also found low coverage in some key diabetes genes using our current exome sequencing approach. Novel variants in the genes ARAP1, GLIS3, MADD, NOTCH2 and WFS1 need further investigation to reveal their possible role in diabetes.Our results demonstrate that exome sequencing can improve molecular diagnostics of MODY when used as a complement to Sanger sequencing. However, improvements will be needed, especially concerning coverage, before the full potential of exome sequencing can be realized.

  11. The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

    Science.gov (United States)

    Shaw, Timothy D; Fairley, D J; Schneiders, T; Pathiraja, M; Hill, R L R; Werner, G; Elborn, J S; McMullan, R

    2018-02-24

    High-throughput sequencing (HTS) has successfully identified novel resistance genes in enterococci and determined clonal relatedness in outbreak analysis. We report the use of HTS to investigate two concurrent outbreaks of glycopeptide-resistant Enterococcus faecium (GRE) with an uncharacterised resistance mechanism to quinupristin-dalfopristin (QD). Seven QD-resistant and five QD-susceptible GRE isolates from a two-centre outbreak were studied. HTS was performed to identify genes or predicted proteins that were associated with the QD-resistant phenotype. MLST and SNP typing on HTS data was used to determine clonal relatedness. Comparative genomic analysis confirmed this GRE outbreak involved two distinct clones (ST80 and ST192). HTS confirmed the absence of known QD resistance genes, suggesting a novel mechanism was conferring resistance. Genomic analysis identified two significant genetic determinants with explanatory power for the high level of QD resistance in the ST80 QD-resistant clone: an additional 56aa leader sequence at the N-terminus of the lsaE gene and a transposon containing seven genes encoding proteins with possible drug or drug-target modification activities. However, HTS was unable to conclusively determine the QD resistance mechanism and did not reveal any genetic basis for QD resistance in the ST192 clone. This study highlights the usefulness of HTS in deciphering the degree of relatedness in two concurrent GRE outbreaks. Although HTS was able to reveal some genetic candidates for uncharacterised QD resistance, this study demonstrates the limitations of HTS as a tool for identifying putative determinants of resistance to QD.

  12. Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

    Science.gov (United States)

    Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

    2017-07-01

    Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.

  13. Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

    Science.gov (United States)

    Egge, Elianne Sirnaes; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

    2015-06-01

    Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

  14. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

    International Nuclear Information System (INIS)

    Kwon, Y.K.; Hecht, N.B.

    1991-01-01

    The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

  15. Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data

    Directory of Open Access Journals (Sweden)

    William H Thiel

    2016-01-01

    Full Text Available Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment. High-throughput sequencing (HTS revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs.

  16. Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences.

    Science.gov (United States)

    Arend, J; Warzecha, H; Stöckigt, J

    2000-01-01

    Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone. A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase. SDS-PAGE showed a M(r) for the enzyme of 52 kDa. Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced. Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.

  17. [Sequence-based typing of enviromental Legionella pneumophila isolates in Guangzhou].

    Science.gov (United States)

    Zhang, Ying; Qu, Pinghua; Zhang, Jian; Chen, Shouyi

    2011-03-01

    To characterize the genes of Legionella pneumophila isolated from different water source in Guangzhou from 2006 to 2009. To genotype the strains by using sequence-based typing (SBT) scheme. In total 44 L. pneumophila strains were identified by SBT with 7 diversifying genes of flaA, asd, mip, pilE, mompS, proA and neuA. Analysis of the amplicons sequence was taken in the European Working Group for Legionella Infections (EWGLI) international SBT database to obtain the allelic profiles and sequence types (STs). Serogroups were typed by latex agglutination test. Data from SBT revealed a high diversity among the strains and ST01 accounts for 30% (13/ 44). Fifteen new STs were discovered from 20 STs and 2 of them were newly assigned (ST887 and ST888) by EWGLI. SBT Phylogenetic tree was generated by SplitsTree and BURST programs. High diversity and specificity were observed of the L. pneumophila strains in Guangzhou. SBT is useful for L. pneumophila genomic study and epidemiological surveillance.

  18. Genetic Diversity of Selected Mangifera Species Revealed by Inter Simple Sequence Repeats Markers

    OpenAIRE

    Ariffin, Zulhairil; Md Sah, Muhammad Shafie; Idris, Salma; Hashim, Nuradni

    2015-01-01

    ISSR markers were employed to reveal genetic diversity and genetic relatedness among 28 Mangifera accessions collected from Yan (Kedah), Bukit Gantang (Perak), Sibuti (Sarawak), and Papar (Sabah). A total of 198 markers were generated using nine anchored primers and one nonanchored primer. Genetic variation among the 28 accessions of Mangifera species including wild relatives, landraces, and clonal varieties is high, with an average degree of polymorphism of 98% and mean Shannon index, H0=7.5...

