Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren
An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects
Full Text Available Abstract Background An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Results Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being
Full Text Available Abstract Background A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. Results The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27% were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements and class II (DNA transposons mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large
Melody S Clark
Full Text Available BACKGROUND: The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. METHODOLOGY/PRINCIPAL FINDINGS: The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp. In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS, providing a resource for population and also gene function studies. CONCLUSIONS: This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding
Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H
The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.
Full Text Available BACKGROUND: Bemisia tabaci (Gennadius is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. METHODOLOGY AND PRINCIPAL FINDINGS: Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45% unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10-5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. CONCLUSIONS: This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the
Full Text Available Abstract Background Common bean (Phaseolus vulgaris is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. Results We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt. These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC end sequences, and a total of 21% of the unigenes (12,724 including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs and transcription factors were also identified in this study. Conclusions This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and
Full Text Available Abstract Background The teleost Zoarces viviparus (eelpout lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. Results In the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. Conclusion Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.
Fujimaki, Kotaro; Fujisawa, Tomochika; Yazawa, Shigenobu; Nishimura, Osamu; Sota, Teiji
Ground beetles of the subgenus Ohomopterus (genus Carabus) show marked divergence in species-specific male and female genital morphologies, which contributes to reproductive isolation among species. Characterizing the genetic basis of species-specific genital morphology is essential for understanding their diversification, but genomic information on Ohomopterus is not yet available. We analyzed mRNA extracted from abdominal sections of the last instar larvae and pupae of two sister species, Carabus (Ohomopterus) iwawakianus and C. (O.) uenoi, which show marked differences in genital morphology, to compare transcriptomic profiles using Roche 454 pyrosequencing. We obtained 1,608,572 high-quality reads and assembled them into 176,278 unique sequences, of which 66,049 sequences were combined into 12,662 clusters. Differential expression analyses for sexed pupae suggested that four and five clusters were differentially expressed between species for males and females, respectively. We also identified orthologous sequences of genes involved in genital development in Drosophila, which potentially affect genital development and species-specific genital morphology in Ohomopterus. This study provides the first large transcriptomic data set for a morphologically diversified beetle group, which can facilitate future studies on the genetic basis of species-specific genitalia.
Full Text Available The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.
Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair
The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.
Full Text Available Abstract Background The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. Results Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication. BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. Conclusion Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.
Bazakos, Christos; Manioudaki, Maria E; Sarropoulou, Elena; Spano, Thodhoraq; Kalaitzis, Panagiotis
Olive (Olea europaea L.) is one of the most important crops in the Mediterranean region. The expansion of cultivation in areas irrigated with low quality and saline water has negative effects on growth and productivity however the investigation of the molecular basis of salt tolerance in olive trees has been only recently initiated. To this end, we investigated the molecular response of cultivar Kalamon to salinity stress using next-generation sequencing technology to explore the transcriptome profile of olive leaves and roots and identify differentially expressed genes that are related to salt tolerance response. Out of 291,958 obtained trimmed reads, 28,270 unique transcripts were identified of which 35% are annotated, a percentage that is comparable to similar reports on non-model plants. Among the 1,624 clusters in roots that comprise more than one read, 24 were differentially expressed comprising 9 down- and 15 up-regulated genes. Respectively, inleaves, among the 2,642 clusters, 70 were identified as differentially expressed, with 14 down- and 56 up-regulated genes. Using next-generation sequencing technology we were able to identify salt-response-related transcripts. Furthermore we provide an annotated transcriptome of olive as well as expression data, which are both significant tools for further molecular studies in olive.
Full Text Available BACKGROUND: The abalone Haliotis diversicolor is a good model for study of the settlement and metamorphosis, which are widespread marine ecological phenomena. However, information on the global gene backgrounds and gene expression profiles for the early development of abalones is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, eight non-normalized and multiplex barcode-labeled transcriptomes were sequenced using a 454 GS system to cover the early developmental stages of the abalone H. diversicolor. The assembly generated 35,415 unigenes, of which 7,566 were assigned GO terms. A global gene expression profile containing 636 scaffolds/contigs was constructed and was proven reliable using qPCR evaluation. It indicated that there may be existing dramatic phase transitions. Bioprocesses were proposed, including the 'lock system' in mature eggs, the collagen shells of the trochophore larvae and the development of chambered extracellular matrix (ECM structures within the earliest postlarvae. CONCLUSION: This study globally details the first 454 sequencing data for larval stages of H. diversicolor. A basic analysis of the larval transcriptomes and cluster of the gene expression profile indicates that each stage possesses a batch of specific genes that are indispensable during embryonic development, especially during the two-cell, trochophore and early postlarval stages. These data will provide a fundamental resource for future physiological works on abalones, revealing the mechanisms of settlement and metamorphosis at the molecular level.
Park, J. E.; Lee, B. T.; Son, A.
Heavy metals leached from exhausted mines have been causing severe environmental problems in nearby soils and groundwater. Environmental mitigation was performed based on the heavy metal stabilization using Calcite and steel slag in Korea. Since the soil stabilization only temporarily immobilizes the contaminants to soil matrix, the potential risk of re-leaching heavy metal still exists. Therefore the follow-up management of stabilized soils and the corresponding evaluation methods are required to avoid the consequent contamination from the stabilized soils. In this study, microbial community analysis using pyrosequencing was performed for assessing the potential leaching of the stabilized soils. As a result of rarefaction curve and Chao1 and Shannon indices, the stabilized soil has shown lower richness and diversity as compared to non-contaminated negative control. At the phyla level, as the degree of contamination increases, most of phyla decreased with only exception of increased proteobacteria. Among proteobacteria, gamma-proteobacteria increased against the heavy metal contamination. At the species level, Methylobacter tundripaludum of gamma-proteobacteria showed the highest relative portion of microbial community, indicating that methanotrophs may play an important role in either solubilization or immobilization of heavy metals in stabilized soils.
El-Sayed Yasser Y
Full Text Available Abstract Background The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the Results We describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques. Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region. Conclusion This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora
Transcriptomic analysis of grain amaranth (Amaranthus hypochondriacus using 454 pyrosequencing: comparison with A. tuberculatus, expression profiling in stems and in response to biotic and abiotic stress
Full Text Available Abstract Background Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs, transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scale transcriptome analysis was performed to generate stem- and (abiotic stress-responsive gene expression profiles in grain amaranth. Results A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs. The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing
Natarajan, Purushothaman; Parani, Madasamy
Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of
Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G
The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.
... Also: Talking Glossary of Genetic Terms Definitions for genetic terms used on this page En Español: Transcriptoma Transcriptome What is a transcriptome? What can a transcriptome tell us? How can transcriptome data be used to explore gene function? What is ...
Full Text Available Abstract Background Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. Results From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. Conclusion The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil
Eiko E Kuramae
Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.
Song, Qinxin; Wei, Guijiang; Zhou, Guohua
A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.
Full Text Available BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.
Tekin, Saban; Dowd, Scot E; Davinic, Marko; Bursali, Ahmet; Keskin, Adem
Ticks continue to be a threat to human and animal health in Turkey, as they are considered important vectors of human and animal diseases. The objectives of this investigation are to characterize the microbial communities of two tick species, Rhipicephalus annulatus and Dermacenter marginatus, analyze patterns of co-occurrence among microbial taxa, identify and compare pathogens contributing human diseases, and determine whether avirulent symbionts could exclude human pathogens from tick communities. Furthermore, this study explores a microbiome of the R. annulatus and D. marginatus via the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to describe their bacterial diversity. Pyrosequencing was performed on adult males and females isolated from humans from two high-risk Turkish provinces, Sivas and Amasya, during tick outbreaks in 2009. A total of 36,253 sequences were utilized for analyses of the 8 tick samples. Several pathogenic genera such as Francisella, Coxiella, Rickettsia, and Shigella were detected in the ticks tested. The most distinguishable difference between the two species of ticks was the lack of known human pathogen Rickettsia in R. annulatus and in samples 9 and 10 of D. marginatus. These samples had higher relative abundance of Flavobacterium sp., Curvibacter sp., Acidovorax sp., and Bacteroidaceae genera mostly representing symbionts which form a large component of normal tick microbiota. The outcome of this study is consistent with the predictions of the community ecological theory that diversity-rich bacteriomes are more resistant to bacterial invasion (and consequent pathogen dissemination) than diversity-deprived ones.
Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S
Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.
Full Text Available The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.
Zhang, Bingchang; Li, Renhui; Xiao, Peng; Su, Yangui; Zhang, Yuanming
Cyanobacteria are the primary colonizers and form a dominant component of soil photosynthetic communities in biological soil crusts. They are crucial in improving soil environments, namely accumulating soil carbon and nitrogen. Many classical studies have examined cyanobacterial diversity in desert crusts, but relatively few comprehensive molecular surveys have been conducted. We used 454 pyrosequencing of 16S rRNA to investigate cyanobacterial composition and distribution on regional scales in the Gurbantunggut Desert. The relationship between cyanobacterial distribution and environmental factors was also explored. A total of 24,973 cyanobacteria partial 16S rRNA gene sequences were obtained, and 507OTUs were selected, as most OTUs had very few reads. Among these, 347 OTU sequences were of cyanobacteria origin, belonging to Oscillatoriales, Nostocales, Chroococcales, and uncultured cyanobacterium clone, respectively. Microcoleus vaginatus, Chroococcidiopsis spp. and M. steenstrupii were the dominant species in most areas of the Gurbantunggut Desert. Compared with other desert, the Gurbantunggut Desert differed in the prominence of Chroococcidiopsis spp. and lack of Pseudanabaenales. Species composition and abundance of cyanobacteria also showed distinct variations. Soil texture, precipitation, and nutrients and salt levels affected cyanobacterial distribution. Increased precipitation was helpful in improving cyanobacterial diversity. A higher content of coarse sand promoted the colonization and growth of Oscillatoriales and some phylotypes of Chroococcales. The fine-textured soil with higher nutrients and salts supported more varied populations of cyanobacteria, namely some heterocystous cyanobacteria. The results suggested that the Gurbantunggut Desert was rich in cyanobacteria and that precipitation was a primary regulating factor for cyanobacterial composition on a regional scale. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Al-Sadi, A M; Al-Mazroui, S S; Phillips, A J L
Potting media and organic fertilizers (OFs) are commonly used in agricultural systems. However, there is a lack of studies on the efficiency of culture-based techniques in assessing the level of fungal diversity in these products. A study was conducted to investigate the efficiency of seven culture-based techniques and pyrosequencing for characterizing fungal diversity in potting media and OFs. Fungal diversity was evaluated using serial dilution, direct plating and baiting with carrot slices, potato slices, radish seeds, cucumber seeds and cucumber cotyledons. Identity of all the isolates was confirmed on the basis of the internal transcribed spacer region of the ribosomal RNA (ITS rRNA) sequence data. The direct plating technique was found to be superior over other culture-based techniques in the number of fungal species detected. It was also found to be simple and the least time consuming technique. Comparing the efficiency of direct plating with 454 pyrosequencing revealed that pyrosequencing detected 12 and 15 times more fungal species from potting media and OFs respectively. Analysis revealed that there were differences between potting media and OFs in the dominant phyla, classes, orders, families, genera and species detected. Zygomycota (52%) and Chytridiomycota (60%) were the predominant phyla in potting media and OFs respectively. The superiority of pyrosequencing over cultural methods could be related to the ability to detect obligate fungi, slow growing fungi and fungi that exist at low population densities. The evaluated methods in this study, especially direct plating and pyrosequencing, may be used as tools to help detect and reduce movement of unwanted fungi between countries and regions. © 2015 The Society for Applied Microbiology.
Bonten Marc JM
Full Text Available Abstract Background The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. Results We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI, which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. Conclusions Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.
Construction of an EST-SSR-based interspecific transcriptome linkage map of fibre development in cotton. CHUANXIANG LIU, DAOJUN YUAN and ZHONGXU LIN. ∗. National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan),. Huazhong Agricultural University, Wuhan ...
Fortino, Vittorio; Smolander, Olli-Pekka; Auvinen, Petri; Tagliaferri, Roberto; Greco, Dario
Inferring operon maps is crucial to understanding the regulatory networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies revealed that in many bacterial species the operon structure vary with the change of environmental conditions. Therefore, new computational solutions that use both static and dynamic data are necessary to create condition specific operon predictions. In this work, we propose a novel classification method that integrates RNA-seq based transcriptome profiles with genomic sequence features to accurately identify the operons that are expressed under a measured condition. The classifiers are trained on a small set of confirmed operons and then used to classify the remaining gene pairs of the organism studied. Finally, by linking consecutive gene pairs classified as operons, our computational approach produces condition-dependent operon maps. We evaluated our approach on various RNA-seq expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica. Our results demonstrate that, using features depending on both transcriptome dynamics and genome sequence characteristics, we can identify operon pairs with high accuracy. Moreover, the combination of DNA sequence and expression data results in more accurate predictions than each one alone. We present a computational strategy for the comprehensive analysis of condition-dependent operon maps in prokaryotes. Our method can be used to generate condition specific operon maps of many bacterial organisms for which high-resolution transcriptome data is available.
Dohmen, Elias; Kremer, Lukas P M; Bornberg-Bauer, Erich; Kemena, Carsten
Genome studies have become cheaper and easier than ever before, due to the decreased costs of high-throughput sequencing and the free availability of analysis software. However, the quality of genome or transcriptome assemblies can vary a lot. Therefore, quality assessment of assemblies and annotations are crucial aspects of genome analysis pipelines. We developed DOGMA, a program for fast and easy quality assessment of transcriptome and proteome data based on conserved protein domains. DOGMA measures the completeness of a given transcriptome or proteome and provides information about domain content for further analysis. DOGMA provides a very fast way to do quality assessment within seconds. DOGMA is implemented in Python and published under GNU GPL v.3 license. The source code is available on https://ebbgit.uni-muenster.de/domainWorld/DOGMA/ CONTACTS: firstname.lastname@example.org or email@example.com Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
Lance B Price
Full Text Available BACKGROUND: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007 and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. CONCLUSIONS/SIGNIFICANCE: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.
Castillo Luis F.
Full Text Available Gene annotation is a process that encompasses multiple approaches on the analysis of nucleic acids or protein sequences in order to assign structural and functional characteristics to gene models. When thousands of gene models are being described in an organism genome, construction and visualization of gene networks impose novel challenges in the understanding of complex expression patterns and the generation of new knowledge in genomics research. In order to take advantage of accumulated text data after conventional gene sequence analysis, this work applied semantics in combination with visualization tools to build transcriptome networks from a set of coffee gene annotations. A set of selected coffee transcriptome sequences, chosen by the quality of the sequence comparison reported by Basic Local Alignment Search Tool (BLAST and Interproscan, were filtered out by coverage, identity, length of the query, and e-values. Meanwhile, term descriptors for molecular biology and biochemistry were obtained along the Wordnet dictionary in order to construct a Resource Description Framework (RDF using Ruby scripts and Methontology to find associations between concepts. Relationships between sequence annotations and semantic concepts were graphically represented through a total of 6845 oriented vectors, which were reduced to 745 non-redundant associations. A large gene network connecting transcripts by way of relational concepts was created where detailed connections remain to be validated for biological significance based on current biochemical and genetics frameworks. Besides reusing text information in the generation of gene connections and for data mining purposes, this tool development opens the possibility to visualize complex and abundant transcriptome data, and triggers the formulation of new hypotheses in metabolic pathways analysis.
Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong
Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.
Wang, Ailin; Yao, Zhichao; Zheng, Weiwei; Zhang, Hongyu
The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%-95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.
Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M
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.
Full Text Available BACKGROUND: Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. CONCLUSIONS: This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.
Full Text Available The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs. A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%-95%. Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices and community structure (PCA analysis varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.
Full Text Available Abstract Background Sweetpotato (Ipomoea batatas (L. Lam., a hexaploid outcrossing crop, is an important staple and food security crop in developing countries in Africa and Asia. The availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. Previously existing sequence data were restricted to around 22,000 expressed sequence tag (EST sequences and ~ 1,500 GenBank sequences. We have used 454 pyrosequencing to augment the available gene sequence information to enhance functional genomics and marker design for this plant species. Results Two quarter 454 pyrosequencing runs used two normalized cDNA collections from stems and leaves from drought-stressed sweetpotato clone Tanzania and yielded 524,209 reads, which were assembled together with 22,094 publically available expressed sequence tags into 31,685 sets of overlapping DNA segments and 34,733 unassembled sequences. Blastx comparisons with the UniRef100 database allowed annotation of 23,957 contigs and 15,342 singletons resulting in 24,657 putatively unique genes. Further, 27,119 sequences had no match to protein sequences of UniRef100database. On the basis of this gene index, we have identified 1,661 gene-based microsatellite sequences, of which 223 were selected for testing and 195 were successfully amplified in a test panel of 6 hexaploid (I. batatas and 2 diploid (I. trifida accessions. Conclusions The sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous EST assemblies. A searchable version of the gene index, including a blastn function, is available at http://www.cipotato.org/sweetpotato_gene_index.
Davenport Russell J
Full Text Available Abstract Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high
Oct 25, 2017 ... (Shanghai, China) following manufacturer's protocols (Illumina, San .... suggests that pathways involved in musk production are expressed at a ..... Strickler S. R., Aureliano B. and Mueller L. A. 2012 Designing a transcriptome.
Full Text Available BACKGROUND: The basally divergent phylogenetic position of amphioxus (Cephalochordata, as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae. RESULTS: Starting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode. Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp. Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species. CONCLUSION: We obtained a high-quality amphioxus (B. lanceolatum reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation
Hong, Pei-Ying; Wheeler, Emily; Cann, Isaac K O; Mackie, Roderick I
Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species--the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E-9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E-14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356-896 OTUs) was >2-fold higher than in the MI (112-567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that
In the present study, we used Illumina HiSeq technology to perform de novo assembly of heart and musk gland transcriptomes from the Chinese forest musk deer. A total of 239,383 transcripts and 176,450 unigenes were obtained, of which 37,329 unigenes were matched to known sequences in the NCBI nonredundant ...
Full Text Available BACKGROUND: Sea cucumbers are a special group of marine invertebrates. They occupy a taxonomic position that is believed to be important for understanding the origin and evolution of deuterostomes. Some of them such as Apostichopus japonicus represent commercially important aquaculture species in Asian countries. Many efforts have been devoted to increasing the number of expressed sequence tags (ESTs for A. japonicus, but a comprehensive characterization of its transcriptome remains lacking. Here, we performed the large-scale transcriptome profiling and characterization by pyrosequencing diverse cDNA libraries from A. japonicus. RESULTS: In total, 1,061,078 reads were obtained by 454 sequencing of eight cDNA libraries representing different developmental stages and adult tissues in A. japonicus. These reads were assembled into 29,666 isotigs, which were further clustered into 21,071 isogroups. Nearly 40% of the isogroups showed significant matches to known proteins based on sequence similarity. Gene ontology (GO and KEGG pathway analyses recovered diverse biological functions and processes. Candidate genes that were potentially involved in aestivation were identified. Transcriptome comparison with the sea urchin Strongylocentrotus purpuratus revealed similar patterns of GO term representation. In addition, 4,882 putative orthologous genes were identified, of which 202 were not present in the non-echinoderm organisms. More than 700 simple sequence repeats (SSRs and 54,000 single nucleotide polymorphisms (SNPs were detected in the A. japonicus transcriptome. CONCLUSION: Pyrosequencing was proven to be efficient in rapidly identifying a large set of genes for the sea cucumber A. japonicus. Through the large-scale transcriptome sequencing as well as public EST data integration, we performed a comprehensive characterization of the A. japonicus transcriptome and identified candidate aestivation-related genes. A large number of potential genetic
Full Text Available Mutations in Human Epidermal Growth Factor Receptors (HER are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS, alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC, ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.
Kumar, Sujai; Blaxter, Mark L
Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis. Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs. Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible
Blaxter Mark L
Full Text Available Abstract Background Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis. Results Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects, which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs. Conclusions Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies
Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.
Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren
Abstract Background An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They b...
Full Text Available Abstract Background The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 → 3-β-linked glucose with a (1 → 6-β-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches. Results Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields. Conclusions The results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and
Omranian, Nooshin; Mueller-Roeber, Bernd; Nikoloski, Zoran
The levels of cellular organization, from gene transcription to translation to protein-protein interaction and metabolism, operate via tightly regulated mutual interactions, facilitating organismal adaptability and various stress responses. Characterizing the mutual interactions between genes, transcription factors, and proteins involved in signaling, termed crosstalk, is therefore crucial for understanding and controlling cells' functionality. We aim at using high-throughput transcriptomics data to discover previously unknown links between signaling networks. We propose and analyze a novel method for crosstalk identification which relies on transcriptomics data and overcomes the lack of complete information for signaling pathways in Arabidopsis thaliana. Our method first employs a network-based transformation of the results from the statistical analysis of differential gene expression in given groups of experiments under different signal-inducing conditions. The stationary distribution of a random walk (similar to the PageRank algorithm) on the constructed network is then used to determine the putative transcripts interrelating different signaling pathways. With the help of the proposed method, we analyze a transcriptomics data set including experiments from four different stresses/signals: nitrate, sulfur, iron, and hormones. We identified promising gene candidates, downstream of the transcription factors (TFs), associated to signaling crosstalk, which were validated through literature mining. In addition, we conduct a comparative analysis with the only other available method in this field which used a biclustering-based approach. Surprisingly, the biclustering-based approach fails to robustly identify any candidate genes involved in the crosstalk of the analyzed signals. We demonstrate that our proposed method is more robust in identifying gene candidates involved downstream of the signaling crosstalk for species for which large transcriptomics data sets
Karthik, Govindasamy-Muralidharan; Rantalainen, Mattias; Stålhammar, Gustav; Lövrot, John; Ullah, Ikram; Alkodsi, Amjad; Ma, Ran; Wedlund, Lena; Lindberg, Johan; Frisell, Jan; Bergh, Jonas; Hartman, Johan
Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.