  19. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

  20. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  1. Genome sequencing reveals complex secondary metabolome in themarine actinomycete Salinispora tropica

    Energy Technology Data Exchange (ETDEWEB)

    Udwary, Daniel W.; Zeigler, Lisa; Asolkar, Ratnakar; Singan,Vasanth; Lapidus, Alla; Fenical, William; Jensen, Paul R.; Moore, BradleyS.

    2007-05-01

    Recent fermentation studies have identified actinomycetes ofthe marine-dwelling genus Salinispora as prolific natural productproducers. To further evaluate their biosynthetic potential, we analyzedall identifiable secondary natural product gene clusters from therecently sequenced 5,184,724 bp S. tropica CNB-440 circular genome. Ouranalysis shows that biosynthetic potential meets or exceeds that shown byprevious Streptomyces genome sequences as well as other naturalproduct-producing actinomycetes. The S. tropica genome features ninepolyketide synthase systems of every known formally classified family,non-ribosomal peptide synthetases and several hybrid clusters. While afew clusters appear to encode molecules previously identified inStreptomyces species,the majority of the 15 biosynthetic loci are novel.Specific chemical information about putative and observed natural productmolecules is presented and discussed. In addition, our bioinformaticanalysis was critical for the structure elucidation of the novelpolyenemacrolactam salinilactam A. This study demonstrates the potentialfor genomic analysis to complement and strengthen traditional naturalproduct isolation studies and firmly establishes the genus Salinispora asa rich source of novel drug-like molecules.

  2. Characterization of promoter sequence of toll-like receptor genes in Vechur cattle

    Directory of Open Access Journals (Sweden)

    R. Lakshmi

    2016-06-01

    Full Text Available Aim: To analyze the promoter sequence of toll-like receptor (TLR genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. Materials and Methods: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. Results: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. Conclusion: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases.

  3. Whole Exome Sequencing for a Patient with Rubinstein-Taybi Syndrome Reveals de Novo Variants besides an Overt CREBBP Mutation

    Directory of Open Access Journals (Sweden)

    Hee Jeong Yoo

    2015-03-01

    Full Text Available Rubinstein-Taybi syndrome (RSTS is a rare condition with a prevalence of 1 in 125,000–720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS and Autism diagnostic interview revised (ADI-R to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.

  4. A theoretical justification for single molecule peptide sequencing.

    Directory of Open Access Journals (Sweden)

    Jagannath Swaminathan

    2015-02-01

    Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

  5. Whole genome sequencing of the monomorphic pathogen Mycobacterium bovis reveals local differentiation of cattle clinical isolates.

    Science.gov (United States)

    Lasserre, Moira; Fresia, Pablo; Greif, Gonzalo; Iraola, Gregorio; Castro-Ramos, Miguel; Juambeltz, Arturo; Nuñez, Álvaro; Naya, Hugo; Robello, Carlos; Berná, Luisa

    2018-01-02

    Bovine tuberculosis (bTB) poses serious risks to animal welfare and economy, as well as to public health as a zoonosis. Its etiological agent, Mycobacterium bovis, belongs to the Mycobacterium tuberculosis complex (MTBC), a group of genetically monomorphic organisms featured by a remarkably high overall nucleotide identity (99.9%). Indeed, this characteristic is of major concern for correct typing and determination of strain-specific traits based on sequence diversity. Due to its historical economic dependence on cattle production, Uruguay is deeply affected by the prevailing incidence of Mycobacterium bovis. With the world's highest number of cattle per human, and its intensive cattle production, Uruguay represents a particularly suited setting to evaluate genomic variability among isolates, and the diversity traits associated to this pathogen. We compared 186 genomes from MTBC strains isolated worldwide, and found a highly structured population in M. bovis. The analysis of 23 new M. bovis genomes, belonging to strains isolated in Uruguay evidenced three groups present in the country. Despite presenting an expected highly conserved genomic structure and sequence, these strains segregate into a clustered manner within the worldwide phylogeny. Analysis of the non-pe/ppe differential areas against a reference genome defined four main sources of variability, namely: regions of difference (RD), variable genes, duplications and novel genes. RDs and variant analysis segregated the strains into clusters that are concordant with their spoligotype identities. Due to its high homoplasy rate, spoligotyping failed to reflect the true genomic diversity among worldwide representative strains, however, it remains a good indicator for closely related populations. This study introduces a comprehensive population structure analysis of worldwide M. bovis isolates. The incorporation and analysis of 23 novel Uruguayan M. bovis genomes, sheds light onto the genomic diversity of this

  6. Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

    Directory of Open Access Journals (Sweden)

    David Jean-Philippe

    2009-11-01

    Full Text Available Abstract Background Genome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s or mutation(s targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito Aedes aegypti with the aim to look for candidate genes involved in resistance to Bacillus thuringiensis subsp. israelensis (Bti insecticidal toxins. Results The genome of a Bti-resistant and a Bti-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the Aedes aegypti genome sequence. Several good candidate genes for Bti-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations, as well as a significant under-expression compared to the susceptible strain. Conclusion Both sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to Bti is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with Bti resistance in Ae. aegypti. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full

  7. High-throughput metagenomic analysis of petroleum-contaminated soil microbiome reveals the versatility in xenobiotic aromatics metabolism.

    Science.gov (United States)

    Bao, Yun-Juan; Xu, Zixiang; Li, Yang; Yao, Zhi; Sun, Jibin; Song, Hui

    2017-06-01

    The soil with petroleum contamination is one of the most studied soil ecosystems due to its rich microorganisms for hydrocarbon degradation and broad applications in bioremediation. However, our understanding of the genomic properties and functional traits of the soil microbiome is limited. In this study, we used high-throughput metagenomic sequencing to comprehensively study the microbial community from petroleum-contaminated soils near Tianjin Dagang oilfield in eastern China. The analysis reveals that the soil metagenome is characterized by high level of community diversity and metabolic versatility. The metageome community is predominated by γ-Proteobacteria and α-Proteobacteria, which are key players for petroleum hydrocarbon degradation. The functional study demonstrates over-represented enzyme groups and pathways involved in degradation of a broad set of xenobiotic aromatic compounds, including toluene, xylene, chlorobenzoate, aminobenzoate, DDT, methylnaphthalene, and bisphenol. A composite metabolic network is proposed for the identified pathways, thus consolidating our identification of the pathways. The overall data demonstrated the great potential of the studied soil microbiome in the xenobiotic aromatics degradation. The results not only establish a rich reservoir for novel enzyme discovery but also provide putative applications in bioremediation. Copyright © 2016. Published by Elsevier B.V.

  8. Exome Sequencing Identifies Potential Risk Variants for Mendelian Disorders at High Prevalence in Qatar

    Science.gov (United States)

    Rodriguez-Flores, Juan L.; Fakhro, Khalid; Hackett, Neil R.; Salit, Jacqueline; Fuller, Jennifer; Agosto-Perez, Francisco; Gharbiah, Maey; Malek, Joel A.; Zirie, Mahmoud; Jayyousi, Amin; Badii, Ramin; Al-Marri, Ajayeb Al-Nabet; Chouchane, Lotfi; Stadler, Dora J.; Hunter-Zinck, Haley; Mezey, Jason G.; Crystal, Ronald G.

    2013-01-01

    Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared to 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Pre-marital genetic screening in Qatar tests for only 4 out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance. PMID:24123366

  9. Permutation Entropy for Random Binary Sequences

    Directory of Open Access Journals (Sweden)

    Lingfeng Liu

    2015-12-01

    Full Text Available In this paper, we generalize the permutation entropy (PE measure to binary sequences, which is based on Shannon’s entropy, and theoretically analyze this measure for random binary sequences. We deduce the theoretical value of PE for random binary sequences, which can be used to measure the randomness of binary sequences. We also reveal the relationship between this PE measure with other randomness measures, such as Shannon’s entropy and Lempel–Ziv complexity. The results show that PE is consistent with these two measures. Furthermore, we use PE as one of the randomness measures to evaluate the randomness of chaotic binary sequences.