Li, Pei; Ji, Guoli; Dong, Min; Schmidt, Emily; Lenox, Douglas; Chen, Liangliang; Liu, Qi; Liu, Lin; Zhang, Jie; Liang, Chun
To address the impending need for exploring rapidly increased transcriptomics data generated for non-model organisms, we developed CBrowse, an AJAX-based web browser for visualizing and analyzing transcriptome assemblies and contigs. Designed in a standard three-tier architecture with a data pre-processing pipeline, CBrowse is essentially a Rich Internet Application that offers many seamlessly integrated web interfaces and allows users to navigate, sort, filter, search and visualize data smoothly. The pre-processing pipeline takes the contig sequence file in FASTA format and its relevant SAM/BAM file as the input; detects putative polymorphisms, simple sequence repeats and sequencing errors in contigs and generates image, JSON and database-compatible CSV text files that are directly utilized by different web interfaces. CBowse is a generic visualization and analysis tool that facilitates close examination of assembly quality, genetic polymorphisms, sequence repeats and/or sequencing errors in transcriptome sequencing projects. CBrowse is distributed under the GNU General Public License, available at http://bioinfolab.muohio.edu/CBrowse/ email@example.com or firstname.lastname@example.org; email@example.com Supplementary data are available at Bioinformatics online.
Liu, Xin; Yang, Haiyan; Zheng, Junwei; Ye, Yanrui; Pan, Li
To expand the repertoire of strong promoters for high level expression of proteins based on the transcriptome of Bacillus licheniformis. The transcriptome of B. licheniformis ATCC14580 grown to the early stationary phase was analyzed and the top 10 highly expressed genes/operons out of the 3959 genes and 1249 operons identified were chosen for study promoter activity. Using beta-galactosidase gene as a reporter, the candidate promoter pBL9 exhibited the strongest activity which was comparable to that of the widely used strong promoter p43. Furthermore, the pro-transglutaminase from Streptomyces mobaraensis (pro-MTG) was expressed under the control of promoter pBL9 and the activity of pro-MTG reached 82 U/ml after 36 h, which is 23% higher than that of promoter p43 (66.8 U/ml). In our analyses of the transcriptome of B. licheniformis, we have identified a strong promoter pBL9, which could be adapted for high level expression of proteins in the host Bacillus subtilis.
Tian, Xin-Jie; Long, Yan; Wang, Jiao; Zhang, Jing-Wen; Wang, Yan-Yan; Li, Wei-Min; Peng, Yu-Fa; Yuan, Qian-Hua; Pei, Xin-Wu
The perennial O. rufipogon (common wild rice), which is considered to be the ancestor of Asian cultivated rice species, contains many useful genetic resources, including drought resistance genes. However, few studies have identified the drought resistance and tissue-specific genes in common wild rice. In this study, transcriptome sequencing libraries were constructed, including drought-treated roots (DR) and control leaves (CL) and roots (CR). Using Illumina sequencing technology, we generated 16.75 million bases of high-quality sequence data for common wild rice and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into 119,332 unigenes with an average length of 715 bp. A total of 88,813 distinct sequences (74.42% of unigenes) significantly matched known genes in the NCBI NT database. Differentially expressed gene (DEG) analysis showed that 3617 genes were up-regulated and 4171 genes were down-regulated in the CR library compared with the CL library. Among the DEGs, 535 genes were expressed in roots but not in shoots. A similar comparison between the DR and CR libraries showed that 1393 genes were up-regulated and 315 genes were down-regulated in the DR library compared with the CR library. Finally, 37 genes that were specifically expressed in roots were screened after comparing the DEGs identified in the above-described analyses. This study provides a transcriptome sequence resource for common wild rice plants and establishes a digital gene expression profile of wild rice plants under drought conditions using the assembled transcriptome data as a reference. Several tissue-specific and drought-stress-related candidate genes were identified, representing a fully characterized transcriptome and providing a valuable resource for genetic and genomic studies in plants.
Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten
DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....
Martin, Jeffrey A.; Wang, Zhong
Transcriptomics studies often rely on partial reference transcriptomes that fail to capture the full catalog of transcripts and their variations. Recent advances in sequencing technologies and assembly algorithms have facilitated the reconstruction of the entire transcriptome by deep RNA sequencing (RNA-seq), even without a reference genome. However, transcriptome assembly from billions of RNA-seq reads, which are often very short, poses a significant informatics challenge. This Review summarizes the recent developments in transcriptome assembly approaches - reference-based, de novo and combined strategies-along with some perspectives on transcriptome assembly in the near future.
Wilks, Jessica C; Kitko, Ryan D; Cleeton, Sarah H; Lee, Grace E; Ugwu, Chinagozi S; Jones, Brian D; BonDurant, Sandra S; Slonczewski, Joan L
Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K(+)/H(+) antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.
Serkebaeva, Yulia M; Kim, Yongkyu; Liesack, Werner; Dedysh, Svetlana N
Northern peatlands play a key role in the global carbon and water budget, but the bacterial diversity in these ecosystems remains poorly described. Here, we compared the bacterial community composition in the surface (0-5 cm depth) and subsurface (45-50 cm) peat layers of an acidic (pH 4.0) Sphagnum-dominated wetland, using pyrosequencing of 16S rRNA genes. The denoised sequences (37,229 reads, average length ∼430 bp) were affiliated with 27 bacterial phyla and corresponded to 1,269 operational taxonomic units (OTUs) determined at 97% sequence identity. Abundant OTUs were affiliated with the Acidobacteria (35.5±2.4% and 39.2±1.2% of all classified sequences in surface and subsurface peat, respectively), Alphaproteobacteria (15.9±1.7% and 25.8±1.4%), Actinobacteria (9.5±2.0% and 10.7±0.5%), Verrucomicrobia (8.5±1.4% and 0.6±0.2%), Planctomycetes (5.8±0.4% and 9.7±0.6%), Deltaproteobacteria (7.1±0.4% and 4.4%±0.3%), and Gammaproteobacteria (6.6±0.4% and 2.1±0.1%). The taxonomic patterns of the abundant OTUs were uniform across all the subsamples taken from each peat layer. In contrast, the taxonomic patterns of rare OTUs were different from those of the abundant OTUs and varied greatly among subsamples, in both surface and subsurface peat. In addition to the bacterial taxa listed above, rare OTUs represented the following groups: Armatimonadetes, Bacteroidetes, Chlamydia, Chloroflexi, Cyanobacteria, Elusimicrobia, Fibrobacteres, Firmicutes, Gemmatimonadetes, Spirochaetes, AD3, WS1, WS4, WS5, WYO, OD1, OP3, BRC1, TM6, TM7, WPS-2, and FCPU426. OTU richness was notably higher in the surface layer (882 OTUs) than in the anoxic subsurface peat (483 OTUs), with only 96 OTUs common to both data sets. Most members of poorly studied phyla, such as the Acidobacteria, Verrucomicrobia, Planctomycetes and the candidate division TM6, showed a clear preference for growth in either oxic or anoxic conditions. Apparently, the bacterial communities in surface and
Full Text Available BACKGROUND: Distinguishing between individuals is critical to those conducting animal/plant breeding, food safety/quality research, diagnostic and clinical testing, and evolutionary biology studies. Classical genetic identification studies are based on marker polymorphisms, but polymorphism-based techniques are time and labor intensive and often cannot distinguish between closely related individuals. Illumina sequencing technologies provide the detailed sequence data required for rapid and efficient differentiation of related species, lines/cultivars, and individuals in a cost-effective manner. Here we describe the use of Illumina high-throughput exome sequencing, coupled with SNP mapping, as a rapid means of distinguishing between related cultivars of the lignocellulosic bioenergy crop giant miscanthus (Miscanthus × giganteus. We provide the first exome sequence database for Miscanthus species complete with Gene Ontology (GO functional annotations. RESULTS: A SNP comparative analysis of rhizome-derived cDNA sequences was successfully utilized to distinguish three Miscanthus × giganteus cultivars from each other and from other Miscanthus species. Moreover, the resulting phylogenetic tree generated from SNP frequency data parallels the known breeding history of the plants examined. Some of the giant miscanthus plants exhibit considerable sequence divergence. CONCLUSIONS: Here we describe an analysis of Miscanthus in which high-throughput exome sequencing was utilized to differentiate between closely related genotypes despite the current lack of a reference genome sequence. We functionally annotated the exome sequences and provide resources to support Miscanthus systems biology. In addition, we demonstrate the use of the commercial high-performance cloud computing to do computational GO annotation.
Bomberg, M.; Nyyssoenen, M.; Itaevaara, M. [VTT Technical Research Centre of Finland, Espoo (Finland)
Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.
Bomberg, M; Nyyssoenen, M; Itaevaara, M [VTT Technical Research Centre of Finland, Espoo (Finland)
Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.
Bomberg, M.; Nyyssoenen, M.; Itaevaara, M.
Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected
Fagerberg, Linn; Hallström, Björn M.; Schwenk, Jochen M.; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia
The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects. PMID:25546435
Full Text Available Abstract Background Escherichia coli has been extensively studied as a prokaryotic model organism whose whole genome was determined in 1997. However, it is difficult to identify all the gene products involved in diverse functions by using whole genome sequencesalone. The high-resolution transcriptome mapping using tiling arrays has proved effective to improve the annotation of transcript units and discover new transcripts of ncRNAs. While abundant tiling array data have been generated, the lack of appropriate visualization tools to accommodate and integrate multiple sources of data has emerged. Findings EcoBrowser is a web-based tool for visualizing genome annotations and transcriptome data of E. coli. Important tiling array data of E. coli from different experimental platforms are collected and processed for query. An AJAX based genome browser is embedded for visualization. Thus, genome annotations can be compared with transcript profiling and genome occupancy profiling from independent experiments, which will be helpful in discovering new transcripts including novel mRNAs and ncRNAs, generating a detailed description of the transcription unit architecture, further providing clues for investigation of prokaryotic transcriptional regulation that has proved to be far more complex than previously thought. Conclusions With the help of EcoBrowser, users can get a systemic view both from the vertical and parallel sides, as well as inspirations for the design of new experiments which will expand our understanding of the regulation mechanism.
Han, Zhengzhou; Ma, Xinye; Wei, Min; Zhao, Tong; Zhan, Ruoting; Chen, Weiwen
Chrysanthemum indicum L., an important ancestral species of the flowering plant chrysanthemum, can be used as medicine and for functional food development. Due to the lack of hereditary information for this species and the difficulty of germplasm identification, we herein provide new genetic insight from the perspective of intraspecific transcriptome comparison and present single sequence repeat (SSR) molecular marker recognition technology. Through the study of a diploid germplasm (DIWNT) and a tetraploid germplasm (DIWT), the following outcome were obtained. (1) A significant difference in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations for specific homologous genes was observed using the OrthoMCL method for the identification of homologous gene families between the two cytotypes. Ka/Ks analysis of common, single-copy homologous family members also revealed a greater difference among genes that experienced positive selection than among those experiencing positive selection. (2) Of more practical value, 2575 SSR markers were predicted and partly verified. We used TaxonGap as a visual tool to inspect genotype uniqueness and screen for high-performance molecular loci; we recommend four primers of 65 randomly selected primers with a combined identification success rate of 88.6% as priorities for further development of DNA fingerprinting of C. indicum germplasm. The SSR technology based on next-generation sequencing was proved to be successful in the identification of C. indicum germplasms. And the information on the intraspecfic genetic divergence generated by transcriptome comparison deepened the understanding of this complex species' nature.
Sun, Zhe; Wang, Ting; Deng, Ke; Wang, Xiao-Feng; Lafyatis, Robert; Ding, Ying; Hu, Ming; Chen, Wei
Single cell transcriptome sequencing (scRNA-Seq) has become a revolutionary tool to study cellular and molecular processes at single cell resolution. Among existing technologies, the recently developed droplet-based platform enables efficient parallel processing of thousands of single cells with direct counting of transcript copies using Unique Molecular Identifier (UMI). Despite the technology advances, statistical methods and computational tools are still lacking for analyzing droplet-based scRNA-Seq data. Particularly, model-based approaches for clustering large-scale single cell transcriptomic data are still under-explored. We developed DIMM-SC, a Dirichlet Mixture Model for clustering droplet-based Single Cell transcriptomic data. This approach explicitly models UMI count data from scRNA-Seq experiments and characterizes variations across different cell clusters via a Dirichlet mixture prior. We performed comprehensive simulations to evaluate DIMM-SC and compared it with existing clustering methods such as K-means, CellTree and Seurat. In addition, we analyzed public scRNA-Seq datasets with known cluster labels and in-house scRNA-Seq datasets from a study of systemic sclerosis with prior biological knowledge to benchmark and validate DIMM-SC. Both simulation studies and real data applications demonstrated that overall, DIMM-SC achieves substantially improved clustering accuracy and much lower clustering variability compared to other existing clustering methods. More importantly, as a model-based approach, DIMM-SC is able to quantify the clustering uncertainty for each single cell, facilitating rigorous statistical inference and biological interpretations, which are typically unavailable from existing clustering methods. DIMM-SC has been implemented in a user-friendly R package with a detailed tutorial available on www.pitt.edu/∼wec47/singlecell.html. firstname.lastname@example.org or email@example.com. Supplementary data are available at Bioinformatics online. © The Author
Zhang Michael Q
Full Text Available Abstract Background Although microarray-based studies have revealed global view of gene expression in cancer cells, we still have little knowledge about regulatory mechanisms underlying the transcriptome. Several computational methods applied to yeast data have recently succeeded in identifying expression modules, which is defined as co-expressed gene sets under common regulatory mechanisms. However, such module discovery methods are not applied cancer transcriptome data. Results In order to decode oncogenic regulatory programs in cancer cells, we developed a novel module discovery method termed EEM by extending a previously reported module discovery method, and applied it to breast cancer expression data. Starting from seed gene sets prepared based on cis-regulatory elements, ChIP-chip data, and gene locus information, EEM identified 10 principal expression modules in breast cancer based on their expression coherence. Moreover, EEM depicted their activity profiles, which predict regulatory programs in each subtypes of breast tumors. For example, our analysis revealed that the expression module regulated by the Polycomb repressive complex 2 (PRC2 is downregulated in triple negative breast cancers, suggesting similarity of transcriptional programs between stem cells and aggressive breast cancer cells. We also found that the activity of the PRC2 expression module is negatively correlated to the expression of EZH2, a component of PRC2 which belongs to the E2F expression module. E2F-driven EZH2 overexpression may be responsible for the repression of the PRC2 expression modules in triple negative tumors. Furthermore, our network analysis predicts regulatory circuits in breast cancer cells. Conclusion These results demonstrate that the gene set-based module discovery approach is a powerful tool to decode regulatory programs in cancer cells.
Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing
Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies
PacBio long-read sequencing technology is increasingly popular in genome sequence assembly and transcriptome cataloguing. Recently, a new-generation pig reference genome was assembled based on long reads from this technology. To finely annotate this genome assembly, transcriptomes of nine tissues fr...
Full Text Available Next generation sequencing (NGS technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric and mammal (ciliary classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic
Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios
Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.
Mendes, Filipa; Sieuwerts, Sander; de Hulster, Erik; Almering, Marinka J H; Luttik, Marijke A H; Pronk, Jack T; Smid, Eddy J; Bron, Peter A; Daran-Lapujade, Pascale
Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.
Martinovic-Weigelt, Dalma; Mehinto, Alvine C.; Ankley, Gerald T.; Denslow, Nancy D.; Barber, Larry B.; Lee, Kathy E.; King, Ryan J.; Schoenfuss, Heiko L.; Schroeder, Anthony L.; Villeneuve, Daniel L.
The present study investigated whether a combination of targeted analytical chemistry information with unsupervised, data-rich biological methodology (i.e., transcriptomics) could be utilized to evaluate relative contributions of wastewater treatment plant (WWTP) effluents to biological effects. The effects of WWTP effluents on fish exposed to ambient, receiving waters were studied at three locations with distinct WWTP and watershed characteristics. At each location, 4 d exposures of male fathead minnows to the WWTP effluent and upstream and downstream ambient waters were conducted. Transcriptomic analyses were performed on livers using 15 000 feature microarrays, followed by a canonical pathway and gene set enrichment analyses. Enrichment of gene sets indicative of teleost brain–pituitary–gonadal–hepatic (BPGH) axis function indicated that WWTPs serve as an important source of endocrine active chemicals (EACs) that affect the BPGH axis (e.g., cholesterol and steroid metabolism were altered). The results indicated that transcriptomics may even pinpoint pertinent adverse outcomes (i.e., liver vacuolization) and groups of chemicals that preselected chemical analytes may miss. Transcriptomic Effects-Based monitoring was capable of distinguishing sites, and it reflected chemical pollution gradients, thus holding promise for assessment of relative contributions of point sources to pollution and the efficacy of pollution remediation.
Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting.
Verbruggen, Erik; Kuramae, Eiko E; Hillekens, Remy; de Hollander, Mattias; Kiers, E Toby; Röling, Wilfred F M; Kowalchuk, George A; van der Heijden, Marcel G A
The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.
Balzer, Susanne; Malde, Ketil; Lanzén, Anders; Sharma, Animesh; Jonassen, Inge
The commercial launch of 454 pyrosequencing in 2005 was a milestone in genome sequencing in terms of performance and cost. Throughout the three available releases, average read lengths have increased to approximately 500 base pairs and are thus approaching read lengths obtained from traditional Sanger sequencing. Study design of sequencing projects would benefit from being able to simulate experiments. We explore 454 raw data to investigate its characteristics and derive empirical distributions for the flow values generated by pyrosequencing. Based on our findings, we implement Flowsim, a simulator that generates realistic pyrosequencing data files of arbitrary size from a given set of input DNA sequences. We finally use our simulator to examine the impact of sequence lengths on the results of concrete whole-genome assemblies, and we suggest its use in planning of sequencing projects, benchmarking of assembly methods and other fields. Flowsim is freely available under the General Public License from http://blog.malde.org/index.php/flowsim/.
Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L
A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.
Taouli, Bachir; Hoshida, Yujin; Chen, Xintong; Sun, Xiaochen; Kojima, Kensuke; Toffanin, Sara; Hirschfield, Hadassa; Kakite, Suguru; Tan, Poh Seng; Kihira, Shingo; Fiel, M.I.; Wagner, Mathilde; Llovet, Josep M.
In this preliminary study, we examined whether imaging-based phenotypes are associated with reported predictive gene signatures in hepatocellular carcinoma (HCC). Thirty-eight patients (M/F 30/8, mean age 61 years) who underwent pre-operative CT or MR imaging before surgery as well as transcriptome profiling were included in this IRB-approved single-centre retrospective study. Eleven qualitative and four quantitative imaging traits (size, enhancement ratios, wash-out ratio, tumour-to-liver contrast ratios) were assessed by three observers and were correlated with 13 previously reported HCC gene signatures using logistic regression analysis. Thirty-nine HCC tumours (mean size 5.7 ± 3.2 cm) were assessed. Significant positive associations were observed between certain imaging traits and gene signatures of aggressive HCC phenotype (G3-Boyault, Proliferation-Chiang profiles, CK19-Villanueva, S1/S2-Hoshida) with odds ratios ranging from 4.44-12.73 (P <0.045). Infiltrative pattern at imaging was significantly associated with signatures of microvascular invasion and aggressive phenotype. Significant but weak associations were also observed between each enhancement ratio and tumour-to-liver contrast ratios and certain gene expression profiles. This preliminary study demonstrates a correlation between phenotypic imaging traits with gene signatures of aggressive HCC, which warrants further prospective validation to establish imaging-based surrogate markers of molecular phenotypes in HCC. (orig.)
Taouli, Bachir [Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, NY (United States); Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, NY (United States); Icahn School of Medicine at Mount Sinai, Liver Cancer Program, Tisch Cancer Institute, New York, NY (United States); Hoshida, Yujin; Chen, Xintong; Sun, Xiaochen; Kojima, Kensuke; Toffanin, Sara; Hirschfield, Hadassa [Icahn School of Medicine at Mount Sinai, Liver Cancer Program, Tisch Cancer Institute, New York, NY (United States); Icahn School of Medicine at Mount Sinai, Division of Liver Diseases, Department of Medicine, New York, NY (United States); Kakite, Suguru [Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, NY (United States); Tottori University, Division of Radiology, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Yonago City (Japan); Tan, Poh Seng [Icahn School of Medicine at Mount Sinai, Liver Cancer Program, Tisch Cancer Institute, New York, NY (United States); Icahn School of Medicine at Mount Sinai, Division of Liver Diseases, Department of Medicine, New York, NY (United States); National University Health System, Division of Gastroenterology and Hepatology, University Medicine Cluster, Singapore (Singapore); Kihira, Shingo [Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, NY (United States); Fiel, M.I. [Icahn School of Medicine at Mount Sinai, Department of Pathology, New York, NY (United States); Wagner, Mathilde [Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, NY (United States); Sorbonne Universites, UPMC, Department of Radiology, Hopital Pitie-Salpetriere, Paris (France); Llovet, Josep M. [Icahn School of Medicine at Mount Sinai, Liver Cancer Program, Tisch Cancer Institute, New York, NY (United States); Icahn School of Medicine at Mount Sinai, Division of Liver Diseases, Department of Medicine, New York, NY (United States); Universitat de Barcelona, HCC Translational Research Laboratory, Barcelona-Clinic Liver Cancer Group Institut d' Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Hospital Clinic de Barcelona, Barcelona (Spain); Institucio Catalana de Recerca i Estudis Avancats, Barcelona (Spain)
In this preliminary study, we examined whether imaging-based phenotypes are associated with reported predictive gene signatures in hepatocellular carcinoma (HCC). Thirty-eight patients (M/F 30/8, mean age 61 years) who underwent pre-operative CT or MR imaging before surgery as well as transcriptome profiling were included in this IRB-approved single-centre retrospective study. Eleven qualitative and four quantitative imaging traits (size, enhancement ratios, wash-out ratio, tumour-to-liver contrast ratios) were assessed by three observers and were correlated with 13 previously reported HCC gene signatures using logistic regression analysis. Thirty-nine HCC tumours (mean size 5.7 ± 3.2 cm) were assessed. Significant positive associations were observed between certain imaging traits and gene signatures of aggressive HCC phenotype (G3-Boyault, Proliferation-Chiang profiles, CK19-Villanueva, S1/S2-Hoshida) with odds ratios ranging from 4.44-12.73 (P <0.045). Infiltrative pattern at imaging was significantly associated with signatures of microvascular invasion and aggressive phenotype. Significant but weak associations were also observed between each enhancement ratio and tumour-to-liver contrast ratios and certain gene expression profiles. This preliminary study demonstrates a correlation between phenotypic imaging traits with gene signatures of aggressive HCC, which warrants further prospective validation to establish imaging-based surrogate markers of molecular phenotypes in HCC. (orig.)