  10. High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

    Science.gov (United States)

    Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

    2012-09-11

    Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

  11. High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

    Directory of Open Access Journals (Sweden)

    Sealfon Rachel

    2012-09-01

    Full Text Available Abstract Background Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x; four of the seven isolates were previously sequenced. Results Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961, 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Conclusions Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

  12. Campbell's monkeys concatenate vocalizations into context-specific call sequences

    Science.gov (United States)

    Ouattara, Karim; Lemasson, Alban; Zuberbühler, Klaus

    2009-01-01

    Primate vocal behavior is often considered irrelevant in modeling human language evolution, mainly because of the caller's limited vocal control and apparent lack of intentional signaling. Here, we present the results of a long-term study on Campbell's monkeys, which has revealed an unrivaled degree of vocal complexity. Adult males produced six different loud call types, which they combined into various sequences in highly context-specific ways. We found stereotyped sequences that were strongly associated with cohesion and travel, falling trees, neighboring groups, nonpredatory animals, unspecific predatory threat, and specific predator classes. Within the responses to predators, we found that crowned eagles triggered four and leopards three different sequences, depending on how the caller learned about their presence. Callers followed a number of principles when concatenating sequences, such as nonrandom transition probabilities of call types, addition of specific calls into an existing sequence to form a different one, or recombination of two sequences to form a third one. We conclude that these primates have overcome some of the constraints of limited vocal control by combinatorial organization. As the different sequences were so tightly linked to specific external events, the Campbell's monkey call system may be the most complex example of ‘proto-syntax’ in animal communication known to date. PMID:20007377

  13. Camelid genomes reveal evolution and adaptation to desert environments.

    Science.gov (United States)

    Wu, Huiguang; Guang, Xuanmin; Al-Fageeh, Mohamed B; Cao, Junwei; Pan, Shengkai; Zhou, Huanmin; Zhang, Li; Abutarboush, Mohammed H; Xing, Yanping; Xie, Zhiyuan; Alshanqeeti, Ali S; Zhang, Yanru; Yao, Qiulin; Al-Shomrani, Badr M; Zhang, Dong; Li, Jiang; Manee, Manee M; Yang, Zili; Yang, Linfeng; Liu, Yiyi; Zhang, Jilin; Altammami, Musaad A; Wang, Shenyuan; Yu, Lili; Zhang, Wenbin; Liu, Sanyang; Ba, La; Liu, Chunxia; Yang, Xukui; Meng, Fanhua; Wang, Shaowei; Li, Lu; Li, Erli; Li, Xueqiong; Wu, Kaifeng; Zhang, Shu; Wang, Junyi; Yin, Ye; Yang, Huanming; Al-Swailem, Abdulaziz M; Wang, Jun

    2014-10-21

    Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedarius) and alpaca (Vicugna pacos) are economically important livestock. Although the Bactrian camel and dromedary are large, typically arid-desert-adapted mammals, alpacas are adapted to plateaus. Here we present high-quality genome sequences of these three species. Our analysis reveals the demographic history of these species since the Tortonian Stage of the Miocene and uncovers a striking correlation between large fluctuations in population size and geological time boundaries. Comparative genomic analysis reveals complex features related to desert adaptations, including fat and water metabolism, stress responses to heat, aridity, intense ultraviolet radiation and choking dust. Transcriptomic analysis of Bactrian camels further reveals unique osmoregulation, osmoprotection and compensatory mechanisms for water reservation underpinned by high blood glucose levels. We hypothesize that these physiological mechanisms represent kidney evolutionary adaptations to the desert environment. This study advances our understanding of camelid evolution and the adaptation of camels to arid-desert environments.

  14. Loss of genetic variability in a hatchery strain of Senegalese sole (Solea senegalensis revealed by sequence data of the mitochondrial DNA control region and microsatellite markers

    Directory of Open Access Journals (Sweden)

    Pablo Sánchez

    2012-06-01

    Full Text Available Comparisons of the levels of genetic variation within and between a hatchery F1 (FAR, n=116 of Senegalese sole, Solea senegalensis, and its wild donor population (ATL, n = 26, both native to the SW Atlantic coast of the Iberian peninsula, as well as between the wild donor population and a wild western Mediterranean sample (MED, n=18, were carried out by characterizing 412 base pairs of the nucleotide sequence of the mitochondrial DNA control region I, and six polymorphic microsatellite loci. FAR showed a substantial loss of genetic variability (haplotypic diversity, h=0.49±0.066; nucleotide diversity, π=0.006±0.004; private allelic richness, pAg=0.28 to its donor population ATL (h=0.69±0.114; π=0.009±0.006; pAg=1.21. Pairwise FST values of microsatellite data were highly significant (P < 0.0001 between FAR and ATL (0.053 and FAR and MED (0.055. The comparison of wild samples revealed higher values of genetic variability in MED than in ATL, but only with mtDNA CR-I sequence data (h=0.948±0.033; π=0.030±0.016. However, pairwise ΦST and FST values between ATL and MED were highly significant (P < 0.0001 with mtDNA CR-I (0.228 and with microsatellite data (0.095, respectively. While loss of genetic variability in FAR could be associated with the sampling error when the broodstock was established, the results of parental and sibship inference suggest that most of these losses can be attributed to a high variance in reproductive success among members of the broodstock, particularly among females.

  15. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

    Science.gov (United States)

    Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

    2004-01-01

    Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

  16. Study on high-resolution sequence stratigraphy framework of uranium-hosting rock series in Qianjiadian sag

    International Nuclear Information System (INIS)

    Chen Fanghong; Zhang Mingyu

    2005-01-01

    The ore-hosting Yaojia Formation is composed of a set of braided stream medium-fine grained sediments. Guided by the basic theory of high-resolution sequence stratigraphy, and based on the core observation, the analysis of chemical composition of rocks, and data of natural potential logging and apparent resistivity logging, the authors have set up the high-resolution sequence stratigraphy framework of the ore-hosting Yaojia Formation, and discussed the relation of the stratigraphic structure of the middle cycle, as well as the paleotopography, the micro-facies to the formation of uranium deposit. (authors)

  17. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  18. Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

    Science.gov (United States)

    Tenedini, E; Bernardis, I; Artusi, V; Artuso, L; Roncaglia, E; Guglielmelli, P; Pieri, L; Bogani, C; Biamonte, F; Rotunno, G; Mannarelli, C; Bianchi, E; Pancrazzi, A; Fanelli, T; Malagoli Tagliazucchi, G; Ferrari, S; Manfredini, R; Vannucchi, A M; Tagliafico, E

    2014-01-01

    With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score. PMID:24150215

  19. Evolutionary dynamics of an expressed MHC class IIβ locus in the Ranidae (Anura) uncovered by genome walking and high-throughput amplicon sequencing

    Science.gov (United States)

    Mulder, Kevin P.; Cortazar-Chinarro, Maria; Harris, D. James; Crottini, Angelica; Grant, Evan H. Campbell; Fleischer, Robert C.; Savage, Anna E.

    2017-01-01

    The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class IIβ chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a range of anuran taxa.

  20. Porcine MYF6 gene: sequence, homology analysis, and variation in the promoter region.

    Science.gov (United States)

    Wyszyńska-Koko, J; Kurył, J

    2004-01-01

    MYF6 gene codes for the bHLH transcription factor belonging to MyoD family. Its expression accompanies the processes of differentiation and maturation of myotubes during embriogenesis and continues on a relatively high level after birth, affecting the muscle phenotype. The porcine MYF6 gene was amplified and sequenced and compared with MYF6 gene sequences of other species. The amino acid sequence was deduced and an interspecies homology analysis was performed. Myf-6 protein shows a high conservation among species of 99 and 97% identity when comparing pig with cow and human, respectively, and of 93% when comparing pig with mouse and rat. The single nucleotide polymorphism (SNP) was revealed within the promoter region, which appeared to be T --> C transition recognized by a MspI restriction enzyme.

  1. Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

    Science.gov (United States)

    Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

    2017-04-15

    RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Plastome Sequence Determination and Comparative Analysis for Members of the Lolium-Festuca Grass Species Complex

    Science.gov (United States)

    Hand, Melanie L.; Spangenberg, German C.; Forster, John W.; Cogan, Noel O. I.

    2013-01-01

    Chloroplast genome sequences are of broad significance in plant biology, due to frequent use in molecular phylogenetics, comparative genomics, population genetics, and genetic modification studies. The present study used a second-generation sequencing approach to determine and assemble the plastid genomes (plastomes) of four representatives from the agriculturally important Lolium-Festuca species complex of pasture grasses (Lolium multiflorum, Festuca pratensis, Festuca altissima, and Festuca ovina). Total cellular DNA was extracted from either roots or leaves, was sequenced, and the output was filtered for plastome-related reads. A comparison between sources revealed fewer plastome-related reads from root-derived template but an increase in incidental bacterium-derived sequences. Plastome assembly and annotation indicated high levels of sequence identity and a conserved organization and gene content between species. However, frequent deletions within the F. ovina plastome appeared to contribute to a smaller plastid genome size. Comparative analysis with complete plastome sequences from other members of the Poaceae confirmed conservation of most grass-specific features. Detailed analysis of the rbcL–psaI intergenic region, however, revealed a “hot-spot” of variation characterized by independent deletion events. The evolutionary implications of this observation are discussed. The complete plastome sequences are anticipated to provide the basis for potential organelle-specific genetic modification of pasture grasses. PMID:23550121

  3. The efficacy of high-throughput sequencing and target enrichment on charred archaeobotanical remains

    DEFF Research Database (Denmark)

    Nistelberger, H. M.; Smith, O.; Wales, Nathan

    2016-01-01

    . It has been suggested that high-throughput sequencing (HTS) technologies coupled with DNA enrichment techniques may overcome some of these limitations. Here we report the findings of HTS and target enrichment on four important archaeological crops (barley, grape, maize and rice) performed in three...... lightly-charred maize cob. Even with target enrichment, this sample failed to yield adequate data required to address fundamental questions in archaeology and biology. We further reanalysed part of an existing dataset on charred plant material, and found all purported endogenous DNA sequences were likely...