Rhee, Sun-Ju; Kwon, Taehyung; Seo, Minseok; Jang, Yoon Jeong; Sim, Tae Yong; Cho, Seoae; Han, Sang-Wook; Lee, Gung Pyo
The whole-genome sequence of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), a valuable horticultural crop worldwide, was released in 2013. Here, we compared a de novo-based approach (DBA) to a reference-based approach (RBA) using RNA-seq data, to aid in efforts to improve the annotation of the watermelon reference genome and to obtain biological insight into male-sterility in watermelon. We applied these techniques to available data from two watermelon lines: the male-sterile line DAH3615-MS and the male-fertile line DAH3615. Using DBA, we newly annotated 855 watermelon transcripts, and found gene functional clusters predicted to be related to stimulus responses, nucleic acid binding, transmembrane transport, homeostasis, and Golgi/vesicles. Among the DBA-annotated transcripts, 138 de novo-exclusive differentially-expressed genes (DEDEGs) related to male sterility were detected. Out of 33 randomly selected newly annotated transcripts and DEDEGs, 32 were validated by RT-qPCR. This study demonstrates the usefulness and reliability of the de novo transcriptome assembly in watermelon, and provides new insights for researchers exploring transcriptional blueprints with regard to the male sterility.
Russo Luis MS
Full Text Available Abstract Background Over the past few years, new massively parallel DNA sequencing technologies have emerged. These platforms generate massive amounts of data per run, greatly reducing the cost of DNA sequencing. However, these techniques also raise important computational difficulties mostly due to the huge volume of data produced, but also because of some of their specific characteristics such as read length and sequencing errors. Among the most critical problems is that of efficiently and accurately mapping reads to a reference genome in the context of re-sequencing projects. Results We present an efficient method for the local alignment of pyrosequencing reads produced by the GS FLX (454 system against a reference sequence. Our approach explores the characteristics of the data in these re-sequencing applications and uses state of the art indexing techniques combined with a flexible seed-based approach, leading to a fast and accurate algorithm which needs very little user parameterization. An evaluation performed using real and simulated data shows that our proposed method outperforms a number of mainstream tools on the quantity and quality of successful alignments, as well as on the execution time. Conclusions The proposed methodology was implemented in a software tool called TAPyR--Tool for the Alignment of Pyrosequencing Reads--which is publicly available from http://www.tapyr.net.
Harder, Christoffer Bugge
carefully selected waterworks (Article IV), where the bacterial metabolic diversity and its important for water purification was described. Building on this, the most important part of the thesis consists of two pyrosequencing analyses of protozoa with newly developed 18S primers. One specifically targets...... Cercozoa, a particularly abundant phylum of protozoa (Article III), on heath land that had been subjected to prolonged artificially induced drought in a Danish free-air climate-manipulation experiment (CLIMAITE). Article III showed that the testate cercozoan forms responded negatively to prolonged drought...
Sherman, Amir; Rubinstein, Mor; Eshed, Ravit; Benita, Miri; Ish-Shalom, Mazal; Sharabi-Schwager, Michal; Rozen, Ada; Saada, David; Cohen, Yuval; Ophir, Ron
Germplasm collections are an important source for plant breeding, especially in fruit trees which have a long duration of juvenile period. Thus, efforts have been made to study the diversity of fruit tree collections. Even though mango is an economically important crop, most of the studies on diversity in mango collections have been conducted with a small number of genetic markers. We describe a de novo transcriptome assembly from mango cultivar 'Keitt'. Variation discovery was performed using Illumina resequencing of 'Keitt' and 'Tommy Atkins' cultivars identified 332,016 single-nucleotide polymorphisms (SNPs) and 1903 simple-sequence repeats (SSRs). Most of the SSRs (70.1%) were of trinucleotide with the preponderance of motif (GGA/AAG)n and only 23.5% were di-nucleotide SSRs with the mostly of (AT/AT)n motif. Further investigation of the diversity in the Israeli mango collection was performed based on a subset of 293 SNPs. Those markers have divided the Israeli mango collection into two major groups: one group included mostly mango accessions from Southeast Asia (Malaysia, Thailand, Indonesia) and India and the other with mainly of Floridian and Israeli mango cultivars. The latter group was more polymorphic (FS=-0.1 on the average) and was more of an admixture than the former group. A slight population differentiation was detected (FST=0.03), suggesting that if the mango accessions of the western world apparently was originated from Southeast Asia, as has been previously suggested, the duration of cultivation was not long enough to develop a distinct genetic background. Whole-transcriptome reconstruction was used to significantly broaden the mango's genetic variation resources, i.e., SNPs and SSRs. The set of SNP markers described in this study is novel. A subset of SNPs was sampled to explore the Israeli mango collection and most of them were polymorphic in many mango accessions. Therefore, we believe that these SNPs will be valuable as they recapitulate and
Tai, Siew Leng; Daran-Lapujade, Pascale; Walsh, Michael C; Pronk, Jack T; Daran, Jean-Marc
Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state chemostats, grown at a fixed specific growth rate (0.03 h(-1)). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30 degrees C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12 degrees C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12 degrees C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the chemostat-based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.
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.
Full Text Available Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects, representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket, a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in
In Chapters 2-5 of this thesis, the applicability of transcriptome analysis to guide metabolic engineering strategies in P. chrysogenum is explored by investigating four cellular processes that are of potential relevance for industrial production of ?-lactam antibiotics: - Regulation of secondary
Cristian O Rohr
Full Text Available Fungi of the genus Pycnoporus are white-rot basidiomycetes widely studied because of their ability to synthesize high added-value compounds and enzymes of industrial interest. Here we report the sequencing, assembly and analysis of the transcriptome of Pycnoporus sanguineus BAFC 2126 grown at stationary phase, in media supplemented with copper sulfate. Using the 454 pyrosequencing platform we obtained a total of 226,336 reads (88,779,843 bases that were filtered and de novo assembled to generate a reference transcriptome of 7,303 transcripts. Putative functions were assigned for 4,732 transcripts by searching similarities of six-frame translated sequences against a customized protein database and by the presence of conserved protein domains. Through the analysis of translated sequences we identified transcripts encoding 178 putative carbohydrate active enzymes, including representatives of 15 families with roles in lignocellulose degradation. Furthermore, we found many transcripts encoding enzymes related to lignin hydrolysis and modification, including laccases and peroxidases, as well as GMC oxidoreductases, copper radical oxidases and other enzymes involved in the generation of extracellular hydrogen peroxide and iron homeostasis. Finally, we identified the transcripts encoding all of the enzymes involved in terpenoid backbone biosynthesis pathway, various terpene synthases related to the biosynthesis of sesquiterpenoids and triterpenoids precursors, and also cytochrome P450 monooxygenases, glutathione S-transferases and epoxide hydrolases with potential functions in the biodegradation of xenobiotics and the enantioselective biosynthesis of biologically active drugs. To our knowledge this is the first report of a transcriptome of genus Pycnoporus and a resource for future molecular studies in P. sanguineus.
Full Text Available BACKGROUND: Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. RESULTS: Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs that provide a resource for gene function studies. CONCLUSION: Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.
Hahn Daniel A
Full Text Available Abstract Background Flesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription. Results We obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (ES. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs. Conclusion Our data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic
Full Text Available BACKGROUND: Sweet potato (Ipomoea batatas L. [Lam.] ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end (PE RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (≥ 100 bp, which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. CONCLUSIONS/SIGNIFICANCE: The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots
Joo Hyun Lee
Full Text Available Rice stripe virus (RSV, the type member of the genus Tenuivirus, transmits by the feeding behavior of small brown planthopper (SBPH, Laodelphax striatellus. To investigate the interactions between the virus and vector insect, total RNA was extracted from RSV-viruliferous SBPH (RVLS and non-viruliferous SBPH (NVLS adults to construct expressed sequence tag databases for comparative transcriptome analysis. Over 30 million bases were sequenced by 454 pyrosequencing to construct 1,538 and 953 of isotigs from the mRNA of RVLS and NVLS, respectively. The gene ontology (GO analysis demonstrated that both libraries have similar GO structures, however, the gene expression pattern analysis revealed that 17.8% and 16.8% of isotigs were up- and down-regulated significantly in the RVLS, respectively. These RSV-dependently regulated genes possibly have important roles in the physiology of SBPH, transmission of RSV, and RSV and SBPH interaction.
Ibáñez, Clara; Pérez-Torrado, Roberto; Morard, Miguel; Toft, Christina; Barrio, Eladio; Querol, Amparo
Transcriptome analyses play a central role in unraveling the complexity of gene expression regulation in Saccharomyces cerevisiae. This species, one of the most important microorganisms for humans given its industrial applications, shows an astonishing degree of genetic and phenotypic variability among different strains adapted to specific environments. In order to gain novel insights into the Saccharomyces cerevisiae biology of strains adapted to different fermentative environments, we analyzed the whole transcriptome of three strains isolated from wine, flor wine or mezcal fermentations. An RNA-seq transcriptome comparison of the different yeasts in the samples obtained during synthetic must fermentation highlighted the differences observed in the genes that encode mannoproteins, and in those involved in aroma, sugar transport, glycerol and alcohol metabolism, which are important under alcoholic fermentation conditions. These differences were also observed in the physiology of the strains after mannoprotein and aroma determinations. This study offers an essential foundation for understanding how gene expression variations contribute to the fermentation differences of the strains adapted to unequal fermentative environments. Such knowledge is crucial to make improvements in fermentation processes and to define targets for the genetic improvement or selection of wine yeasts. Copyright © 2017 Elsevier B.V. All rights reserved.
Shen, Di; Wang, Haiping; Wu, Qingjun; Lu, Peng; Qiu, Yang; Song, Jiangping; Zhang, Youjun; Li, Xixiang
Background The diamondback moth (DBM, Plutella xylostella) is a crucifer-specific pest that causes significant crop losses worldwide. Barbarea vulgaris (Brassicaceae) can resist DBM and other herbivorous insects by producing feeding-deterrent triterpenoid saponins. Plant breeders have long aimed to transfer this insect resistance to other crops. However, a lack of knowledge on the biosynthetic pathways and regulatory networks of these insecticidal saponins has hindered their practical application. A pyrosequencing-based transcriptome analysis of B. vulgaris during DBM larval feeding was performed to identify genes and gene networks responsible for saponin biosynthesis and its regulation at the genome level. Principal Findings Approximately 1.22, 1.19, 1.16, 1.23, 1.16, 1.20, and 2.39 giga base pairs of clean nucleotides were generated from B. vulgaris transcriptomes sampled 1, 4, 8, 12, 24, and 48 h after onset of P. xylostella feeding and from non-inoculated controls, respectively. De novo assembly using all data of the seven transcriptomes generated 39,531 unigenes. A total of 37,780 (95.57%) unigenes were annotated, 14,399 of which were assigned to one or more gene ontology terms and 19,620 of which were assigned to 126 known pathways. Expression profiles revealed 2,016–4,685 up-regulated and 557–5188 down-regulated transcripts. Secondary metabolic pathways, such as those of terpenoids, glucosinolates, and phenylpropanoids, and its related regulators were elevated. Candidate genes for the triterpene saponin pathway were found in the transcriptome. Orthological analysis of the transcriptome with four other crucifer transcriptomes identified 592 B. vulgaris-specific gene families with a P-value cutoff of 1e−5. Conclusion This study presents the first comprehensive transcriptome analysis of B. vulgaris subjected to a series of DBM feedings. The biosynthetic and regulatory pathways of triterpenoid saponins and other DBM deterrent metabolites in this plant were
Full Text Available Beetles (Coleoptera are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp length (approximately 700 million bp sequence information with about 30× transcriptome coverage confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity
Huang, Jianzi; Lu, Xiang; Yan, Hao; Chen, Shouyi; Zhang, Wanke; Huang, Rongfeng; Zheng, Yizhi
Semi-mangroves form a group of transitional species between glycophytes and halophytes, and hold unique potential for learning molecular mechanisms underlying plant salt tolerance. Millettia pinnata is a semi-mangrove plant that can survive a wide range of saline conditions in the absence of specialized morphological and physiological traits. By employing the Illumina sequencing platform, we generated ~192 million short reads from four cDNA libraries of M. pinnata and processed them into 108,598 unisequences with a high depth of coverage. The mean length and total length of these unisequences were 606 bp and 65.8 Mb, respectively. A total of 54,596 (50.3%) unisequences were assigned Nr annotations. Functional classification revealed the involvement of unisequences in various biological processes related to metabolism and environmental adaptation. We identified 23,815 candidate salt-responsive genes with significantly differential expression under seawater and freshwater treatments. Based on the reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses, we verified the changes in expression levels for a number of candidate genes. The functional enrichment analyses for the candidate genes showed tissue-specific patterns of transcriptome remodelling upon salt stress in the roots and the leaves. The transcriptome of M. pinnata will provide valuable gene resources for future application in crop improvement. In addition, this study sets a good example for large-scale identification of salt-responsive genes in non-model organisms using the sequencing-based approach.
Der Haupthistokompatibilitätskomplex ist durch seine biologische Funktion eine für die Diagnostik und Forschung äußerst wichtige Region im humanen Genom. Die Untersuchung von HLA-Genorten stellt ein wichtiges Instrument in der molekulargenetischen Praxis dar. Die Pyrosequencing-Technik ist gut geeignet, um kurze DNA-Abschnitte mit weitgehend bekannter Sequenz schnell und effizient zu untersuchen. Ziel dieser Arbeit war die Entwicklung von Pyrosequencing-basierten Methoden zur HLA-Typisierung....
Background: The barnacle Balanus amphitrite is a globally distributed biofouler and a model species in intertidal ecology and larval settlement studies. However, a lack of genomic information has hindered the comprehensive elucidation of the molecular mechanisms coordinating its larval settlement. The pyrosequencing-based transcriptomic approach is thought to be useful to identify key molecular changes during larval settlement. Methodology and Principal Findings: Using 454 pyrosequencing, we collected totally 630,845 reads including 215,308 from the larval stages and 415,537 from the adults; 23,451 contigs were generated while 77,785 remained as singletons. We annotated 31,720 of the 92,322 predicted open reading frames, which matched hits in the NCBI NR database, and identified 7,954 putative genes that were differentially expressed between the larval and adult stages. Of these, several genes were further characterized with quantitative real-time PCR and in situ hybridization, revealing some key findings: 1) vitellogenin was uniquely expressed in late nauplius stage, suggesting it may be an energy source for the subsequent non-feeding cyprid stage; 2) the locations of mannose receptors suggested they may be involved in the sensory system of cyprids; 3) 20 kDa-cement protein homologues were expressed in the cyprid cement gland and probably function during attachment; and 4) receptor tyrosine kinases were expressed higher in cyprid stage and may be involved in signal perception during larval settlement. Conclusions: Our results provide not only the basis of several new hypotheses about gene functions during larval settlement, but also the availability of this large transcriptome dataset in B. amphitrite for further exploration of larval settlement and developmental pathways in this important marine species. © 2011 Chen et al.
Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B
Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map. PMID:25060816
Full Text Available Most previous studies have focused on analgesic and anti-cancer activities for the conotoxins identified from piscivorous and molluscivorous cone snails, but little attention has been devoted to insecticidal activity of conotoxins from the dominant vermivorous species. As a representative vermivorous cone snail, the Chinese tubular cone snail (Conus betulinus is the dominant Conus species inhabiting the South China Sea. We sequenced related venom transcriptomes from C. betulinus using both the next-generation sequencing and traditional Sanger sequencing technologies, and a comprehensive library of 215 conotoxin transcripts was constructed. In our current study, six conotoxins with potential insecticidal activity were screened out from our conotoxin library by homologous search with a reported positive control (alpha-conotoxin ImI from C. imperialis as the query. Subsequently, these conotoxins were synthesized by chemical solid-phase and oxidative folding for further insecticidal activity validation, such as MTT assay, insect bioassay and homology modeling. The final results proved insecticidal activities of our achieved six conotoxins from the transcriptome-based dataset. Interestingly, two of them presented a lot of high insecticidal activity, which supports their usefulness for a trial as insecticides in field investigations. In summary, our present work provides a good example for high throughput development of biological insecticides on basis of the accumulated genomic resources.
Ismail, Ku Syahidah Ku; Sakamoto, Takatoshi; Hasunuma, Tomohisa; Kondo, Akihiko
Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.
Hou, X-L; Cao, Q-Y; Jia, H-Y; Chen, Z
Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long ( approximately1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 10(4) CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.
Kunin, Victor; Engelbrektson, Anna; Ochman, Howard; Hugenholtz, Philip
Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the 'rare biosphere', is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere.
Yang, Minglei; Wu, Ying; Jin, Shan; Hou, Jinyan; Mao, Yingji; Liu, Wenbo; Shen, Yangcheng; Wu, Lifang
Sapium sebiferum (Linn.) Roxb. (Chinese Tallow Tree) is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.
Tanase, Koji; Nishitani, Chikako; Hirakawa, Hideki; Isobe, Sachiko; Tabata, Satoshi; Ohmiya, Akemi; Onozaki, Takashi
Carnation (Dianthus caryophyllus L.), in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST) database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology. We constructed a normalized cDNA library and a 3'-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380) of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO) and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs) in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway. We present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.
Full Text Available Abstract Background Carnation (Dianthus caryophyllus L., in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology. Results We constructed a normalized cDNA library and a 3’-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380 of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway. Conclusions We present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.
Ciraulo, Maristela Boaceff; Santos, Daiene Souza; Rodrigues, Ana Claudia de Freitas Oliveira; de Oliveira, Marcus Vinícius; Rodrigues, Tiago; de Oliveira, Regina Costa; Nunes, Luiz R
Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierce's disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.
Machado, Daniel; Herrgard, Markus
of these methods has not been critically evaluated and compared. This work presents a survey of recently published methods that use transcript levels to try to improve metabolic flux predictions either by generating flux distributions or by creating context-specific models. A subset of these methods...... is then systematically evaluated using published data from three different case studies in E. coli and S. cerevisiae. The flux predictions made by different methods using transcriptomic data are compared against experimentally determined extracellular and intracellular fluxes (from 13C-labeling data). The sensitivity...... of the results to method-specific parameters is also evaluated, as well as their robustness to noise in the data. The results show that none of the methods outperforms the others for all cases. Also, it is observed that for many conditions, the predictions obtained by simple flux balance analysis using growth...
Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the
Full Text Available Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs, 41,690 into 58 gene ontology (GO terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.
Non-model organisms represent the majority of life forms in our planet. However, the lack of genetic information hinders us to understand the unique biological phenomena in non-model organisms at the molecular level. In this study, we applied a tandem transcriptome and proteome profiling on a non-model marine fouling organism, Bugula neritina. Using a 454 pyrosequencing platform with the updated titanium reagents, we generated a total of 48M bp transcriptome data consisting of 131 450 high-quality reads. Of these, 122 650 reads (93%) were assembled to produce 6392 contigs with an average length of 538 bases and the remaining 8800 reads were singletons. Of the total 15 192 unigenes, 13 863 ORFs were predicated, of which 6917 were functionally annotated based on gene ontology and eukaryotic orthologous groups. Subsequent proteome analysis identified and quantified 882 proteins from B. neritina. These results would provide fundamental and important information for the subsequent studies of molecular mechanism in larval biology, development, antifouling research. Furthermore, we demonstrated, for the first time, the combined use of two high-throughput technologies as a powerful approach for accelerating the studies of non-model but otherwise important species. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Schooten, Frederik J. van; Piersma, Aldert H.
The embryonic stem cell test (EST) predicts developmental toxicity based on the inhibition of cardiomyocyte differentiation of embryonic stem cells (ESC). The subjective endpoint, the long culture duration together with the undefined applicability domain and related predictivity need further improvement to facilitate implementation of the EST into regulatory strategies. These aspects may be improved by studying gene expression changes in the ESC differentiation cultures and their modulation by compound exposure using transcriptomics. Here, we tested the developmental toxicants monobutyl phthalate and 6-aminonicotinamide. ESC were allowed to differentiated, and cardiomyocyte differentiation was assessed after 10 days of culture. RNA of solvent controls was collected after 0, 24, 48, 72 and 96 h of exposure, and RNA of developmental-toxicant-exposed cultures was collected after 24 and 96 h. Samples were hybridized to DNA microarrays, and 1355 genes were found differentially expressed among the unexposed experimental groups. These regulated genes were involved in differentiation-related processes, and Principal Component Analysis (PCA) based on these genes showed that the unexposed experimental groups appeared in chronological order in the PCA, which can therefore be regarded as a continuous representation of the differentiation track. The developmental-toxicant-exposed cultures appeared to deviate significantly from this differentiation track, which confirms the compound-modulating effects on the differentiation process. The incorporation of transcriptomics in the EST is expected to provide a more informative and improved endpoint in the EST as compared with morphology, allowing early detection of differentiation modulation. Furthermore, this approach may improve the definition of the applicability domain and predictivity of the EST.