  4. Algorithms for mapping high-throughput DNA sequences

    DEFF Research Database (Denmark)

    Frellsen, Jes; Menzel, Peter; Krogh, Anders

    2014-01-01

    of data generation, new bioinformatics approaches have been developed to cope with the large amount of sequencing reads obtained in these experiments. In this chapter, we first introduce HTS technologies and their usage in molecular biology and discuss the problem of mapping sequencing reads...... to their genomic origin. We then in detail describe two approaches that offer very fast heuristics to solve the mapping problem in a feasible runtime. In particular, we describe the BLAT algorithm, and we give an introduction to the Burrows-Wheeler Transform and the mapping algorithms based on this transformation....

  5. The complete chloroplast genome sequence of Mahonia bealei (Berberidaceae) reveals a significant expansion of the inverted repeat and phylogenetic relationship with other angiosperms.

    Science.gov (United States)

    Ma, Ji; Yang, Bingxian; Zhu, Wei; Sun, Lianli; Tian, Jingkui; Wang, Xumin

    2013-10-10

    Mahonia bealei (Berberidaceae) is a frequently-used traditional Chinese medicinal plant with efficient anti-inflammatory ability. This plant is one of the sources of berberine, a new cholesterol-lowering drug with anti-diabetic activity. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of M. bealei. The complete cp genome of M. bealei is 164,792 bp in length, and has a typical structure with large (LSC 73,052 bp) and small (SSC 18,591 bp) single-copy regions separated by a pair of inverted repeats (IRs 36,501 bp) of large size. The Mahonia cp genome contains 111 unique genes and 39 genes are duplicated in the IR regions. The gene order and content of M. bealei are almost unarranged which is consistent with the hypothesis that large IRs stabilize cp genome and reduce gene loss-and-gain probabilities during evolutionary process. A large IR expansion of over 12 kb has occurred in M. bealei, 15 genes (rps19, rpl22, rps3, rpl16, rpl14, rps8, infA, rpl36, rps11, petD, petB, psbH, psbN, psbT and psbB) have expanded to have an additional copy in the IRs. The IR expansion rearrangement occurred via a double-strand DNA break and subsequence repair, which is different from the ordinary gene conversion mechanism. Repeat analysis identified 39 direct/inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Analysis also revealed 75 simple sequence repeat (SSR) loci and almost all are composed of A or T, contributing to a distinct bias in base composition. Comparison of protein-coding sequences with ESTs reveals 9 putative RNA edits and 5 of them resulted in non-synonymous modifications in rpoC1, rps2, rps19 and ycf1. Phylogenetic analysis using maximum parsimony (MP) and maximum likelihood (ML) was performed on a dataset composed of 65 protein-coding genes from 25 taxa, which yields an identical tree topology as previous plastid-based trees, and provides strong support for the sister relationship between Ranunculaceae and Berberidaceae

  6. Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3.

    Science.gov (United States)

    Sánchez-Navarro, J A; Pallás, V

    1997-01-01

    The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic alpha-helical structure preceding the conserved motif of the '30K superfamily' proposed by Mushegian and Koonin [26] for MP's. Within this '30K' motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP's of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses.

  7. Predator-prey interaction reveals local effects of high-altitude insect migration

    Science.gov (United States)

    High-altitude nocturnal insect migrations represent significant pulses of resources, yet are difficult to study and poorly understood. Predator-prey interactions, specifically migratory moth consumption by high-flying bats, potentially reveal flows of migratory insects across a landscape. In North...

  8. Mycobacterium malmesburyense sp. nov., a non-tuberculous species of the genus Mycobacterium revealed by multiple gene sequence characterization.