Peng, Hua; He, Xiujing; Gao, Jian; Ma, Haixia; Zhang, Zhiming; Shen, Yaou; Pan, Guangtang; Lin, Haijian
The heavy metal cadmium (Cd), acts as a widespread environmental contaminant, which has shown to adversely affect human health, food safety and ecosystem safety in recent years. However, research on how plant respond to various kinds of heavy metal stress is scarcely reported, especially for understanding of complex molecular regulatory mechanisms and elucidating the gene networks of plant respond to Cd stress. Here, transcriptomic changes during Mo17 and B73 seedlings development responsive to Cd pollution were investigated and comparative RNAseq-based approach in both genotypes were performed. 115 differential expression genes (DEGs) with significant alteration in expression were found co-modulated in both genotypes during the maize seedling development; of those, most of DGEs were found comprised of stress and defense responses proteins, transporters, as well as transcription factors, such as thaumatin-like protein, ZmOPR2 and ZmOPR5. More interestingly, genotype-specific transcriptional factors changes induced by Cd stress were found contributed to the regulatory mechanism of Cd sensitivity in both different genotypes. Moreover, 12 co-expression modules associated with specific biological processes or pathways (M1 to M12) were identified by consensus co-expression network. These results will expand our understanding of complex molecular mechanism of response and defense to Cd exposure in maize seedling roots. - Highlights: • Transcriptomic changes responsive to Cd pollution using comparative RNAseq-based approach. • 115 differential expression genes (DEGs) were found co-modulated in both genotypes. • Most of DGEs belong to stress and defense responses proteins, transporters, transcription factors. • 12 co-expression modules associated with specific biological processes or pathways. • Genotype-specific transcriptional factors changes induced by Cd stress were found
Full Text Available With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA. Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.
Peng, Hua [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130 (China); Sichuan Tourism College, Chengdu, 610000, Sichuan (China); He, Xiujing [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130 (China); Gao, Jian [Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing (China); Ma, Haixia; Zhang, Zhiming; Shen, Yaou [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130 (China); Pan, Guangtang, E-mail: firstname.lastname@example.org [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130 (China); Lin, Haijian, E-mail: email@example.com [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130 (China)
The heavy metal cadmium (Cd), acts as a widespread environmental contaminant, which has shown to adversely affect human health, food safety and ecosystem safety in recent years. However, research on how plant respond to various kinds of heavy metal stress is scarcely reported, especially for understanding of complex molecular regulatory mechanisms and elucidating the gene networks of plant respond to Cd stress. Here, transcriptomic changes during Mo17 and B73 seedlings development responsive to Cd pollution were investigated and comparative RNAseq-based approach in both genotypes were performed. 115 differential expression genes (DEGs) with significant alteration in expression were found co-modulated in both genotypes during the maize seedling development; of those, most of DGEs were found comprised of stress and defense responses proteins, transporters, as well as transcription factors, such as thaumatin-like protein, ZmOPR2 and ZmOPR5. More interestingly, genotype-specific transcriptional factors changes induced by Cd stress were found contributed to the regulatory mechanism of Cd sensitivity in both different genotypes. Moreover, 12 co-expression modules associated with specific biological processes or pathways (M1 to M12) were identified by consensus co-expression network. These results will expand our understanding of complex molecular mechanism of response and defense to Cd exposure in maize seedling roots. - Highlights: • Transcriptomic changes responsive to Cd pollution using comparative RNAseq-based approach. • 115 differential expression genes (DEGs) were found co-modulated in both genotypes. • Most of DGEs belong to stress and defense responses proteins, transporters, transcription factors. • 12 co-expression modules associated with specific biological processes or pathways. • Genotype-specific transcriptional factors changes induced by Cd stress were found.
Hand, Daniel; Wallis, Corrin; Colyer, Alison; Penn, Charles W.
Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5′ region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a “core microbiota”. Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs) at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs. PMID:23382835
Full Text Available Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5' region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a "core microbiota". Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs.
Park, O-J; Yi, H; Jeon, J H; Kang, S-S; Koo, K-T; Kum, K-Y; Chun, J; Yun, C-H; Han, S H
Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases. © International & American Associations for Dental Research 2015.
Full Text Available Fungi are important soil components as both decomposers and plant symbionts and play a major role in ecological and biogeochemical processes. However, little is known about the richness and structure of fungal communities. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. We therefore used 454 pyrosequencing to investigate the fungal communities in the rhizosphere of Xinjiang jujube. We obtained no less than 40,488 internal transcribed spacer (ITS rDNA reads, the number of each sample was 6943, 6647, 6584, 6550, 6860, and 6904, and we used bioinformatics and multivariate statistics to analyze the results. The index of diversity showed greater richness in the rhizosphere fungal community of a 3-year-old jujube than in that of an 8-year-old jujube. Most operational taxonomic units belonged to Ascomycota, and taxonomic analyses identified Hypocreales as the dominant fungal order. Our results demonstrated that the fungal orders are present in different proportions in different sampling areas. Redundancy analysis (RDA revealed a significant correlation between soil properties and the abundance of fungal phyla. Our results indicated lower fungal diversity in the rhizosphere of Xinjiang jujube than that reported in other studies, and we hope our findings provide a reference for future research.
Shcherbik, Svetlana V; Pearce, Nicholas C; Levine, Marnie L; Klimov, Alexander I; Villanueva, Julie M; Bousse, Tatiana L
Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.
Svetlana V Shcherbik
Full Text Available BACKGROUND: Live attenuated influenza vaccine viruses (LAIVs can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV and a seasonal wild-type (wt virus. The vaccine candidates contain hemagglutinin (HA and neuraminidase (NA genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. METHODOLOGY/PRINCIPAL FINDINGS: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2 (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012 or influenza A (H7N9 (A/Anhui/1/2013 wt viruses with the MDV A/Leningrad/134/17/57(H2N2. Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.
Full Text Available Sapium sebiferum (Linn. Roxb. (Chinese Tallow Tree is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA and triacylglycerol (TAG biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.
Hong, Bo-Young; Lee, Tae-Kwon; Lim, Sang-Min; Chang, Seok Woo; Park, Joonhong; Han, Seung Hyun; Zhu, Qiang; Safavi, Kamran E; Fouad, Ashraf F; Kum, Kee Yeon
The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.
Moreira, Nathalia R; Cardoso, Christiane; Dias, Renata O; Ferreira, Clelia; Terra, Walter R
Physiological data showed that T. molitor midgut is buffered at pH 5.6 at the two anterior thirds and at 7.9 at the posterior third. Furthermore, water is absorbed and secreted at the anterior and posterior midgut, respectively, driving a midgut counter flux of fluid. To look for the molecular mechanisms underlying these phenomena and nutrient absorption as well, a transcriptomic approach was used. For this, 11 types of transporters were chosen from the midgut transcriptome obtained by pyrosequencing (Roche 454). After annotation with the aid of databanks and manual curation, the sequences were validated by RT-PCR. The expression level of each gene at anterior, middle and posterior midgut and carcass (larva less midgut) was evaluated by RNA-seq taking into account reference sequences based on 454 contigs and reads obtained by Illumina sequencing. The data showed that sugar and amino acid uniporters and symporters are expressed along the whole midgut. In the anterior midgut are found transporters for NH 3 and NH 4 + that with a chloride channel may be responsible for acidifying the lumen. At the posterior midgut, bicarbonate-Cl - antiporter with bicarbonate supplied by carbonic anhydrase may alkalinize the lumen. Water absorption caused mainly by an anterior Na + -K + -2Cl - symporter and water secretion caused by a posterior K + -Cl - may drive the midgut counter flux. Transporters that complement the action of those described were also found. Copyright © 2017 Elsevier Ltd. All rights reserved.
Lee, Jungeun; Noh, Eun Kyeung; Choi, Hyung-Seok; Shin, Seung Chul; Park, Hyun; Lee, Hyoungseok
Antarctic hairgrass (Deschampsia antarctica Desv.) is the only natural grass species in the maritime Antarctic. It has been studied as an extremophile that has successfully adapted to marginal land with the harshest environment for terrestrial plants. However, limited genetic research has focused on this species due to the lack of genomic resources. Here, we present the first de novo assembly of its transcriptome by massive parallel sequencing and its expression profile using D. antarctica grown under various stress conditions. Total sequence reads generated by pyrosequencing were assembled into 60,765 unigenes (28,177 contigs and 32,588 singletons). A total of 29,173 unique protein-coding genes were identified based on sequence similarities to known proteins. The combined results from all three stress conditions indicated differential expression of 3,110 genes. Quantitative reverse transcription polymerase chain reaction showed that several well-known stress-responsive genes encoding late embryogenesis abundant protein, dehydrin 1, and ice recrystallization inhibition protein were induced dramatically and that genes encoding U-box-domain-containing protein, electron transfer flavoprotein-ubiquinone, and F-box-containing protein were induced by abiotic stressors in a manner conserved with other plant species. We identified more than 2,000 simple sequence repeats that can be developed as functional molecular markers. This dataset is the most comprehensive transcriptome resource currently available for D. antarctica and is therefore expected to be an important foundation for future genetic studies of grasses and extremophiles.
Li, Ke; Cheng, Jun; Lu, Hongxiang; Yang, Weijuan; Zhou, Junhu; Cen, Kefa
To elucidate the mechanism underlying the enhanced growth rate in the Haematococcus pluvialis mutated with 60 Co-γ rays and domesticated with 15% CO 2 , transcriptome sequencing was conducted to clarify the carbon metabolic pathways of mutant cells. The CO 2 fixation rate of mutant cells increased to 2.57gL -1 d -1 under 15% CO 2 due to the enhanced photosynthesis, carbon fixation, glycolysis pathways. The upregulation of PetH, ATPF0A and PetJ related to photosynthetic electron transport, ATP synthase and NADPH generation promoted the photosynthesis. The upregulation of genes related to Calvin cycle and ppdK promoted carbon fixation in both C3 and C4 photosynthetic pathways. The reallocation of carbon was also enhanced under 15% CO 2 . The 19-, 14- and 3.5-fold upregulation of FBA, TPI and PK genes, respectively, remarkably promoted the glycolysis pathways. This accelerated the conversion of photosynthetic carbon to pyruvate, which was an essential precursor for astaxanthin and lipids biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.
Full Text Available Fish venom remains a virtually untapped resource. There are so few fish toxin sequences for reference, which increases the difficulty to study toxins from venomous fish and to develop efficient and fast methods to dig out toxin genes or proteins. Here, we utilized Chinese yellow catfish (Pelteobagrus fulvidraco as our research object, since it is a representative species in Siluriformes with its venom glands embedded in the pectoral and dorsal fins. In this study, we set up an in-house toxin database and a novel toxin-discovering protocol to dig out precise toxin genes by combination of transcriptomic and proteomic sequencing. Finally, we obtained 15 putative toxin proteins distributed in five groups, namely Veficolin, Ink toxin, Adamalysin, Za2G and CRISP toxin. It seems that we have developed a novel bioinformatics method, through which we could identify toxin proteins with high confidence. Meanwhile, these toxins can also be useful for comparative studies in other fish and development of potential drugs.
Full Text Available Intramuscular fat (IMF content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs between the higher (H and lower (L IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG enrichment analysis indicated that the DEG perilipin-1 (PLIN1 was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG level and lipid droplet (LD size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation.
Li, Bojiang; Weng, Qiannan; Dong, Chao; Zhang, Zengkai; Li, Rongyang; Liu, Jingge; Jiang, Aiwen; Li, Qifa; Jia, Chao; Wu, Wangjun; Liu, Honglin
Intramuscular fat (IMF) content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs) between the higher (H) and lower (L) IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEG perilipin-1 ( PLIN1 ) was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR) signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG) level and lipid droplet (LD) size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation.
Yuan, Can; Peng, Fang; Yang, Ze-Mao; Zhong, Wen-Juan; Mou, Fang-Sheng; Gong, Yi-Yun; Ji, Pei-Cheng; Pu, De-Qiang; Huang, Hai-Yan; Yang, Xiao; Zhang, Chao
Ligusticum chuanxiong is a well-known traditional Chinese medicine plant. The study on its molecular markers development and germplasm resources is very important. In this study, we obtained 24 422 unigenes by assembling transcriptome sequencing reads of L. chuanxiong root. EST-SSR was detected and 4 073 SSR loci were identified. EST-SSR distribution and characteristic analysis results showed that the mono-nucleotide repeats were the main repeat types, accounting for 41.0%. In addition, the sequences containing SSR were functionally annotated in Gene Ontology (GO) and KEGG pathway and were assigned to 49 GO categories, 242 KEGG pathways, among them 2 201 sequences were annotated against Nr database. By validating 235 EST-SSRs,74 primer pairs were ultimately proved to have high quality amplification. Subsequently, genetic diversity analysis, UPGMA cluster analysis, PCoA analysis and population structure analysis of 34 L. chuanxiong germplasm resources were carried out with 74 primer pairs. In both UPGMA tree and PCoA results, L. chuanxiong resources were clustered into two groups, which are believed to be partial related to their geographical distribution. In this study, EST-SSRs in L. chuanxiong was firstly identified, and newly developed molecular markers would contribute significantly to further genetic diversity study, the purity detection, gene mapping, and molecular breeding. Copyright© by the Chinese Pharmaceutical Association.
Maristela Boaceff Ciraulo
Full Text Available Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC and grapevine Pierce's disease (PD. Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW, the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.
Wong, Kim; Fernández Navarro, José; Bergenstråhle, Ludvig; Ståhl, Patrik L; Lundeberg, Joakim
Spatial transcriptomics (ST) is a method which combines high resolution tissue imaging with high throughput transcriptome sequencing data. This data must be aligned with the images for correct visualisation, a process that involves several manual steps. Here we present ST Spot Detector, a web tool that automates and facilitates this alignment through a user friendly interface. Open source under the MIT license, available from https://github.com/SpatialTranscriptomicsResearch/st_spot_detector. firstname.lastname@example.org. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: email@example.com
Robinson, Leslie C; Santagata, Sandro; Hankinson, Todd C
The recent genomic and transcriptomic characterization of human craniopharyngiomas has provided important insights into the pathogenesis of these tumors and supports that these tumor types are distinct entities. Critically, the insights provided by these data offer the potential for the introduction of novel therapies and surgical treatment paradigms for these tumors, which are associated with high morbidity rates and morbid conditions. Mutations in the CTNNB1 gene are primary drivers of adamantinomatous craniopharyngioma (ACP) and lead to the accumulation of β-catenin protein in a subset of the nuclei within the neoplastic epithelium of these tumors. Dysregulation of epidermal growth factor receptor (EGFR) and of sonic hedgehog (SHH) signaling in ACP suggest that paracrine oncogenic mechanisms may underlie ACP growth and implicate these signaling pathways as potential targets for therapeutic intervention using directed therapies. Recent work shows that ACP cells have primary cilia, further supporting the potential importance of SHH signaling in the pathogenesis of these tumors. While further preclinical data are needed, directed therapies could defer, or replace, the need for radiation therapy and/or allow for less aggressive surgical interventions. Furthermore, the prospect for reliable control of cystic disease without the need for surgery now exists. Studies of papillary craniopharyngioma (PCP) are more clinically advanced than those for ACP. The vast majority of PCPs harbor the BRAF v600e mutation. There are now 2 reports of patients with PCP that had dramatic therapeutic responses to targeted agents. Ongoing clinical and research studies promise to not only advance our understanding of these challenging tumors but to offer new approaches for patient management.
Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere
BACKGROUND: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis
Panitz, Frank; Nielsen, Rasmus Ory; Andersen, Pernille K
abundance. Our objective was to investigate animals previously not affected by lung disease and those that had been affected. To this end lung tissue samples were collected, separately pooled and tagged before sequencing using the Roche/454 FLX platform. We sequenced about one million reads that were...... clustered and mapped to the current pig genome reference sequence. Identified genes or clusters were annotated for functional classes and mined for singe nucleotide polymorphisms. In addition, we compared gene expression between sample groups in order to investigate possible changes in the lung...
Chen, Jie; Pei, Zhihua; Dai, Lunjin; Wang, Bo; Liu, Lijun; An, Xia; Peng, Dingxiang
Ramie (Boehmeria nivea L.), popularly known as "China grass", is one of the oldest crops in China and the second most important fiber crop in terms of area sown. Ramie fiber, extracted from the plant bast, is important in the textile industry. However, the molecular mechanism of ramie fiber development remains unknown. A whole sequencing run was performed on the 454 GS FLX + platform using four separately pooled parts of ramie bast. This generated 1,030,057 reads with an average length of 457 bp. Among the 58,369 unigenes (13,386 contigs and 44,983 isotigs) that were generated through de novo assembly, 780 were differentially expressed. As a result, 13 genes that belong to the cellulose synthase gene family (four), the expansin gene family (three) and the xyloglucan endotransglucosylase/hydrolase (XTH) gene family (six) were up-regulated in the top part of the bast, which was in contrast to the other three parts. The identification of these 13 concurrently up-regulated unigenes indicated that the early stage (represented by the top part of the bast) might be important for the molecular regulation of ramie fiber development. Further analysis indicated that four of the 13 unigenes from the expansin (two) and XTH (two) families shared a coincident expression pattern during the whole growth season, which implied they were more relevant to ramie fiber development (fiber quality, etc.) during the different seasons than the other genes. To the best of our knowledge, this study is the first to characterize ramie fiber development at different developmental stages. The identified transcripts will improve our understanding of the molecular mechanisms involved in ramie fiber development. Moreover, the identified differentially expressed genes will accelerate molecular research on ramie fiber growth and the breeding of ramie with better fiber yields and quality.
Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre
HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.
Full Text Available Abstract Background We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture. Results We compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website. Conclusions This comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management.
Background We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture. Results We compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website. Conclusions This comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management. PMID:22747707
Fonseca, Fernando Campos de Assis; Firmino, Alexandre Augusto Pereira; de Macedo, Leonardo Lima Pepino; Coelho, Roberta Ramos; de Souza Júnior, José Dijair Antonino; de Sousa Júnior, José Dijair Antonino; Silva-Junior, Orzenil Bonfim; Togawa, Roberto Coiti; Pappas, Georgios Joannis; de Góis, Luiz Avelar Brandão; da Silva, Maria Cristina Mattar; Grossi-de-Sá, Maria Fátima
Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.
Fernando Campos de Assis Fonseca
Full Text Available Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus, a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB transcriptome, a number of aminopeptidase N (APN cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.
Sheveleva, Anna; Kudryavtseva, Anna; Speranskaya, Anna; Belenikin, Maxim; Melnikova, Natalia; Chirkov, Sergei
The near-complete (99.7 %) genome sequence of a novel Russian Plum pox virus (PPV) isolate Pk, belonging to the strain Winona (W), has been determined by 454 pyrosequencing with the exception of the thirty-one 5'-terminal nucleotides. This region was amplified using 5'RACE kit and sequenced by the Sanger method. Genomic RNA released from immunocaptured PPV particles was employed for generation of cDNA library using TransPlex Whole transcriptome amplification kit (WTA2, Sigma-Aldrich). The entire Pk genome has identity level of 92.8-94.5 % when compared to the complete nucleotide sequences of other PPV-W isolates (W3174, LV-141pl, LV-145bt, and UKR 44189), confirming a high degree of variability within the PPV-W strain. The isolates Pk and LV-141pl are most closely related. The Pk has been found in a wild plum (Prunus domestica) in a new region of Russia indicating widespread dissemination of the PPV-W strain in the European part of the former USSR.
Full Text Available Tea plant (Camellia sinensis (L. O. Kuntze is affected by abiotic stress during its growth and development. DNA-binding with one finger (Dof transcription factors (TFs play important roles in abiotic stress tolerance of plants. In this study, a total of 29 putative Dof TFs were identified based on transcriptome of tea plant, and the conserved domains and common motifs of these CsDof TFs were predicted and analyzed. The 29 CsDof proteins were divided into 7 groups (A, B1, B2, C1, C2.1, C2.2, and D2, and the interaction networks of Dof proteins in C. sinensis were established according to the data in Arabidopsis. Gene expression was analyzed in “Yingshuang” and “Huangjinya” under four experimental stresses by qRT-PCR. CsDof genes were expressed differentially and related to different abiotic stress conditions. In total, our results might suggest that there is a potential relationship between CsDof factors and tea plant stress resistance.
Full Text Available Vaccinia virus (VACV encodes the soluble type I interferon (IFN binding protein B18 that is secreted from infected cells and also attaches to the cell surface, as an immunomodulatory strategy to inhibit the host IFN response. By using next generation sequencing technologies, we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFN based host response by VACV and B18. Transcriptome profiling of L929 cells after incubation with purified recombinant B18 protein showed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation. Consistent with its ability to bind type I IFN, B18 completely inhibited the IFN-mediated modulation of host gene expression. Addition of UV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes, including genes involved in innate immunity. Differential gene expression analyses of cells infected with replication competent VACV identified the activation of a broad range of host genes involved in multiple cellular pathways. Interestingly, we did not detect an IFN-mediated response among the transcriptional changes induced by VACV, even after the addition of IFN to cells infected with a mutant VACV lacking B18. This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection.
Nookaew, Intawat; Papini, Marta; Pornputtapong, Natapol
RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated with the I......RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated...... gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays...
Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko
The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.
Young, Jason A; Fivelman, Quinton L; Blair, Peter L; de la Vega, Patricia; Le Roch, Karine G; Zhou, Yingyao; Carucci, Daniel J; Baker, David A; Winzeler, Elizabeth A
... a full-genome high-density oligonucleotide microarray. The interpretation of this transcriptional data was aided by applying a novel knowledge-based data-mining algorithm termed ontology-based pattern identification (OPI...