    Science.gov (United States)

    Gcebe, Nomakorinte; Rutten, Victor; Pittius, Nicolaas Gey van; Naicker, Brendon; Michel, Anita

    2017-04-01

    Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment, and an increasing number of NTM species have been isolated and characterized from both humans and animals, highlighting the zoonotic potential of these bacteria. Host exposure to NTM may impact on cross-reactive immune responsiveness, which may affect diagnosis of bovine tuberculosis and may also play a role in the variability of the efficacy of Mycobacterium bovis BCG vaccination against tuberculosis. In this study we characterized 10 NTM isolates originating from water, soil, nasal swabs of cattle and African buffalo as well as bovine tissue samples. These isolates were previously identified during an NTM survey and were all found, using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. A polyphasic approach that included phenotypic characterization, antibiotic susceptibility profiling, mycolic acid profiling and phylogenetic analysis of four gene loci, 16S rRNA, hsp65, sodA and rpoB, was employed to characterize these isolates. Sequence data analysis of the four gene loci revealed that these isolates belong to a unique species of the genus Mycobacterium. This evidence was further supported by several differences in phenotypic characteristics between the isolates and the closely related species. We propose the name Mycobacterium malmesburyense sp. nov. for this novel species. The type strain is WCM 7299T (=ATCC BAA-2759T=CIP 110822T).

  9. Information decomposition method to analyze symbolical sequences

    International Nuclear Information System (INIS)

    Korotkov, E.V.; Korotkova, M.A.; Kudryashov, N.A.

    2003-01-01

    The information decomposition (ID) method to analyze symbolical sequences is presented. This method allows us to reveal a latent periodicity of any symbolical sequence. The ID method is shown to have advantages in comparison with application of the Fourier transformation, the wavelet transform and the dynamic programming method to look for latent periodicity. Examples of the latent periods for poetic texts, DNA sequences and amino acids are presented. Possible origin of a latent periodicity for different symbolical sequences is discussed

  10. Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate

    Science.gov (United States)

    Mangold, Elisabeth; Böhmer, Anne C.; Ishorst, Nina; Hoebel, Ann-Kathrin; Gültepe, Pinar; Schuenke, Hannah; Klamt, Johanna; Hofmann, Andrea; Gölz, Lina; Raff, Ruth; Tessmann, Peter; Nowak, Stefanie; Reutter, Heiko; Hemprich, Alexander; Kreusch, Thomas; Kramer, Franz-Josef; Braumann, Bert; Reich, Rudolf; Schmidt, Gül; Jäger, Andreas; Reiter, Rudolf; Brosch, Sibylle; Stavusis, Janis; Ishida, Miho; Seselgyte, Rimante; Moore, Gudrun E.; Nöthen, Markus M.; Borck, Guntram; Aldhorae, Khalid A.; Lace, Baiba; Stanier, Philip; Knapp, Michael; Ludwig, Kerstin U.

    2016-01-01

    Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10−2). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10−5; ORallelic = 2.46 [95% CI 1.6–3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10−9). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO. PMID:27018475

  11. Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira

    Science.gov (United States)

    Xu, Yinghua; Zhu, Yongzhang; Wang, Yuezhu; Chang, Yung-Fu; Zhang, Ying; Jiang, Xiugao; Zhuang, Xuran; Zhu, Yongqiang; Zhang, Jinlong; Zeng, Lingbing; Yang, Minjun; Li, Shijun; Wang, Shengyue; Ye, Qiang; Xin, Xiaofang; Zhao, Guoping; Zheng, Huajun; Guo, Xiaokui; Wang, Junzhi

    2016-01-01

    Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp. PMID:26833181

  12. Development of a multilocus sequence typing scheme for Ureaplasma.

    Science.gov (United States)

    Zhang, J; Kong, Y; Feng, Y; Huang, J; Song, T; Ruan, Z; Song, J; Jiang, Y; Yu, Y; Xie, X

    2014-04-01

    Ureaplasma is a commensal of the human urogenital tract but is always associated with invasive diseases such as non-gonococcal urethritis and infertility adverse pregnancy outcomes. To better understand the molecular epidemiology and population structure of Ureaplasma, a multilocus sequence typing (MLST) scheme based on four housekeeping genes (ftsH, rpL22, valS, thrS) was developed and validated using 283 isolates, including 14 serovars of reference strains and 269 strains obtained from clinical patients. A total of 99 sequence types (STs) were revealed: the 14 type strains of the Ureaplasma serovars were assigned to 12 STs, and 87 novel and special STs appeared among the clinical isolates. ST1 and ST22 were the predominant STs, which contained 68 and 70 isolates, respectively. Two clonal lineages (CC1 and CC2) were shown by eBURST analysis, and linkage disequilibrium was revealed through a standardized index of association (I A (S)). The neighbor-joining tree results of 14 Ureaplasma serovars showed two genetically significantly distant clusters, which was highly congruent with the species taxonomy of ureaplasmas [Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR)]. Analysis of the biotypes of 269 clinical isolates revealed that all the isolates of CC1 were UPA and those of CC2 were UUR. Additionally, CC2 was found more often in symptomatic patients with vaginitis, tubal obstruction, and cervicitis. In conclusion, this MLST scheme is adequate for investigations of molecular epidemiology and population structure with highly discriminating capacity.