Full Text Available Drought has become a critical environmental stress affecting on plant in temperate area. As one of the promising bio-energy crops to sustainable biomass production, the genus Miscanthus has been widely studied around the world. However, the most widely used hybrid cultivar among this genus, Miscanthus × giganteus is proved poor drought tolerance compared to some parental species. Here we mainly focused on Miscanthus sinensis, which is one of the progenitors of M. × giganteus providing a comparable yield and well abiotic stress tolerance in some places. The main objectives were to characterize the physiological and photosynthetic respond to drought stress and to develop simple sequence repeats (SSRs markers associated with drought tolerance by transcriptome sequencing within an originally collection of 44 Miscanthus genotypes from southwest China. Significant phenotypic differences were observed among genotypes, and the average of leaf relative water content (RWC were severely affected by drought stress decreasing from 88.27 to 43.21%, which could well contribute to separating the drought resistant and drought sensitive genotype of Miscanthus. Furthermore, a total of 16,566 gene-associated SSRs markers were identified based on Illumina RNA sequencing under drought conditions, and 93 of them were randomly selected to validate. In total, 70 (75.3% SSRs were successfully amplified and the generated loci from 30 polymorphic SSRs were used to estimate the genetic differentiation and population structure. Finally, two optimum subgroups of the population were determined by structure analysis and based on association analysis, seven significant associations were identified including two markers with leaf RWC and five markers with photosynthetic traits. With the rich sequencing resources annotation, such associations would serve an efficient tool for Miscanthus drought response mechanism study and facilitate genetic improvement of drought resistant for
Illumina HiSeq technology was used to sequence the transcriptome from various dissected tissues and life stages from the horn fly, Haematobia irritans. These samples include eggs (0, 2, 4, and 9 hours post-oviposition), adult fly gut, adult fly legs, adult fly malpighian tubule, adult fly ovary, adu...
Ballerstedt, H.; Volkers, R.J.M.; Mars, A.E.; Hallsworth, J.E.; Santos, V.A.M.D.; Puchalka, J.; Duuren, J. van; Eggink, G.; Timmis, K.N.; Bont, J.A.M. de; Wery, J.
Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for
Full Text Available Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC and medial smooth muscle cells (SMC from canine vein grafts, 2 hours (H to 30 days (D following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12-24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1 signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1, a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.
Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E
Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid
Farmerie William G
Full Text Available Abstract Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20 System (454 Life Sciences Corporation, to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae and Platanus occidentalis (Platanaceae. Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy
Full Text Available A number of transcriptome datasets for differential expression (DE genes have been widely used for understanding organismal biology, but these datasets also contain untapped information that can be used to develop more precise analytical tools. With the use of transcriptome data generated from poplar/canker disease interaction system, we describe a methodology to identify candidate reference genes from high-throughput sequencing data. This methodology will improve the accuracy of RT-qPCR and will lead to better standards for the normalization of expression data. Expression stability analysis from xylem and phloem of Populus bejingensis inoculated with the fungal canker pathogen Botryosphaeria dothidea revealed that 729 poplar transcripts (1.11% were stably expressed, at a threshold level of coefficient of variance (CV of FPKM < 20% and maximum fold change (MFC of FPKM < 2.0. Expression stability and bioinformatics analysis suggested that commonly used house-keeping (HK genes were not the most appropriate internal controls: 70 of the 72 commonly used HK genes were not stably expressed, 45 of the 72 produced multiple isoform transcripts, and some of their reported primers produced unspecific amplicons in PCR amplification. RT-qPCR analysis to compare and evaluate the expression stability of 10 commonly used poplar HK genes and 20 of the 729 newly-identified stably expressed transcripts showed that some of the newly-identified genes (such as SSU_S8e, LSU_L5e, and 20S_PSU had higher stability ranking than most of commonly used HK genes. Based on these results, we recommend a pipeline for deriving reference genes from transcriptome data. An appropriate candidate gene should have a unique transcript, constitutive expression, CV value of expression < 20% (or possibly 30% and MFC value of expression <2, and an expression level of 50–1,000 units. Lastly, when four of the newly identified HK genes were used in the normalization of expression data for 20
Full Text Available Abstract Background Biodiesel or ethanol derived from lipids or starch produced by microalgae may overcome many of the sustainability challenges previously ascribed to petroleum-based fuels and first generation plant-based biofuels. The paucity of microalgae genome sequences, however, limits gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for the non-model microalgae species, Dunaliella tertiolecta, and identify pathways and genes of importance related to biofuel production. Results Next generation DNA pyrosequencing technology applied to D. tertiolecta transcripts produced 1,363,336 high quality reads with an average length of 400 bases. Following quality and size trimming, ~ 45% of the high quality reads were assembled into 33,307 isotigs with a 31-fold coverage and 376,482 singletons. Assembled sequences and singletons were subjected to BLAST similarity searches and annotated with Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology (KO identifiers. These analyses identified the majority of lipid and starch biosynthesis and catabolism pathways in D. tertiolecta. Conclusions The construction of metabolic pathways involved in the biosynthesis and catabolism of fatty acids, triacylglycrols, and starch in D. tertiolecta as well as the assembled transcriptome provide a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock.
Full Text Available High-throughput mRNA sequencing (RNA-Seq is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA, the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.
2008). Analyses of the genetic structure .... The annotations and classifications for the transcriptome ... Based on the Pfam classification, the predic- ..... J. Lipid. Res. 33, 251–262. Eckert C. G., Samis K. E. and Lougheed S. C. 2008 Genetic vari-.
Wilks, Jessica C.; Kitko, Ryan D.; Cleeton, Sarah H.; Lee, Grace E.; Ugwu, Chinagozi S.; Jones, Brian D.; BonDurant, Sandra S.; Slonczewski, Joan L.
Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K+/H+ antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids. PMID:19114526
Christopher N LaRock
Full Text Available The etiologic agent of bubonic plague, Yersinia pestis, senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.
Brown, David S; Jarman, Simon N; Symondson, William O C
Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms (N=80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles. © 2011 Blackwell Publishing Ltd.
Full Text Available BACKGROUND: Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. METHODOLOGY/PRINCIPAL FINDINGS: A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. CONCLUSIONS/SIGNIFICANCE: In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.
Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G
Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.
Barker, Christopher J; Gillett, Amber; Polkinghorne, Adam; Timms, Peter
As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut. Copyright © 2013 Elsevier B.V. All rights reserved.
Dobroff, Andrey S; D'Angelo, Sara; Eckhardt, Bedrich L; Ferrara, Fortunato; Staquicini, Daniela I; Cardó-Vila, Marina; Staquicini, Fernanda I; Nunes, Diana N; Kim, Kisu; Driessen, Wouter H P; Hajitou, Amin; Lomo, Lesley C; Barry, Marc; Krishnamurthy, Savitri; Sahin, Aysegul; Woodward, Wendy A; Prossnitz, Eric R; Anderson, Robin L; Dias-Neto, Emmanuel; Brown-Glaberman, Ursa A; Royce, Melanie E; Ueno, Naoto T; Cristofanilli, Massimo; Hortobagyi, Gabriel N; Marchiò, Serena; Gelovani, Juri G; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata
Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.
Campbell, Malcolm G; Kohane, Isaac S; Kong, Sek Won
signal, and showed that outlier groups were distinct for each implicated pathway. Moreover, our results suggest that by seeking heterogeneity, pathway-based outlier analysis can reveal expression signals that are not apparent when considering only shared group differences.
Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar
The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. © 2017 Scandinavian Plant Physiology Society.
Salvador, Ricardo; Príncipi, Darío; Berretta, Marcelo; Fernández, Paula; Paniego, Norma; Sciocco-Cap, Alicia; Hopp, Esteban
Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.
How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg
Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.
Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai
Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530
Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.
Castro-Carrera, T; Toral, P G; Frutos, P; McEwan, N R; Hervás, G; Abecia, L; Pinloche, E; Girdwood, S E; Belenguer, A
Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition
Nejat, Naghmeh; Cahill, David M; Vadamalai, Ganesan; Ziemann, Mark; Rookes, James; Naderali, Neda
Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host-phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.
Hwang, Hwan-Su; Lee, Hyoshin; Choi, Yong Eui
Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.
Garg, Rohini; Jain, Mukesh
Chickpea is a very important crop legume plant, which provides a protein-rich supplement to cereal-based diets and has the ability to fix atmospheric nitrogen. Despite its economic importance, the functional genomic resources for chickpea are very limited. Recently, we reported the complete transcriptome of chickpea using next generation sequencing technologies. We analyzed the tissue-specific expression of chickpea transcripts based on RNA-seq data. In addition, we identified two sets of lin...
Zhang, Xia; Liao, Maoseng; Chang, Dan; Zhang, Fuchun
Much attention has been given to the potential of halophytes as sources of tolerance traits for introduction into cereals. However, a great deal remains unknown about the diverse mechanisms employed by halophytes to cope with salinity. To characterize salt tolerance mechanisms underlying Karelinia caspica, an Asteraceae halophyte, we performed Large-scale transcriptomic analysis using a high-throughput Illumina sequencing platform. Comparative gene expression analysis was performed to correlate the effects of salt stress and ABA regulation at the molecular level. Total sequence reads generated by pyrosequencing were assembled into 287,185 non-redundant transcripts with an average length of 652 bp. Using the BLAST function in the Swiss-Prot, NCBI nr, GO, KEGG, and KOG databases, a total of 216,416 coding sequences associated with known proteins were annotated. Among these, 35,533 unigenes were classified into 69 gene ontology categories, and 18,378 unigenes were classified into 202 known pathways. Based on the fold changes observed when comparing the salt stress and control samples, 60,127 unigenes were differentially expressed, with 38,122 and 22,005 up- and down-regulated, respectively. Several of the differentially expressed genes are known to be involved in the signaling pathway of the plant hormone ABA, including ABA metabolism, transport, and sensing as well as the ABA signaling cascade. Transcriptome profiling of K. caspica contribute to a comprehensive understanding of K. caspica at the molecular level. Moreover, the global survey of differentially expressed genes in this species under salt stress and analyses of the effects of salt stress and ABA regulation will contribute to the identification and characterization of genes and molecular mechanisms underlying salt stress responses in Asteraceae plants.
Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian
To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, PNested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, Pnested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.
Sarkar, Soumyadev; Chakravorty, Somnath; Mukherjee, Avishek; Bhattacharya, Debanjana; Bhattacharya, Semantee; Gachhui, Ratan
Nitrogen is a key nutrient for all cell forms. Most organisms respond to nitrogen scarcity by slowing down their growth rate. On the contrary, our previous studies have shown that Papiliotrema laurentii strain RY1 has a robust growth under nitrogen starvation. To understand the global regulation that leads to such an extraordinary response, we undertook a de novo approach for transcriptome analysis of the yeast. Close to 33 million sequence reads of high quality for nitrogen limited and enriched condition were generated using Illumina NextSeq500. Trinity analysis and clustered transcripts annotation of the reads produced 17,611 unigenes, out of which 14,157 could be annotated. Gene Ontology term analysis generated 44.92% cellular component terms, 39.81% molecular function terms and 15.24% biological process terms. The most over represented pathways in general were translation, carbohydrate metabolism, amino acid metabolism, general metabolism, folding, sorting, degradation followed by transport and catabolism, nucleotide metabolism, replication and repair, transcription and lipid metabolism. A total of 4256 Single Sequence Repeats were identified. Differential gene expression analysis detected 996 P-significant transcripts to reveal transmembrane transport, lipid homeostasis, fatty acid catabolism and translation as the enriched terms which could be essential for Papiliotrema laurentii strain RY1 to adapt during nitrogen deprivation. Transcriptome data was validated by quantitative real-time PCR analysis of twelve transcripts. To the best of our knowledge, this is the first report of Papiliotrema laurentii strain RY1 transcriptome which would play a pivotal role in understanding the biochemistry of the yeast under acute nitrogen stress and this study would be encouraging to initiate extensive investigations into this Papiliotrema system. Copyright © 2017 Elsevier B.V. All rights reserved.
Transcriptomics is part of a family of disciplines focussing on high throughput molecular biology experiments. In the case of transcriptomics, scientists study the expression of genes resulting in transcripts. These transcripts can either perform a biological function themselves or function as
Full Text Available To better understand the molecular functions of the master stress-response regulator AtfA in Aspergillus nidulans, transcriptomic analyses of the atfA null mutant and the appropriate control strains exposed to menadione sodium bisulfite- (MSB-, t-butylhydroperoxide- and diamide-induced oxidative stresses were performed. Several elements of oxidative stress response were differentially expressed. Many of them, including the downregulation of the mitotic cell cycle, as the MSB stress-specific upregulation of FeS cluster assembly and the MSB stress-specific downregulation of nitrate reduction, tricarboxylic acid cycle, and ER to Golgi vesicle-mediated transport, showed AtfA dependence. To elucidate the potential global regulatory role of AtfA governing expression of a high number of genes with very versatile biological functions, we devised a model based on the comprehensive transcriptomic data. Our model suggests that an important function of AtfA is to modulate the transduction of stress signals. Although it may regulate directly only a limited number of genes, these include elements of the signaling network, for example, members of the two-component signal transduction systems. AtfA acts in a stress-specific manner, which may increase further the number and diversity of AtfA-dependent genes. Our model sheds light on the versatility of the physiological functions of AtfA and its orthologs in fungi.
Kawakami, Takeshi; Darby, Brian J; Ungerer, Mark C
Next-generation sequencing (NGS) technologies provide a rapid means to generate genomic resources for species exhibiting interesting ecological and evolutionary variation but for which such resources are scant or nonexistent. In the current report, we utilize 454 pyrosequencing to obtain transcriptome information for multiple individuals and tissue types from geographically disparate and ecologically differentiated populations of the perennial sunflower species Helianthus maximiliani. A total of 850 275 raw reads were obtained averaging 355 bp in length. Reads were assembled, postprocessing, into 16 681 unique contigs with an N50 of 898 bp and a total length of 13.6 Mb. A majority (67%) of these contigs were annotated based on comparison with the Arabidopsis thaliana genome (TAIR10). Contigs were identified that exhibit high similarity to genes associated with natural variation in flowering time and freezing tolerance in other plant species and will facilitate future studies aimed at elucidating the molecular basis of clinal life history variation and adaptive differentiation in H. maximiliani. Large numbers of gene-associated simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNPs) also were identified that can be deployed in mapping and population genomic analyses. © 2014 John Wiley & Sons Ltd.
Full Text Available Avian pathogenic Escherichia coli (APEC leads to economic losses in poultry production and is also a threat to human health. The goal of this study was to characterize the chicken spleen transcriptome and to identify candidate genes for response and resistance to APEC infection using Solexa sequencing. We obtained 14422935, 14104324, and 14954692 Solexa read pairs for non-challenged (NC, challenged-mild pathology (MD, and challenged-severe pathology (SV, respectively. A total of 148197 contigs and 98461 unigenes were assembled, of which 134949 contigs and 91890 unigenes match the chicken genome. In total, 12272 annotated unigenes take part in biological processes (11664, cellular components (11927, and molecular functions (11963. Summing three specific contrasts, 13650 significantly differentially expressed unigenes were found in NC Vs. MD (6844, NC Vs. SV (7764, and MD Vs. SV (2320. Some unigenes (e.g. CD148, CD45 and LCK were involved in crucial pathways, such as the T cell receptor (TCR signaling pathway and microbial metabolism in diverse environments. This study facilitates understanding of the genetic architecture of the chicken spleen transcriptome, and has identified candidate genes for host response to APEC infection.
Simon, Matthew J; Murchison, Charles; Iliff, Jeffrey J
Astrocytes play a critical role in regulating the interface between the cerebral vasculature and the central nervous system. Contributing to this is the astrocytic endfoot domain, a specialized structure that ensheathes the entirety of the vasculature and mediates signaling between endothelial cells, pericytes, and neurons. The astrocytic endfoot has been implicated as a critical element of the glymphatic pathway, and changes in protein expression profiles in this cellular domain are linked to Alzheimer's disease pathology. Despite this, basic physiological properties of this structure remain poorly understood including the developmental timing of its formation, and the protein components that localize there to mediate its functions. Here we use human transcriptome data from male and female subjects across several developmental stages and brain regions to characterize the gene expression profile of the dystrophin-associated complex (DAC), a known structural component of the astrocytic endfoot that supports perivascular localization of the astroglial water channel aquaporin-4. Transcriptomic profiling is also used to define genes exhibiting parallel expression profiles to DAC elements, generating a pool of candidate genes that encode gene products that may contribute to the physiological function of the perivascular astrocytic endfoot domain. We found that several genes encoding transporter proteins are transcriptionally associated with DAC genes. © 2017 Wiley Periodicals, Inc.
Sugiyama, Akifumi; Ueda, Yoshikatsu; Takase, Hisabumi; Yazaki, Kazufumi
Soil fungal communities play essential roles in soil ecosystems, affecting plant growth and health. Rhizosphere bacterial communities have been shown to undergo dynamic changes during plant growth. This study utilized 454 pyrosequencing to analyze rhizosphere fungal communities during soybean growth. Members of the Ascomycota and Basiodiomycota dominated in all soils. There were no statistically significant changes at the phylum level among growth stages or between bulk and rhizosphere soils. In contrast, the relative abundance of small numbers of operational taxonomic units, 4 during growth and 28 between bulk and rhizosphere soils, differed significantly. Clustering analysis revealed that rhizosphere fungal communities were different from bulk fungal communities during growth stages of soybeans. Taken together, these results suggest that in contrast to rhizosphere bacterial communities, most constituents of rhizosphere fungal communities remained stable during soybean growth.
Kuo, Alan; Grigoriev, Igor
Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentous ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.
Pyrosequencing of the samples resulted in a total of 18520 sequences (3000 per sample, approximately with an average length of 325 bp (base pairs. The taxonomic assignment of sequences allowed the identification to the genus level, being observed a large bacterial diversity with over 110 genera. The most prevalent genera in the samples were Hydrotalea, Acinetobacter, Delftia, Sediminibacter and Pseudomonas, among others. Differences in the microbial communities were observed among the samples, and the PCoA analysis allowed their separation by means on their gender and if they proceed from sampling before or after the spawning. Nevertheless, the rarefaction curves obtained for each sample failed to reach a saturation phase, indicating that more sequencing effort would be necessary.
Full Text Available Both the promises and pitfalls of the cell reprogramming research platform rest on human genetic variation, making the measurement of its impact one of the most urgent issues in the field. Harnessing large transcriptomics datasets of induced pluripotent stem cells (iPSC, we investigate the implications of this variability for iPSC-based disease modeling. In particular, we show that the widespread use of more than one clone per individual in combination with current analytical practices is detrimental to the robustness of the findings. We then proceed to identify methods to address this challenge and leverage multiple clones per individual. Finally, we evaluate the specificity and sensitivity of different sample sizes and experimental designs, presenting computational tools for power analysis. These findings and tools reframe the nature of replicates used in disease modeling and provide important resources for the design, analysis, and interpretation of iPSC-based studies.
Lee, Young-Sang; Ku, Kang-Mo; Becker, Talon M; Juvik, John A
Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market.
Full Text Available The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones, and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to
Assou, Said; Le Carrour, Tanguy; Tondeur, Sylvie; Ström, Susanne; Gabelle, Audrey; Marty, Sophie; Nadal, Laure; Pantesco, Véronique; Réme, Thierry; Hugnot, Jean-Philippe; Gasca, Stéphan; Hovatta, Outi; Hamamah, Samir; Klein, Bernard; De Vos, John
Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.
Full Text Available Abstract Background The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.. While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. Results We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold under salt stress in both organs, witnessing a differential organ-specific response to stress. Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 Uni
Wong Melissa ML
Full Text Available Abstract Background Next Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP discovery in Acacia auriculiformis and Acacia mangium. Like other non-model species, SNP detection and genotyping in Acacia are challenging due to lack of genome sequences. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of A. auriculiformis x A. mangium hybrids. Results We identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two Acacia hybrid mapping populations using Bowtie against 7,839 de novo transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our in silico SNP detection approach is highly accurate (100% compared to the traditional in vitro approach (44%. Further validation of 96 SNPs using Illumina GoldenGate Assay gave an overall assay success rate of 89.6% and conversion rate of 37.5%. We explored possible factors lowering assay success rate by predicting exon-intron boundaries and paralogous genes of Acacia contigs using Medicago truncatula genome as reference. This assessment revealed that presence of exon-intron boundary is the main cause (50% of assay failure. Subsequent SNPs filtering and improved assay design resulted in assay success and conversion rate of 92.4% and 57.4%, respectively based on 768 SNPs genotyping. Analysis of clustering patterns revealed that 27.6% of the assays were not reproducible and flanking sequence might play a role in determining cluster compression. In addition, we identified a total of 258 and 319 polymorphic SNPs in A. auriculiformis and A. mangium natural germplasms, respectively. Conclusion We have successfully discovered a large number of SNP markers in A. auriculiformis x A. mangium hybrids using next generation transcriptome sequencing. By using a reference genome from the most closely
Full Text Available Elms, especially Ulmus minor and Ulmus americana, are carrying out a hard battle against Dutch elm disease (DED. This vascular wilt disease, caused by Ophiostoma ulmi and O. novo-ulmi, appeared in the twentieth century and killed millions of elms across North America and Europe. Elm breeding and conservation programmes have identified a reduced number of DED tolerant genotypes. In this study, three U. minor genotypes with contrasted levels of tolerance to DED were exposed to several biotic and abiotic stresses in order to (i obtain a de novo assembled transcriptome of U. minor using 454 pyrosequencing, (ii perform a functional annotation of the assembled transcriptome, (iii identify genes potentially involved in the molecular response to environmental stress, and (iv develop gene-based markers to support breeding programmes. A total of 58,429 putative unigenes were identified after assembly and filtering of the transcriptome. 32,152 of these unigenes showed homology with proteins identified in the genome from the most common plant model species. Well-known family proteins and transcription factors involved in abiotic, biotic or both stresses were identified after functional annotation. A total of 30,693 polymorphisms were identified in 7,125 isotigs, a large number of them corresponding to SNPs (27,359. In a subset randomly selected for validation, 87 % of the SNPs were confirmed. The material generated may be valuable for future Ulmus gene expression, population genomics and association genetics studies, especially taking into account the scarce molecular information available for this genus and the great impact that DED has on elm populations.
Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W
The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.
David J. Rooks
Full Text Available Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785 were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%. The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases.
Lee, Onon; Wang, Yong; Yang, Jiangke; Lafi, Feras Fawzi; Al-Suwailem, Abdulaziz M.; Qian, Peiyuan
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
Kostovčík, Martin; Bateman, C.C.; Kolařík, Miroslav; Stelinski, L.L.; Jordal, B.H.; Hulcr, J.
Roč. 9, č. 1 (2015), s. 126-138 ISSN 1751-7362 Institutional support: RVO:61388971 Keywords : ambrosia symbiosis * pyrosequencing Subject RIV: EE - Microbiology, Virology Impact factor: 9.328, year: 2015
Harrison, Peter W; Mank, Judith E; Wedell, Nina
Males and females experience differences in gene dose for loci in the nonrecombining region of heteromorphic sex chromosomes. If not compensated, this leads to expression imbalances, with the homogametic sex on average exhibiting greater expression due to the doubled gene dose. Many organisms with heteromorphic sex chromosomes display global dosage compensation mechanisms, which equalize gene expression levels between the sexes. However, birds and Schistosoma have been previously shown to lack chromosome-wide dosage compensation mechanisms, and the status in other female heterogametic taxa including Lepidoptera remains unresolved. To further our understanding of dosage compensation in female heterogametic taxa and to resolve its status in the lepidopterans, we assessed the Indian meal moth, Plodia interpunctella. As P. interpunctella lacks a complete reference genome, we conducted de novo transcriptome assembly combined with orthologous genomic location prediction from the related silkworm genome, Bombyx mori, to compare Z-linked and autosomal gene expression levels for each sex. We demonstrate that P. interpunctella lacks complete Z chromosome dosage compensation, female Z-linked genes having just over half the expression level of males and autosomal genes. This finding suggests that the Lepidoptera and possibly all female heterogametic taxa lack global dosage compensation, although more species will need to be sampled to confirm this assertion.
Naumenko, Sergey A; Logacheva, Maria D; Popova, Nina V; Klepikova, Anna V; Penin, Aleksey A; Bazykin, Georgii A; Etingova, Anna E; Mugue, Nikolai S; Kondrashov, Alexey S; Yampolsky, Lev Y
Endemic species flocks inhabiting ancient lakes, oceanic islands and other long-lived isolated habitats are often interpreted as adaptive radiations. Yet molecular evidence for directional selection during species flocks radiation is scarce. Using partial transcriptomes of 64 species of Lake Baikal (Siberia, Russia) endemic amphipods and two nonendemic outgroups, we report a revised phylogeny of this species flock and analyse evidence for positive selection within the endemic lineages. We confirm two independent invasions of amphipods into Baikal and demonstrate that several morphological features of Baikal amphipods, such as body armour and reduction in appendages and sensory organs, evolved in several lineages in parallel. Radiation of Baikal amphipods has been characterized by short phylogenetic branches and frequent episodes of positive selection which tended to be more frequent in the early phase of the second invasion of amphipods into Baikal when the most intensive diversification occurred. Notably, signatures of positive selection are frequent in genes encoding mitochondrial membrane proteins with electron transfer chain and ATP synthesis functionality. In particular, subunits of both the membrane and substrate-level ATP synthases show evidence of positive selection in the plankton species Macrohectopus branickii, possibly indicating adaptation to active plankton lifestyle and to survival under conditions of low temperature and high hydrostatic pressures known to affect membranes functioning. Other functional categories represented among genes likely to be under positive selection include Ca-binding muscle-related proteins, possibly indicating adaptation to Ca-deficient low mineralization Baikal waters. © 2016 John Wiley & Sons Ltd.
Reverón, Inés; Plaza-Vinuesa, Laura; Franch, Mónica; de Las Rivas, Blanca; Muñoz, Rosario; López de Felipe, Félix
This study was undertaken to expand our insights into the mechanisms involved in the tolerance to resveratrol (RSV) that operate at system-level in gut microorganisms and advance knowledge on new RSV-responsive gene circuits. Whole genome transcriptional profiling was used to characterize the molecular response of Lactobacillus plantarum WCFS1 to RSV. DNA repair mechanisms were induced by RSV and responses were triggered to decrease the load of copper, a metal required for RSV-mediated DNA cleavage, and H 2 S, a genotoxic gas. To counter the effects of RSV, L. plantarum strongly up- or downregulated efflux systems and ABC transporters pointing to transport control of RSV across the membrane as a key mechanism for RSV tolerance. L. plantarum also downregulated tRNAs, induced chaperones, and reprogrammed its transcriptome to tightly control ammonia levels. RSV induced a probiotic effector gene and a likely deoxycholate transporter, two functions that improve the host health status. Our data identify novel protective mechanisms involved in RSV tolerance operating at system level in a gut microbe. These insights could influence the way RSV is used for a better management of gut microbial ecosystems to obtain associated health benefits. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Effects of the total replacement of fish-based diet with plant-based diet on the hepatic transcriptome of two European sea bass (Dicentrarchus labrax half-sibfamilies showing different growth rates with the plant-based diet
Full Text Available Abstract Background Efforts towards utilisation of diets without fish meal (FM or fish oil (FO in finfish aquaculture have been being made for more than two decades. Metabolic responses to substitution of fishery products have been shown to impact growth performance and immune system of fish as well as their subsequent nutritional value, particularly in marine fish species, which exhibit low capacity for biosynthesis of long-chain poly-unsaturated fatty acids (LC-PUFA. The main objective of the present study was to analyse the effects of a plant-based diet on the hepatic transcriptome of European sea bass (Dicentrarchus labrax. Results We report the first results obtained using a transcriptomic approach on the liver of two half-sibfamilies of the European sea bass that exhibit similar growth rates when fed a fish-based diet (FD, but significantly different growth rates when fed an all-plant diet (VD. Overall gene expression was analysed using oligo DNA microarrays (GPL9663. Statistical analysis identified 582 unique annotated genes differentially expressed between groups of fish fed the two diets, 199 genes regulated by genetic factors, and 72 genes that exhibited diet-family interactions. The expression of several genes involved in the LC-PUFA and cholesterol biosynthetic pathways was found to be up-regulated in fish fed VD, suggesting a stimulation of the lipogenic pathways. No significant diet-family interaction for the regulation of LC-PUFA biosynthesis pathways could be detected by microarray analysis. This result was in agreement with LC-PUFA profiles, which were found to be similar in the flesh of the two half-sibfamilies. In addition, the combination of our transcriptomic data with an analysis of plasmatic immune parameters revealed a stimulation of complement activity associated with an immunodeficiency in the fish fed VD, and different inflammatory status between the two half-sibfamilies. Biological processes related to protein
Full Text Available Acidithiobacillus caldus (A. caldus is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox system (omitting SoxCD, non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR. The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system.An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor was created and its growth abilities were measured in media using elemental sulfur (S(0 and tetrathionate (K(2S(4O(6 as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR of the wild type and the Δsor mutant in S(0 and K(2S(4O(6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO and heterodisulfide reductase (HDR, the truncated Sox pathway, and the S(4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media.An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.
Full Text Available Although invertebrates are incapable of adaptive immunity, immunal reactions which are functionally similar to the adaptive immunity of vertebrates have been described in many studies of invertebrates including insects. The phenomenon was termed immune priming. In order to understand the molecular mechanism of immune priming, we employed Illumina/Solexa platform to investigate the transcriptional changes of the hemocytes and fat body of Helicoverpa armigera larvae immune-primed with the pathogenic bacteria Photorhabdus luminescens TT01. A total of 43.6 and 65.1 million clean reads with 4.4 and 6.5 gigabase sequence data were obtained from the TT01 (the immune-primed and PBS (non-primed cDNA libraries and assembled into 35,707 all-unigenes (non-redundant transcripts, which has a length varied from 201 to 16,947 bp and a N50 length of 1,997 bp. For 35,707 all-unigenes, 20,438 were functionally annotated and 2,494 were differentially expressed after immune priming. The differentially expressed genes (DEGs are mainly related to immunity, detoxification, development and metabolism of the host insect. Analysis on the annotated immune related DEGs supported a hypothesis that we proposed previously: the immune priming phenomenon observed in H. armigera larvae was achieved by regulation of key innate immune elements. The transcriptome profiling data sets (especially the sequences of 1,022 unannotated DEGs and the clues (such as those on immune-related signal and regulatory pathways obtained from this study will facilitate immune-related novel gene discovery and provide valuable information for further exploring the molecular mechanism of immune priming of invertebrates. All these will increase our understanding of invertebrate immunity which may provide new approaches to control insect pests or prevent epidemic of infectious diseases in economic invertebrates in the future.
Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere
Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.
Gao, Yue; Kim, Kyungmun; Kwon, Deok Ho; Jeong, In Hong; Clark, J Marshall; Lee, Si Hyeock
When the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, were pretreated with sublethal doses (LC 10 ) and then subsequently exposed to lethal doses (LC 50 ) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. To identify genes that commonly respond to the treatment of different insecticides and are responsible for the tolerance enhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared with that of untreated control. A total of 117,181 transcripts with a mean length of 662bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined to be up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealed little differences in their GO profiles between treatments with different insecticides except for spinosad. Finally, the DEGs commonly responding to all insecticides were selected for further characterization, and some of their over-transcription levels were confirmed by quantitative PCR. The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantly down-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested, the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in the tolerance/resistance development were discussed. Copyright © 2017 Elsevier
Fu, Yuanshuai; Jia, Liang; Shi, Zhiyi; Zhang, Junling; Li, Wenjuan
The Japanese flounder (Paralichthys olivaceus) is one of the most important commercial and biological marine fishes. However, the molecular biology involved during embryogenesis and early development of the Japanese flounder remains largely unknown due to a lack of genomic resources. A comprehensive and integrated transcriptome is necessary to study the molecular mechanisms of early development and to allow for the detailed characterization of gene expression patterns during embryogenesis; this approach is critical to understanding the processes that occur prior to mesectoderm formation during early embryonic development. In this study, more than 117.8 million 100bp PE reads were generated from pooled RNA extracted from unfertilized eggs to 41dph (days post-hatching) embryos and were sequenced using Illumina pair-end sequencing technology. In total, 121,513 transcripts (≥200bp) were obtained using de novo assembly. A sequence similarity search indicated that 52,338 transcripts show significant similarity to 22,462 known proteins from the NCBI non-redundant database and the Swiss-Prot protein database and were annotated using Blast2GO. GO terms were assigned to 44,627 transcripts with 12,006 functional terms, and 10,024 transcripts were assigned to 133 KEGG pathways. Furthermore, gene expression differences between the unfertilized egg and the gastrula embryo were analysed using Illumina RNA-Seq with single-read sequencing technology, and 24,837 differentially and specifically expressed transcripts were identified and included 5,286 annotated transcripts and 19,569 non-annotated transcripts. All of the expressed transcripts in the unfertilized egg and gastrula embryo were further classified as maternal, zygotic, or maternal-zygotic transcripts, which may help us to understand the roles of these transcripts during the embryonic development of the Japanese flounder. Thus, the results will contribute to an improved understanding of the gene expression patterns and
Feng, Bing; Yi, Soojin V; Zhang, Manman; Zhou, Xiaoyun
The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus.
Xiao, Yong; Zhou, Lixia; Xia, Wei; Mason, Annaliese S; Yang, Yaodong; Ma, Zilong; Peng, Ming
The oil palm (Elaeis guineensis, 2n = 32) has the highest oil yield of any crop species, as well as comprising the richest dietary source of provitamin A. For the tropical species, the best mean growth temperature is about 27°C, with a minimal growth temperature of 15°C. Hence, the plantation area is limited into the geographical ranges of 10°N to 10°S. Enhancing cold tolerance capability will increase the total cultivation area and subsequently oil productivity of this tropical species. Developing molecular markers related to cold tolerance would be helpful for molecular breeding of cold tolerant Elaeis guineensis. In total, 5791 gene-based SSRs were identified in 51,452 expressed sequences from Elaeis guineensis transcriptome data: approximately one SSR was detected per 10 expressed sequences. Of these 5791 gene-based SSRs, 916 were derived from expressed sequences up- or down-regulated at least two-fold in response to cold stress. A total of 182 polymorphic markers were developed and characterized from 442 primer pairs flanking these cold-responsive SSR repeats. The polymorphic information content (PIC) of these polymorphic SSR markers across 24 lines of Elaeis guineensis varied from 0.08 to 0.65 (mean = 0.31 ± 0.12). Using in-silico mapping, 137 (75.3%) of the 182 polymorphic SSR markers were located onto the 16 Elaeis guineensis chromosomes. Total coverage of 473 Mbp was achieved, with an average physical distance of 3.4 Mbp between adjacent markers (range 96 bp - 20.8 Mbp). Meanwhile, Comparative analysis of transcriptome under cold stress revealed that one ICE1 putative ortholog, five CBF putative orthologs, 19 NAC transcription factors and four cold-induced orhologs were up-regulated at least two fold in response to cold stress. Interestingly, 5' untranslated region of both Unigene21287 (ICE1) and CL2628.Contig1 (NAC) both contained an SSR markers. In the present study, a series of SSR markers were developed based on sequences
Ewen-Campen, Ben; Shaner, Nathan; Panfilio, Kristen A; Suzuki, Yuichiro; Roth, Siegfried; Extavour, Cassandra G
Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery. We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses. Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high
Full Text Available Abstract Background Most evolutionary developmental biology ("evo-devo" studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery. Results We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies and Hymenoptera (including honeybees, and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses. Conclusions Our sequencing, assembly and annotation framework
Bendele Kylie G
Full Text Available Abstract Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be
Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts
Full Text Available The white-backed planthopper, Sogatella furcifera, a notorious rice pest in Asia, employs host plant volatiles as cues for host location. In insects, odor detection is mediated by two types of olfactory receptors: odorant receptors (ORs and ionotropic receptors (IRs. In this study, we identified 63 SfurORs and 14 SfurIRs in S. furcifera based on sequences obtained from the head transcriptome and bioinformatics analysis. The motif-pattern of 130 hemiptera ORs indicated an apparent differentiation in this order. Phylogenetic trees of the ORs and IRs were constructed using neighbor-joining estimates. Most of the ORs had orthologous genes, but a specific OR clade was identified in S. furcifera, which suggests that these ORs may have specific olfactory functions in this species. Our results provide a basis for further investigations of how S. furcifera coordinates its olfactory receptor genes with its plant hosts, thereby providing a foundation for novel pest management approaches based on these genes.
SSRs developed from high-throughput transcriptome data will facilitate marker-assisted selection for all traits in Sudan grass.
Full Text Available Parasteatoda tepidariorum is an increasingly popular model for the study of spider development and the evolution of development more broadly. However, fully understanding the regulation and evolution of P. tepidariorum development in comparison to other animals requires a genomic perspective. Although research on P. tepidariorum has provided major new insights, gene analysis to date has been limited to candidate gene approaches. Furthermore, the few available EST collections are based on embryonic transcripts, which have not been systematically annotated and are unlikely to contain transcripts specific to post-embryonic stages of development. We therefore generated cDNA from pooled embryos representing all described embryonic stages, as well as post-embryonic stages including nymphs, larvae and adults, and using Illumina HiSeq technology obtained a total of 625,076,514 100-bp paired end reads. We combined these data with 24,360 ESTs available in GenBank, and 1,040,006 reads newly generated from 454 pyrosequencing of a mixed-stage embryo cDNA library. The combined sequence data were assembled using a custom de novo assembly strategy designed to optimize assembly product length, number of predicted transcripts, and proportion of raw reads incorporated into the assembly. The de novo assembly generated 446,427 contigs with an N50 of 1,875 bp. These sequences obtained 62,799 unique BLAST hits against the NCBI non-redundant protein data base, including putative orthologs to 8,917 Drosophila melanogaster genes based on best reciprocal BLAST hit identity compared with the D. melanogaster proteome. Finally, we explored the utility of the transcriptome for RNA-Seq studies, and showed that this resource can be used as a mapping scaffold to detect differential gene expression in different cDNA libraries. This resource will therefore provide a platform for future genomic, gene expression and functional approaches using P. tepidariorum.
Smith, Drake M; Snow, David E; Rees, Eric; Zischkau, Ann M; Hanson, J Delton; Wolcott, Randall D; Sun, Yan; White, Jennifer; Kumar, Shashi; Dowd, Scot E
Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities. Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics. Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.
Wolcott Randall D
Full Text Available Abstract Background Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories. Methods Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP, a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities. Results Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics. Conclusions Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.
Suchodolski, Jan S; Dowd, Scot E; Wilke, Vicky; Steiner, Jörg M; Jergens, Albert E
Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, pmicrobial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.
Neng Fei eWang
Full Text Available This study assessed the diversity and composition of bacterial communities in four different soils (human-, penguin-, seal-colony impacted soils and pristine soil in the Fildes Region (King George Island, Antarctica using 454 pyrosequencing with bacterial-specific primers targeting the 16S rRNA gene. Proteobacteria, Actinobacteria, Acidobacteria, and Verrucomicrobia were abundant phyla in almost all the soil samples. The four types of soils were significantly different in geochemical properties and bacterial community structure. Thermotogae, Cyanobacteria, Fibrobacteres, Deinococcus-Thermus, and Chlorobi obviously varied in their abundance among the 4 soil types. Considering all the samples together, members of the genera Gaiella, Chloracidobacterium, Nitrospira, Polaromonas, Gemmatimonas, Sphingomonas and Chthoniobacter were found to predominate, whereas members of the genera Chamaesiphon, Herbaspirillum, Hirschia, Nevskia, Nitrosococcus, Rhodococcus, Rhodomicrobium, and Xanthomonas varied obviously in their abundance among the four soil types. Distance-based redundancy analysis revealed that pH (p < 0.01, phosphate phosphorus (p < 0.01, organic carbon (p < 0.05, and organic nitrogen (p < 0.05 were the most significant factors that correlated with the community distribution of soil bacteria. To our knowledge, this is the first study to explore the soil bacterial communities in human-, penguin-, and seal- colony impacted soils from ice-free areas in maritime Antarctica using high-throughput pyrosequencing.
Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR mutations, especially in-frame deletions in exon 19 (ΔLRE and a point mutation in exon 21 (L858R predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. Methods We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. Results This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%. In the prospective analysis of 213 samples, 7 (3.3% samples were not analyzed and EGFR mutations were detected in 18 (8.7% patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. Conclusions pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.
Oki, Kaihei; Dugersuren, Jamyan; Demberel, Shirchin; Watanabe, Koichi
Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare's milk), 5 Khoormog (fermented camel's milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 phyla. The genus Lactobacillus is a core bacterial component of Mongolian fermented milks, and Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus delbrueckii were the predominant species of lactic acid bacteria (LAB) in the Airag, Khoormog and Tarag samples, respectively. By using this pyrosequencing approach, we successfully detected most LAB species that have been isolated as well as seven LAB species that have not been found in our previous culture-based study. A subsequent analysis of the principal components of the samples revealed that L. delbrueckii, L. helveticus, L. kefiranofaciens and Streptococcus thermophilus were the main factors influencing the microbial diversity of these Mongolian traditional fermented dairy products and that this diversity correlated with the animal species from which the milk was sourced.
He, Peng; Zhang, Yun-Fei; Hong, Duan-Yang; Wang, Jun; Wang, Xing-Liang; Zuo, Ling-Hua; Tang, Xian-Fu; Xu, Wei-Ming; He, Ming
comprehensive gene data set of sex pheromone biosynthesis and degradation enzyme related genes in DBM created by genome- and transcriptome-wide identification, characterization and expression profiling. Our findings provide a basis to better understand the function of genes with tissue enriched expression. The results also provide information on the genes involved in sex pheromone biosynthesis and degradation, and may be useful to identify potential gene targets for pest control strategies by disrupting the insect-insect communication using pheromone-based behavioral antagonists.
Li, Ou; Xiao, Rong; Sun, Lihua; Guan, Chenglin; Kong, Dedong; Hu, Xiufang
As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39-38.42%), Burkholderia (2.71-15.98%), Escherichia/Shigella (4.90-25.12%), Pseudomonas (2.68-30.72%) and Sphingomonas (1.83-2.05%) dominated in four planting bases. Pseudomonas (17.94-22.06%), Escherichia/Shigella (6.59-11.59%), Delftia (9.65-22.14%) and Burkholderia (3.12-11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.
Full Text Available As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ to 96.08% (YN, and Delftia (10.39-38.42%, Burkholderia (2.71-15.98%, Escherichia/Shigella (4.90-25.12%, Pseudomonas (2.68-30.72% and Sphingomonas (1.83-2.05% dominated in four planting bases. Pseudomonas (17.94-22.06%, Escherichia/Shigella (6.59-11.59%, Delftia (9.65-22.14% and Burkholderia (3.12-11.05% dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.
Li, Ou; Sun, Lihua; Guan, Chenglin; Kong, Dedong
As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39–38.42%), Burkholderia (2.71–15.98%), Escherichia/Shigella (4.90–25.12%), Pseudomonas (2.68–30.72%) and Sphingomonas (1.83–2.05%) dominated in four planting bases. Pseudomonas (17.94–22.06%), Escherichia/Shigella (6.59–11.59%), Delftia (9.65–22.14%) and Burkholderia (3.12–11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples. PMID:28931073
Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.
After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642
Zhang, Jinpeng; Liu, Weihua; Lu, Yuqing; Liu, Qunxing; Yang, Xinming; Li, Xiuquan; Li, Lihui
Agropyron cristatum is a wild grass of the tribe Triticeae and serves as a gene donor for wheat improvement. However, very few markers can be used to monitor A. cristatum chromatin introgressions in wheat. Here, we reported a resource of large-scale molecular markers for tracking alien introgressions in wheat based on transcriptome sequences. By aligning A. cristatum unigenes with the Chinese Spring reference genome sequences, we designed 9602 A. cristatum expressed sequence tag-sequence-tagged site (EST-STS) markers for PCR amplification and experimental screening. As a result, 6063 polymorphic EST-STS markers were specific for the A. cristatum P genome in the single-receipt wheat background. A total of 4956 randomly selected polymorphic EST-STS markers were further tested in eight wheat variety backgrounds, and 3070 markers displaying stable and polymorphic amplification were validated. These markers covered more than 98% of the A. cristatum genome, and the marker distribution density was approximately 1.28 cM. An application case of all EST-STS markers was validated on the A. cristatum 6 P chromosome. These markers were successfully applied in the tracking of alien A. cristatum chromatin. Altogether, this study provided a universal method of large-scale molecular marker development to monitor wild relative chromatin in wheat.