  13. The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences

    Directory of Open Access Journals (Sweden)

    Yandell Mark

    2010-07-01

    Full Text Available Abstract Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24. The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity elsewhere in the genome, but only 23% have identical copies (99% identity. The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is

  14. High-throughput sequencing reveals microbial communities in drinking water treatment sludge from six geographically distributed plants, including potentially toxic cyanobacteria and pathogens.

    Science.gov (United States)

    Xu, Hangzhou; Pei, Haiyan; Jin, Yan; Ma, Chunxia; Wang, Yuting; Sun, Jiongming; Li, Hongmin

    2018-04-10

    The microbial community structures of drinking water treatment sludge (DWTS) generated for raw water (RW) from different locations and with different source types - including river water, lake water and reservoir water -were investigated using high-throughput sequencing. Because the unit operations in the six DWTPs were similar, community composition in fresh sludge may be determined by microbial community in the corresponding RW. Although Proteobacteria, Cyanobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia, and Planctomycetes were the dominant phyla among the six DWTS samples, no single phylum exhibited similar abundance across all the samples, owing to differences in total phosphorus, chemical oxygen demand, Al, Fe, and chloride in RW. Three genera of potentially toxic cyanobacteria (Planktothrix, Microcystis and Cylindrospermopsis), and four potential pathogens (Escherichia coli, Bacteroides ovatus, Prevotella copri and Rickettsia) were found in sludge samples. Because proliferation of potentially toxic cyanobacteria and Rickettsia in RW was mainly affected by nutrients, while growth of Escherichia coli, Bacteroides ovatus and Prevotella copri in RW may be influenced by Fe, control of nutrients and Fe in RW is essential to decrease toxic cyanobacteria and pathogens in DWTS. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Sequence elements correlating with circulating viral load in genotype 1b hepatitis C virus infection

    International Nuclear Information System (INIS)

    Watanabe, Hideki; Nagayama, Kazuyoshi; Enomoto, Nobuyuki; Itakura, Jun; Tanabe, Yoko; Hamano, Kosei; Izumi, Namiki; Sato, Chifumi; Watanabe, Mamoru

    2003-01-01

    The correlation between hepatitis C virus (HCV) genomic sequences and circulating HCV RNA levels was assessed to investigate the genetic elements affecting viral load. The interferon sensitivity-determining region (ISDR) sequence and the serum viral load were strongly correlated in 226 patients examined. Analysis of the entire HCV genome from six patients (three with a high and the others with a low viral load) with similar ISDR sequences identified several candidate residues associated with viral load. The amino acid (aa) sequences of these candidate residues and flanking regions in 67 additional patients revealed that only the residue at aa 962 varied significantly between the HCV patients with low and high serum loads (P 0.042). At this position, alanine was observed more frequently in the patients with a high viral load. In conclusion, our results strongly suggest that serum HCV RNA loads are inversely correlated with amino acid substitutions in the ISDR, and aa 962 was identified as a possible second determinant of serum HCV RNA load

  16. MicroRNAs in Amoebozoa: deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs.

    Science.gov (United States)

    Avesson, Lotta; Reimegård, Johan; Wagner, E Gerhart H; Söderbom, Fredrik

    2012-10-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  17. Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming.

    Science.gov (United States)

    Harwig, Alex; Herrera-Carrillo, Elena; Jongejan, Aldo; van Kampen, Antonius Hubertus; Berkhout, Ben

    2015-07-14

    The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

  18. Sequence periodicity in nucleosomal DNA and intrinsic curvature.

    Science.gov (United States)

    Nair, T Murlidharan

    2010-05-17

    Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures. Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature. The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

  19. High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs.

    Science.gov (United States)

    Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus; Morling, Niels

    2016-01-01

    Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

  20. Using high-throughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A(H1N1 pandemic.

    Directory of Open Access Journals (Sweden)

    Juan Téllez-Sosa

    Full Text Available BACKGROUND: Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The "deep sequencing" approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples. METHODOLOGY AND PRINCIPAL FINDINGS: We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1 pandemic (A(H1N1pdm virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n  =  299 taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July to second wave (September-November of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance. CONCLUSIONS: NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that