Full Text Available Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks, relative to non-diabetic control, in the presence and absence of short-term (seven-day treatment with the angiotensin receptor blocker, losartan (n = 3-6 biological replicates per condition. We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (p52-fold, encoded by the cationic amino acid transporter Slc7a12, and the gene product most highly downregulated by diabetes (>99%--encoded by the "pseudogene" Gm6300--are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential.
Wei, Ling; Yang, Chao; Tao, Wenjing; Wang, Deshou
The Sox transcription factor family is characterized with the presence of a Sry-related high-mobility group (HMG) box and plays important roles in various biological processes in animals, including sex determination and differentiation, and the development of multiple organs. In this study, 27 Sox genes were identified in the genome of the Nile tilapia (Oreochromis niloticus), and were classified into seven groups. The members of each group of the tilapia Sox genes exhibited a relatively conserved exon-intron structure. Comparative analysis showed that the Sox gene family has undergone an expansion in tilapia and other teleost fishes following their whole genome duplication, and group K only exists in teleosts. Transcriptome-based analysis demonstrated that most of the tilapia Sox genes presented stage-specific and/or sex-dimorphic expressions during gonadal development, and six of the group B Sox genes were specifically expressed in the adult brain. Our results provide a better understanding of gene structure and spatio-temporal expression of the Sox gene family in tilapia, and will be useful for further deciphering the roles of the Sox genes during sex determination and gonadal development in teleosts.
Full Text Available The Sox transcription factor family is characterized with the presence of a Sry-related high-mobility group (HMG box and plays important roles in various biological processes in animals, including sex determination and differentiation, and the development of multiple organs. In this study, 27 Sox genes were identified in the genome of the Nile tilapia (Oreochromis niloticus, and were classified into seven groups. The members of each group of the tilapia Sox genes exhibited a relatively conserved exon-intron structure. Comparative analysis showed that the Sox gene family has undergone an expansion in tilapia and other teleost fishes following their whole genome duplication, and group K only exists in teleosts. Transcriptome-based analysis demonstrated that most of the tilapia Sox genes presented stage-specific and/or sex-dimorphic expressions during gonadal development, and six of the group B Sox genes were specifically expressed in the adult brain. Our results provide a better understanding of gene structure and spatio-temporal expression of the Sox gene family in tilapia, and will be useful for further deciphering the roles of the Sox genes during sex determination and gonadal development in teleosts.
Hu, Yulong; Huang, Meng; Wang, Weiji; Guan, Jiantao; Kong, Jie
The mechanisms underlying sexual reproduction and sex ratio determination remains unclear in turbot, a flatfish of great commercial value. And there is limited information in the turbot database regarding genes related to the reproductive system. Here, we conducted high-throughput transcriptome profiling of turbot gonad tissues to better understand their reproductive functions and to supply essential gene sequence information for marker-assisted selection programs in the turbot industry. In this study, two gonad libraries representing sex differences in Scophthalmus maximus yielded 453 818 high-quality reads that were assembled into 24 611 contigs and 33 713 singletons by using 454 pyrosequencing, 13 936 contigs and singletons (CS) of which were annotated using BLASTx. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that various biological functions and processes were associated with many of the annotated CS. Expression analyses showed that 510 genes were differentially expressed in males versus females; 80% of these genes were annotated. In addition, 6484 and 6036 single nucleotide polymorphisms (SNPs) were identified in male and female libraries, respectively. This transcriptome resource will serve as the foundation for cDNA or SNP microarray construction, gene expression characterization, and sex-specific linkage mapping in turbot.
Jiang, Wen; Zhang, Jie; Chen, Hui
Severe early childhood caries are a prevalent public health problem among preschool children throughout the world. However, little is known about the microbiota found in association with severe early childhood caries. Our study aimed to explore the bacterial microbiota of dental plaques to study the etiology of severe early childhood caries through pyrosequencing analysis based on 16S rRNA gene V1-V3 hypervariable regions. Forty participants were enrolled in the study, and we obtained twenty samples of supragingival plaque from caries-free subjects and twenty samples from subjects with severe early childhood caries. A total of 175,918 reads met the quality control standards, and the bacteria found belonged to fourteen phyla and sixty-three genera. Our results show the overall structure and microbial composition of oral bacterial communities, and they suggest that these bacteria may present a core microbiome in the dental plaque microbiota. Three genera, Streptococcus, Granulicatella, and Actinomyces, were increased significantly in children with severe dental cavities. These data may facilitate improvements in the prevention and treatment of severe early childhood caries.
Full Text Available Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering. Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales. These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.
Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y H; Maguire, Anita J; Kienzle, Marco; Klieve, Athol V
Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.
Preusser, Matthias; Berghoff, Anna S.; Manzl, Claudia; Filipits, Martin; Weinhäusel, Andreas; Pulverer, Walter; Dieckmann, Karin; Widhalm, Georg; Wöhrer, Adelheid; Knosp, Engelbert; Marosi, Christine; Hainfellner, Johannes A.
Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing. PMID:24359605
Im, Sungoh; Lee, Ha-Nul; Jung, Hyun Shin; Yang, Sunghwan; Park, Eun-Jeong; Hwang, Mi Sook; Jeong, Won-Joong; Choi, Dong-Woog
Pyropia tenera (Kjellman) are marine red algae that grow in the intertidal zone and lose more than 90% of water during hibernal low tides every day. In order to identify the desiccation response gene (DRG) in P. tenera, we generated 1,444,210 transcriptome sequences using the 454-FLX platform from the gametophyte under control and desiccation conditions. De novo assembly of the transcriptome reads generated 13,170 contigs, covering about 12 Mbp. We selected 1160 differentially expressed genes (DEGs) in response to desiccation stress based on reads per kilobase per million reads (RPKM) expression values. As shown in green higher plants, DEGs under desiccation are composed of two groups of genes for gene regulation networks and functional proteins for carbohydrate metabolism, membrane perturbation, compatible solutes, and specific proteins similar to higher plants. DEGs that show no significant homology with known sequences in public databases were selected as DRGs in P. tenera. PtDRG2 encodes a novel polypeptide of 159 amino acid residues locating chloroplast. When PtDRG2 was overexpressed in Chlamydomonas, the PtDRG2 confer mannitol and salt tolerance in transgenic cells. These results suggest that Pyropia may possess novel genes that differ from green plants, although the desiccation tolerance mechanism in red algae is similar to those of higher green plants. These transcriptome sequences will facilitate future studies to understand the common processes and novel mechanisms involved in desiccation stress tolerance in red algae.
Full Text Available Abstract Background Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy. Results A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated. Conclusions The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome
Alexandre Augusto Pereira Firmino
Full Text Available Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families' data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.
Firmino, Alexandre Augusto Pereira; Fonseca, Fernando Campos de Assis; de Macedo, Leonardo Lima Pepino; Coelho, Roberta Ramos; Antonino de Souza, José Dijair; Togawa, Roberto Coiti; Silva-Junior, Orzenil Bonfim; Pappas, Georgios Joannis; da Silva, Maria Cristina Mattar; Engler, Gilbert; Grossi-de-Sa, Maria Fatima
Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families' data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.
Full Text Available Ash (Fraxinus spp. is a dominant tree species throughout urban and forested landscapes of North America (NA. The rapid invasion of NA by emerald ash borer (Agrilus planipennis, a wood-boring beetle endemic to Eastern Asia, has resulted in the death of millions of ash trees and threatens billions more. Larvae feed primarily on phloem tissue, which girdles and kills the tree. While NA ash species including black (F. nigra, green (F. pennsylvannica and white (F. americana are highly susceptible, the Asian species Manchurian ash (F. mandshurica is resistant to A. planipennis perhaps due to their co-evolutionary history. Little is known about the molecular genetics of ash. Hence, we undertook a functional genomics approach to identify the repertoire of genes expressed in ash phloem.Using 454 pyrosequencing we obtained 58,673 high quality ash sequences from pooled phloem samples of green, white, black, blue and Manchurian ash. Intriguingly, 45% of the deduced proteins were not significantly similar to any sequences in the GenBank non-redundant database. KEGG analysis of the ash sequences revealed a high occurrence of defense related genes. Expression analysis of early regulators potentially involved in plant defense (i.e. transcription factors, calcium dependent protein kinases and a lipoxygenase 3 revealed higher mRNA levels in resistant ash compared to susceptible ash species. Lastly, we predicted a total of 1,272 single nucleotide polymorphisms and 980 microsatellite loci, among which seven microsatellite loci showed polymorphism between different ash species.The current transcriptomic data provide an invaluable resource for understanding the genetic make-up of ash phloem, the target tissue of A. planipennis. These data along with future functional studies could lead to the identification/characterization of defense genes involved in resistance of ash to A. planipennis, and in future ash breeding programs for marker development.
Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc
Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex
Soderlund, Carol; Nelson, William; Willer, Mark; Gang, David R
The analysis of transcriptome data involves many steps and various programs, along with organization of large amounts of data and results. Without a methodical approach for storage, analysis and query, the resulting ad hoc analysis can lead to human error, loss of data and results, inefficient use of time, and lack of verifiability, repeatability, and extensibility. The Transcriptome Computational Workbench (TCW) provides Java graphical interfaces for methodical analysis for both single and comparative transcriptome data without the use of a reference genome (e.g. for non-model organisms). The singleTCW interface steps the user through importing transcript sequences (e.g. Illumina) or assembling long sequences (e.g. Sanger, 454, transcripts), annotating the sequences, and performing differential expression analysis using published statistical programs in R. The data, metadata, and results are stored in a MySQL database. The multiTCW interface builds a comparison database by importing sequence and annotation from one or more single TCW databases, executes the ESTscan program to translate the sequences into proteins, and then incorporates one or more clusterings, where the clustering options are to execute the orthoMCL program, compute transitive closure, or import clusters. Both singleTCW and multiTCW allow extensive query and display of the results, where singleTCW displays the alignment of annotation hits to transcript sequences, and multiTCW displays multiple transcript alignments with MUSCLE or pairwise alignments. The query programs can be executed on the desktop for fastest analysis, or from the web for sharing the results. It is now affordable to buy a multi-processor machine, and easy to install Java and MySQL. By simply downloading the TCW, the user can interactively analyze, query and view their data. The TCW allows in-depth data mining of the results, which can lead to a better understanding of the transcriptome. TCW is freely available from www.agcol.arizona.edu/software/tcw.
Marcus J Claesson
Full Text Available BACKGROUND: Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. METHODS AND FINDINGS: Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400-1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. CONCLUSIONS: The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate
Xu, Zhifeng; Zhu, Wenyi; Liu, Yanchao; Liu, Xing; Chen, Qiushuang; Peng, Miao; Wang, Xiangzun; Shen, Guangmao; He, Lin
The carmine spider mite (CSM), Tetranychus cinnabarinus, is an important pest mite in agriculture, because it can develop insecticide resistance easily. To gain valuable gene information and molecular basis for the future insecticide resistance study of CSM, the first transcriptome analysis of CSM was conducted. A total of 45,016 contigs and 25,519 unigenes were generated from the de novo transcriptome assembly, and 15,167 unigenes were annotated via BLAST querying against current databases, including nr, SwissProt, the Clusters of Orthologous Groups (COGs), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Aligning the transcript to Tetranychus urticae genome, the 19255 (75.45%) of the transcripts had significant (e-value insecticide resistance in arthropod were generated from CSM transcriptome, including 53 P450-, 22 GSTs-, 23 CarEs-, 1 AChE-, 7 GluCls-, 9 nAChRs-, 8 GABA receptor-, 1 sodium channel-, 6 ATPase- and 12 Cyt b genes. We developed significant molecular resources for T. cinnabarinus putatively involved in insecticide resistance. The transcriptome assembly analysis will significantly facilitate our study on the mechanism of adapting environmental stress (including insecticide) in CSM at the molecular level, and will be very important for developing new control strategies against this pest mite.
Full Text Available Cymbidium ensifolium belongs to the genus Cymbidium of the orchid family. Owing to its spectacular flower morphology, C. ensifolium has considerable ecological and cultural value. However, limited genetic data is available for this non-model plant, and the molecular mechanism underlying floral organ identity is still poorly understood. In this study, we characterize the floral transcriptome of C. ensifolium and present, for the first time, extensive sequence and transcript abundance data of individual floral organs. After sequencing, over 10 Gb clean sequence data were generated and assembled into 111,892 unigenes with an average length of 932.03 base pairs, including 1,227 clusters and 110,665 singletons. Assembled sequences were annotated with gene descriptions, gene ontology, clusters of orthologous group terms, the Kyoto Encyclopedia of Genes and Genomes, and the plant transcription factor database. From these annotations, 131 flowering-associated unigenes, 61 CONSTANS-LIKE (COL unigenes and 90 floral homeotic genes were identified. In addition, four digital gene expression libraries were constructed for the sepal, petal, labellum and gynostemium, and 1,058 genes corresponding to individual floral organ development were identified. Among them, eight MADS-box genes were further investigated by full-length cDNA sequence analysis and expression validation, which revealed two APETALA1/AGL9-like MADS-box genes preferentially expressed in the sepal and petal, two AGAMOUS-like genes particularly restricted to the gynostemium, and four DEF-like genes distinctively expressed in different floral organs. The spatial expression of these genes varied distinctly in different floral mutant corresponding to different floral morphogenesis, which validated the specialized roles of them in floral patterning and further supported the effectiveness of our in silico analysis. This dataset generated in our study provides new insights into the molecular mechanisms
Full Text Available Radish (Raphanus sativus L. is an important worldwide root vegetable crop with high nutrient values and is adversely affected by non-essential heavy metals including chromium (Cr. Little is known about the molecular mechanism underlying Cr stress response in radish. In this study, RNA-Seq technique was employed to identify differentially expressed genes (DEGs under Cr stress. Based on de novo transcriptome assembly, there were 30,676 unigenes representing 60,881 transcripts isolated from radish root under Cr stress. Differential gene analysis revealed that 2,985 uingenes were significantly differentially expressed between Cr-free (CK and Cr-treated (Cr600 libraries, among which 1,424 were up-regulated and 1,561 down-regulated. Gene ontology (GO analysis revealed that these DEGs were mainly involved in primary metabolic process, response to abiotic stimulus, cellular metabolic process and small molecule metabolic process. Kyoto encyclopedia of genes and genomes (KEGG enrichment analysis showed that the DEGs were mainly involved in protein processing in endoplasmic reticulum, starch and sucrose metabolism, amino acid metabolism, glutathione metabolism, drug and xenobiotics by cytochrome P450 metabolism. RT-qPCR analysis showed that the expression patterns of 12 randomly selected DEGs were highly accordant with the results from RNA-seq. Furthermore, many candidate genes including signaling protein kinases, transcription factors and metal transporters, chelate compound biosynthesis and antioxidant system, were involved in defense and detoxification mechanisms of Cr stress response regulatory networks. These results would provide novel insight into molecular mechanism underlying plant responsiveness to Cr stress and facilitate further genetic manipulation on Cr uptake and accumulation in radish.
Full Text Available Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000-20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.
Vannini, Andrea; Bruni, Natalia; Tomassini, Alessia; Franceschini, Selma; Vettraino, Anna Maria
Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Harrison, C.; Musgrave-Brown, E.; Bender, K.
Analysing minute amounts of DNA is a routine challenge in forensics in part due to the poor sensitivity of an instrument and its inability to detect results from forensic samples. In this study, the sensitivity of the Pyrosequencing method is investigated using varying concentrations of DNA and f...
Wolcott Benjamin M
Full Text Available Abstract Background Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D, venous leg ulcers (V, and pressure ulcers (P. Results There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. Conclusion If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in
Full Text Available Breast cancer is the most common malignancy in women worldwide. With the increasing awareness of heterogeneity in breast cancers, better prediction of breast cancer prognosis is much needed for more personalized treatment and disease management. Towards this goal, we have developed a novel computational model for breast cancer prognosis by combining the Pathway Deregulation Score (PDS based pathifier algorithm, Cox regression and L1-LASSO penalization method. We trained the model on a set of 236 patients with gene expression data and clinical information, and validated the performance on three diversified testing data sets of 606 patients. To evaluate the performance of the model, we conducted survival analysis of the dichotomized groups, and compared the areas under the curve based on the binary classification. The resulting prognosis genomic model is composed of fifteen pathways (e.g., P53 pathway that had previously reported cancer relevance, and it successfully differentiated relapse in the training set (log rank p-value = 6.25e-12 and three testing data sets (log rank p-value < 0.0005. Moreover, the pathway-based genomic models consistently performed better than gene-based models on all four data sets. We also find strong evidence that combining genomic information with clinical information improved the p-values of prognosis prediction by at least three orders of magnitude in comparison to using either genomic or clinical information alone. In summary, we propose a novel prognosis model that harnesses the pathway-based dysregulation as well as valuable clinical information. The selected pathways in our prognosis model are promising targets for therapeutic intervention.
Chen, Xiaoping; Zhu, Wei; Azam, Sarwar; Li, Heying; Zhu, Fanghe; Li, Haifen; Hong, Yanbin; Liu, Haiyan; Zhang, Erhua; Wu, Hong; Yu, Shanlin; Zhou, Guiyuan; Li, Shaoxiong; Zhong, Ni; Wen, Shijie; Li, Xingyu; Knapp, Steve J; Ozias-Akins, Peggy; Varshney, Rajeev K; Liang, Xuanqiang
The failure of peg penetration into the soil leads to seed abortion in peanut. Knowledge of genes involved in these processes is comparatively deficient. Here, we used RNA-seq to gain insights into transcriptomes of aerial and subterranean pods. More than 2 million transcript reads with an average length of 396 bp were generated from one aerial (AP) and two subterranean (SP1 and SP2) pod libraries using pyrosequencing technology. After assembly, sets of 49 632, 49 952 and 50 494 from a total of 74 974 transcript assembly contigs (TACs) were identified in AP, SP1 and SP2, respectively. A clear linear relationship in the gene expression level was observed between these data sets. In brief, 2194 differentially expressed TACs with a 99.0% true-positive rate were identified, among which 859 and 1068 TACs were up-regulated in aerial and subterranean pods, respectively. Functional analysis showed that putative function based on similarity with proteins catalogued in UniProt and gene ontology term classification could be determined for 59 342 (79.2%) and 42 955 (57.3%) TACs, respectively. A total of 2968 TACs were mapped to 174 KEGG pathways, of which 168 were shared by aerial and subterranean transcriptomes. TACs involved in photosynthesis were significantly up-regulated and enriched in the aerial pod. In addition, two senescence-associated genes were identified as significantly up-regulated in the aerial pod, which potentially contribute to embryo abortion in aerial pods, and in turn, to cessation of swelling. The data set generated in this study provides evidence for some functional genes as robust candidates underlying aerial and subterranean pod development and contributes to an elucidation of the evolutionary implications resulting from fruit development under light and dark conditions. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen
Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.
Carvalho, Ana Sofia; Ribeiro, Helena; Voabil, Paula
We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We...... questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed....... Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking....
Full Text Available BACKGROUND: Plants selectively attract particular soil microorganisms, in particular consumers of root-excreted compounds. It is unclear to what extent cultivar type and/or growth stage affect this process. METHODOLOGY/PRINCIPAL FINDINGS: DNA-based pyrosequencing was used to characterize the structure of bacterial communities in a field cropped with potato. The rhizospheres of six cultivars denoted Aveka, Aventra, Karnico, Modena, Premiere and Desiree, at three growth stages (young, flowering and senescence were examined, in addition to corresponding bulk soils. Around 350,000 sequences were obtained (5,700 to 38,000 per sample. Across all samples, rank abundance distributions best fitted the power law model, which indicates a community composed of a few highly dominant species next to numerous rare species. Grouping of the sequences showed that members of the Actinobacteria, Alphaproteobacteria, next to as-yet-unclassified bacteria, dominated. Other groups that were consistently found, albeit at lower abundance, were Beta-, Gamma- and Deltaproteobacteria and Acidobacteria. Principal components analyses revealed that rhizosphere samples were significantly different from corresponding bulk soil in each growth stage. Furthermore, cultivar effects were found in the young plant stage, whereas these became insignificant in the flowering and senescence stages. Besides, an effect of time of season was observed for both rhizosphere and bulk soils. The analyzed rhizosphere samples of the potato cultivars were grouped into two groups, in accordance with the allocation of carbon to starch in their tubers, i.e. Aveka, Aventra and Karnico (high versus Premiere and Desiree (low and thus replicates per group were established. CONCLUSIONS: Across all potato cultivars, the young plant stages revealed cultivar-dependent bacterial community structures, which disappeared in the flowering and senescence stages. Furthermore, Pseudomonas, Beta-, Alpha- and
Full Text Available The commercial availability of next generation sequencing (NGS technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of PQQ-dependent methanol dehydrogenase (mxaF has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2, of the NAD-dependent MDH (mdh, of the methanol oxidoreductase of Actinobacteria (mdo, of the fungal FAD-dependent alcohol oxidase (mod1, mod2, and homologues, and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle.
Zhu, Qin; Fisher, Stephen A; Dueck, Hannah; Middleton, Sarah; Khaladkar, Mugdha; Kim, Junhyong
Many R packages have been developed for transcriptome analysis but their use often requires familiarity with R and integrating results of different packages requires scripts to wrangle the datatypes. Furthermore, exploratory data analyses often generate multiple derived datasets such as data subsets or data transformations, which can be difficult to track. Here we present PIVOT, an R-based platform that wraps open source transcriptome analysis packages with a uniform user interface and graphical data management that allows non-programmers to interactively explore transcriptomics data. PIVOT supports more than 40 popular open source packages for transcriptome analysis and provides an extensive set of tools for statistical data manipulations. A graph-based visual interface is used to represent the links between derived datasets, allowing easy tracking of data versions. PIVOT further supports automatic report generation, publication-quality plots, and program/data state saving, such that all analysis can be saved, shared and reproduced. PIVOT will allow researchers with broad background to easily access sophisticated transcriptome analysis tools and interactively explore transcriptome datasets.
Full Text Available BACKGROUND: The analysis of transcriptome data involves many steps and various programs, along with organization of large amounts of data and results. Without a methodical approach for storage, analysis and query, the resulting ad hoc analysis can lead to human error, loss of data and results, inefficient use of time, and lack of verifiability, repeatability, and extensibility. METHODOLOGY: The Transcriptome Computational Workbench (TCW provides Java graphical interfaces for methodical analysis for both single and comparative transcriptome data without the use of a reference genome (e.g. for non-model organisms. The singleTCW interface steps the user through importing transcript sequences (e.g. Illumina or assembling long sequences (e.g. Sanger, 454, transcripts, annotating the sequences, and performing differential expression analysis using published statistical programs in R. The data, metadata, and results are stored in a MySQL database. The multiTCW interface builds a comparison database by importing sequence and annotation from one or more single TCW databases, executes the ESTscan program to translate the sequences into proteins, and then incorporates one or more clusterings, where the clustering options are to execute the orthoMCL program, compute transitive closure, or import clusters. Both singleTCW and multiTCW allow extensive query and display of the results, where singleTCW displays the alignment of annotation hits to transcript sequences, and multiTCW displays multiple transcript alignments with MUSCLE or pairwise alignments. The query programs can be executed on the desktop for fastest analysis, or from the web for sharing the results. CONCLUSION: It is now affordable to buy a multi-processor machine, and easy to install Java and MySQL. By simply downloading the TCW, the user can interactively analyze, query and view their data. The TCW allows in-depth data mining of the results, which can lead to a better understanding of the
Full Text Available The cattle tick of Australia, Rhipicephalus australis, is a vector for microbial parasites that cause serious bovine diseases. The Haller's organ, located in the tick's forelegs, is crucial for host detection and mating. To facilitate the development of new technologies for better control of this agricultural pest, we aimed to sequence and annotate the transcriptome of the R. australis forelegs and associated tissues, including the Haller's organ. As G protein-coupled receptors (GPCRs are an important family of eukaryotic proteins studied as pharmaceutical targets in humans, we prioritized the identification and classification of the GPCRs expressed in the foreleg tissues. The two forelegs from adult R. australis were excised, RNA extracted, and pyrosequenced with 454 technology. Reads were assembled into unigenes and annotated by sequence similarity. Python scripts were written to find open reading frames (ORFs from each unigene. These ORFs were analyzed by different GPCR prediction approaches based on sequence alignments, support vector machines, hidden Markov models, and principal component analysis. GPCRs consistently predicted by multiple methods were further studied by phylogenetic analysis and 3D homology modeling. From 4,782 assembled unigenes, 40,907 possible ORFs were predicted. Using Blastp, Pfam, GPCRpred, TMHMM, and PCA-GPCR, a basic set of 46 GPCR candidates were compiled and a phylogenetic tree was constructed. With further screening of tertiary structures predicted by RaptorX, 6 likely GPCRs emerged and the strongest candidate was classified by PCA-GPCR to be a GABAB receptor.
Jan S Suchodolski
Full Text Available BACKGROUND: Canine idiopathic inflammatory bowel disease (IBD is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6 and dogs with moderate IBD (n = 7 or severe IBD (n = 7 as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001. Proportions of Fusobacteria (p = 0.010, Bacteroidaceae (p = 0.015, Prevotellaceae (p = 0.022, and Clostridiales (p = 0.019 were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044 and Acinetobacter (p = 0.040, were either more abundant or more frequently identified in IBD dogs. CONCLUSIONS/SIGNIFICANCE: In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.
Full Text Available Transcriptomics meta-analysis aims at re-using existing data to derive novel biological hypotheses, and is motivated by the public availability of a large number of independent studies. Current methods are based on breaking down studies into multiple comparisons between phenotypes (e.g. disease vs. healthy, based on the studies' experimental designs, followed by computing the overlap between the resulting differential expression signatures. While useful, in this methodology each study yields multiple independent phenotype comparisons, and connections are established not between studies, but rather between subsets of the studies corresponding to phenotype comparisons. We propose a rank-based statistical meta-analysis framework that establishes global connections between transcriptomics studies without breaking down studies into sets of phenotype comparisons. By using a rank product method, our framework extracts global features from each study, corresponding to genes that are consistently among the most expressed or differentially expressed genes in that study. Those features are then statistically modelled via a term-frequency inverse-document frequency (TF-IDF model, which is then used for connecting studies. Our framework is fast and parameter-free; when applied to large collections of Homo sapiens and Streptococcus pneumoniae transcriptomics studies, it performs better than similarity-based approaches in retrieving related studies, using a Medical Subject Headings gold standard. Finally, we highlight via case studies how the framework can be used to derive novel biological hypotheses regarding related studies and the genes that drive those connections. Our proposed statistical framework shows that it is possible to perform a meta-analysis of transcriptomics studies with arbitrary experimental designs by deriving global expression features rather than decomposing studies into multiple phenotype comparisons.
Full Text Available The carmine spider mite (CSM, Tetranychus cinnabarinus, is an important pest mite in agriculture, because it can develop insecticide resistance easily. To gain valuable gene information and molecular basis for the future insecticide resistance study of CSM, the first transcriptome analysis of CSM was conducted. A total of 45,016 contigs and 25,519 unigenes were generated from the de novo transcriptome assembly, and 15,167 unigenes were annotated via BLAST querying against current databases, including nr, SwissProt, the Clusters of Orthologous Groups (COGs, Kyoto Encyclopedia of Genes and Genomes (KEGG and Gene Ontology (GO. Aligning the transcript to Tetranychus urticae genome, the 19255 (75.45% of the transcripts had significant (e-value <10-5 matches to T. urticae DNA genome, 19111 sequences matched to T. urticae proteome with an average protein length coverage of 42.55%. Core Eukaryotic Genes Mapping Approach (CEGMA analysis identified 435 core eukaryotic genes (CEGs in the CSM dataset corresponding to 95% coverage. Ten gene categories that relate to insecticide resistance in arthropod were generated from CSM transcriptome, including 53 P450-, 22 GSTs-, 23 CarEs-, 1 AChE-, 7 GluCls-, 9 nAChRs-, 8 GABA receptor-, 1 sodium channel-, 6 ATPase- and 12 Cyt b genes. We developed significant molecular resources for T. cinnabarinus putatively involved in insecticide resistance. The transcriptome assembly analysis will significantly facilitate our study on the mechanism of adapting environmental stress (including insecticide in CSM at the molecular level, and will be very important for developing new control strategies against this pest mite.
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
Gross, Joshua B.; Furterer, Allison; Carlson, Brian M.; Stahl, Bethany A.
Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptome analysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F2 pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected in
Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.
Hasegawa, Noriko; Sekizuka, Tsuyoshi; Sugi, Yutaka; Kawakami, Nobuhiro; Ogasawara, Yumiko; Kato, Kengo; Yamashita, Akifumi; Takeuchi, Fumihiko; Kuroda, Makoto
Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin β1 (PSMβ1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections. Copyright © 2017 American Society for Microbiology.
Brulle, Franck [Univ Lille Nord de France, F59000 Lille (France); LGCgE-Lille 1, Ecologie Numerique et Ecotoxicologie, F-59650 Villeneuve d' Ascq (France); Morgan, A. John [Cardiff School of Biosciences, Cardiff University, P.O. Box 915, Cardiff, CF10 3US Wales (United Kingdom); Cocquerelle, Claude [Univ Lille Nord de France, F59000 Lille (France); LGCgE-Lille 1, Ecologie Numerique et Ecotoxicologie, F-59650 Villeneuve d' Ascq (France); Vandenbulcke, Franck, E-mail: firstname.lastname@example.org [Univ Lille Nord de France, F59000 Lille (France); LGCgE-Lille 1, Ecologie Numerique et Ecotoxicologie, F-59650 Villeneuve d' Ascq (France)
Diverse anthropogenic activities often lead to the accumulation of inorganic and organic residues in topsoils. Biota living in close contact with contaminated soils may experience stress at different levels of biological organisation throughout the continuum from the molecular-genetic to ecological and community levels. To date, the relationship between changes at the molecular (mRNA expression) and biochemical/physiological levels evoked by exposures to chemical compounds has been partially established in a limited number of terrestrial invertebrate species. Recently, the advent of a family of transcriptomic tools (e.g. Real-time PCR, Subtractive Suppressive Hybridization, Expressed Sequence Tag sequencing, pyro-sequencing technologies, Microarray chips), together with supporting informatic and statistical procedures, have permitted the robust analyses of global gene expression changes within an ecotoxicological context. This review focuses on how transcriptomics is enlightening our understanding of the molecular-genetic responses of three contrasting terrestrial macroinvertebrate taxa (nematodes, earthworms, and springtails) to inorganics, organics, and agrochemicals. - Environmental toxicology and transcriptomics in soil macroinvertebrates.
Brulle, Franck; Morgan, A. John; Cocquerelle, Claude; Vandenbulcke, Franck
Diverse anthropogenic activities often lead to the accumulation of inorganic and organic residues in topsoils. Biota living in close contact with contaminated soils may experience stress at different levels of biological organisation throughout the continuum from the molecular-genetic to ecological and community levels. To date, the relationship between changes at the molecular (mRNA expression) and biochemical/physiological levels evoked by exposures to chemical compounds has been partially established in a limited number of terrestrial invertebrate species. Recently, the advent of a family of transcriptomic tools (e.g. Real-time PCR, Subtractive Suppressive Hybridization, Expressed Sequence Tag sequencing, pyro-sequencing technologies, Microarray chips), together with supporting informatic and statistical procedures, have permitted the robust analyses of global gene expression changes within an ecotoxicological context. This review focuses on how transcriptomics is enlightening our understanding of the molecular-genetic responses of three contrasting terrestrial macroinvertebrate taxa (nematodes, earthworms, and springtails) to inorganics, organics, and agrochemicals. - Environmental toxicology and transcriptomics in soil macroinvertebrates.
Rask, Thomas Salhøj; Petersen, Bent; Chen, Donald S.
. The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data....... This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene...... family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon...
Carda Diéguez, Miguel; Mira, Alex; Fouz Rodríguez, Belén
The routine use of chemotherapy to control bacterial diseases in aquatic populations has resulted in the development and spread of antibiotic resistance. The inclusion of immunostimulants in fish diets (functional diets) is one of the main strategies to solve this threat. This study aimed to analyse the intestinal microbiota of cultured European sea bass (Dicentrarchus labrax) fed two functional diets applying pyrosequencing of PCR-amplified 16S rRNA gene. Quality-filtered reads were assigned...
Fournier, D.; Hanus, Robert; Roisin, Y.
Roč. 7, č. 2 (2015), s. 521-524 ISSN 1877-7252 Institutional support: RVO:61388963 Keywords : Cavitermes tuberosus * termite * microsatellite * pyrosequencing * population genetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.446, year: 2015
Lee, O. O.; Yang, J.; Bougouffa, S.; Wang, Y.; Batang, Zenon B.; Tian, R.; Al-Suwailem, A.; Qian, P.-Y.
-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than
Baudo, María Marcela; Powers, Stephen J.; Mitchell, Rowan A. C.; Shewry, Peter R.
Regulatory authorities in Western Europe require transgenic crops to be substantially equivalent to conventionally bred forms if they are to be approved for commercial production. One way to establish substantial equivalence is to compare the transcript profiles of developing grain and other tissues of transgenic and conventionally bred lines, in order to identify any unintended effects of the transformation process. We present detailed protocols for transcriptomic comparisons of developing wheat grain and leaf material, and illustrate their use by reference to our own studies of lines transformed to express additional gluten protein genes controlled by their own endosperm-specific promoters. The results show that the transgenes present in these lines (which included those encoding marker genes) did not have any significant unpredicted effects on the expression of endogenous genes and that the transgenic plants were therefore substantially equivalent to the corresponding parental lines.
Rodd F Helen
Full Text Available Abstract Background Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. Results We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs, and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in
A Mark Ibekwe
Full Text Available Current microbial source tracking (MST methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs, recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP, Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%, agricultural runoff sediment (6.52%, and Prado Park sediment (6.00%, respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78-4.08%. Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.
Keskin, Cangül; Demiryürek, Ebru Özsezer; Onuk, Ertan Emek
This study aimed to characterize the microbial communities of primary and secondary/persistent endodontic infections using high-throughput pyrosequencing from the pulverized samples. The roots of 20 extracted human teeth with primary endodontic infection and 20 teeth with secondary/persistent endodontic infection were collected. The outer surfaces of the roots were disinfected, and whole roots were cryopulverized. 16S amplicon pyrosequencing data from the DNA extracted from the pulverized root powders were obtained, and microorganism abundance and diversity were calculated. Data were analyzed using statistical and bioinformatic methods. Pyrosequencing analysis resulted a total of 2,606,128 sequences from 40 samples. A total of 15 phyla, 160 genera, and 368 species were detected. No significant difference between primary and secondary/persistent endodontic infections was found regarding the diversity and richness of operational taxonomic units at the phyla, genera, and species levels (P > .005). The present study revealed that the microbial diversity of secondary/persistent endodontic infections did not differ than those of primary endodontic infections. A new archaeal species, Candidatus Nitrosoarchaeum limnia, was detected in root canals of 1 patient with primary endodontic infection for the first time. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.
Heyland, Andreas; Vue, Zer; Voolstra, Christian R.; Medina, Mó nica; Moroz, Leonid L.
developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms
Phylogenetic studies of concrete biofilms using 16SrRNA-based approaches have demonstrated that concrete surfaces harbor a diverse microbial community. These approaches can provide information on the general taxonomical groups present in a sample but cannot shed light on the func...
Full Text Available Next generation sequencing (NGS technologies have greatly changed the landscape of transcriptomic studies of non-model organisms. Since there is no reference genome available, de novo assembly methods play key roles in the analysis of these data sets. Because of the huge amount of data generated by NGS technologies for each run, many assemblers, e.g., ABySS, Velvet and Trinity, are developed based on a de Bruijn graph due to its time- and space-efficiency. However, most of these assemblers were developed initially for the Illumina/Solexa platform. The performance of these assemblers on 454 transcriptomic data is unknown. In this study, we evaluated and compared the relative performance of these de Bruijn graph based assemblers on both simulated and real 454 transcriptomic data. The results suggest that Trinity, the Illumina/Solexa-specialized transcriptomic assembler, performs the best among the multiple de Bruijn graph assemblers, comparable to or even outperforming the standard 454 assembler Newbler which is based on the overlap-layout-consensus algorithm. Our evaluation is expected to provide helpful guidance for researchers to choose assemblers when analyzing 454 transcriptomic data.
Martins, Maria João F
Euphausia superba is a keystone species in Antarctic food webs. However, the continued decrease in stock density raises concerns over the resilience and adaptive potential of krill to withstand the current rate of environmental change. We undertook a transcriptome-scale approach (454 pyrosequencing) as a baseline for future studies addressing the physiological response of krill to short-term food shortage and natural UV-B stress. The final assembly resulted in a total of 26,415 contigs, 39.8% of which were putatively annotated. Exploratory analyses indicate an overall reduction in protein synthesis under food shortage while UV stress resulted in the activation of photo-protective mechanisms. © 2015.
Martins, Maria Joã o F; Lago-Leston, Asuncion; Anjos, Antonio; Duarte, Carlos M.; Agusti, Susana; Serrã o, Ester A.; Pearson, Gareth A.
Euphausia superba is a keystone species in Antarctic food webs. However, the continued decrease in stock density raises concerns over the resilience and adaptive potential of krill to withstand the current rate of environmental change. We undertook a transcriptome-scale approach (454 pyrosequencing) as a baseline for future studies addressing the physiological response of krill to short-term food shortage and natural UV-B stress. The final assembly resulted in a total of 26,415 contigs, 39.8% of which were putatively annotated. Exploratory analyses indicate an overall reduction in protein synthesis under food shortage while UV stress resulted in the activation of photo-protective mechanisms. © 2015.
Full Text Available Elettaria cardamomum (L. Maton, known as ‘queen of spices, is a perennial herbaceous monocot of the family Zingiberaceae, native to southern India. Cardamom is an economically valuable spice crop and used widely in culinary and medicinal purposes. In the present study, using Ion Proton RNA sequencing technology, we performed transcriptome sequencing and de novo transcriptome assembly of a wild and five cultivar genotypes of cardamom. RNA-seq generated a total of 22,811,983 (92 base and 24,889,197 (75 base raw reads accounting for approximately 8.21GB and 7.65GB of sequence data for wild and cultivar genotypes of cardamom respectively. The raw data were submitted to SRA database of NCBI under the accession numbers SRX1141272 (wild and SRX1141276 (cultivars. The raw reads were quality filtered and assembled using MIRA assembler resulted with 112,208 and 264,161contigs having N50 value 616 and 664 for wild and cultivar cardamom respectively. The assembled unigenes were functionally annotated using several databases including PlantCyc for pathway annotation. This work represents the first report on cardamom transcriptome sequencing. In order to generate a comprehensive reference transcriptome, we further assembled the raw reads of wild and cultivar genotypes which might enrich the plant transcriptome database and trigger advanced research in cardamom genomics.
Torales Susana L
Full Text Available Abstract Background Nothofagus nervosa is one of the most emblematic native tree species of Patagonian temperate forests. Here, the shotgun RNA-sequencing (RNA-Seq of the transcriptome of N. nervosa, including de novo assembly, functional annotation, and in silico discovery of potential molecular markers to support population and associations genetic studies, are described. Results Pyrosequencing of a young leaf cDNA library generated a total of 111,814 high quality reads, with an average length of 447 bp. De novo assembly using Newbler resulted into 3,005 tentative isotigs (including alternative transcripts. The non-assembled sequences (singletons were clustered with CD-HIT-454 to identify natural and artificial duplicates from pyrosequencing reads, leading to 21,881 unique singletons. 15,497 out of 24,886 non-redundant sequences or unigenes, were successfully annotated against a plant protein database. A substantial number of simple sequence repeat markers (SSRs were discovered in the assembled and annotated sequences. More than 40% of the SSR sequences were inside ORF sequences. To confirm the validity of these predicted markers, a subset of 73 SSRs selected through functional annotation evidences were successfully amplified from six seedlings DNA samples, being 14 polymorphic. Conclusions This paper is the first report that shows a highly precise representation of the mRNAs diversity present in young leaves of a native South American tree, N. nervosa, as well as its in silico deduced putative functionality. The reported Nothofagus transcriptome sequences represent a unique resource for genetic studies and provide a tool to discover genes of interest and genetic markers that will greatly aid questions involving evolution, ecology, and conservation using genetic and genomic approaches in the genus.
Full Text Available Abstract Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays, implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile, useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene
Turpin, Williams; Weiman, Marion; Guyot, Jean-Pierre; Lajus, Aurélie; Cruveiller, Stéphane; Humblot, Christèle
The objective of this work was to investigate the nutritional potential of Lactobacillus plantarum A6 in a food matrix using next generation sequencing. To this end, we characterized the genome of the A6 strain for a complete overview of its potential. We then compared its transcriptome when grown in a food matrix made from pearl millet to and its transcriptome when cultivated in a laboratory medium. Genomic comparison of the strain L. plantarum A6 with the strains WCFS1, ST-III, JDM1 and ATCC14917 led to the identification of five regions of genomic plasticity. More specifically, 362 coding sequences, mostly annotated as coding for proteins of unknown functions, were specific to L. plantarum A6. A total of 1201 genes were significantly differentially expressed in laboratory medium and food matrix. Among them, 821 genes were up-regulated in the food matrix compared to the laboratory medium, representing 23% of whole genomic objects. In the laboratory medium, the expression of 380 genes, representing 11% of the all genomic objects was at least double than in the food matrix. Genes encoding important functions for the nutritional quality of the food were identified. Considering its efficiency as an amylolytic strain, we investigated all genes involved in carbohydrate metabolism, paying particular attention to starch metabolism. An extracellular alpha amylase, a neopullulanase and maltodextrin transporters were identified, all of which were highly expressed in the food matrix. In addition, genes involved in alpha-galactoside metabolism were identified but only two of them were induced in food matrix than in laboratory medium. This may be because alpha galactosides were already eliminated during soaking. Different biosynthetic pathways involved in the synthesis of vitamin B (folate, riboflavin, and cobalamin) were identified. They allowed the identification of a potential of vitamin synthesis, which should be confirmed through biochemical analysis in further work
Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie
Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.
Boissy, Robert J; Romberger, Debra J; Roughead, William A; Weissenburger-Moser, Lisa; Poole, Jill A; LeVan, Tricia D
Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses
Robert J Boissy
Full Text Available Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance. The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70% at the phylum level, Clostridia (44% at the Class level, and Clostridiales at the Order level (41%. In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the
Domínguez-Contreras, J. F.; Munguía-Vega, A.; Ceballos-Vázquez, B. P.; Arellano-Martínez, M.; Culver, Melanie
We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum, respectively.
Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W
We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.
The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.
Lopes, Ana R; Manaia, Célia M; Nunes, Olga C
Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Milanović, Vesna; Osimani, Andrea; Taccari, Manuela; Garofalo, Cristiana; Butta, Alessandro; Clementi, Francesca; Aquilanti, Lucia
The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.
Takeshita, Toru; Yasui, Masaki; Shibata, Yukie; Furuta, Michiko; Saeki, Yoji; Eshima, Nobuoki; Yamashita, Yoshihisa
Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience.
Kang, Jung-Ha; Park, Jung-Youn; Jo, Hyun-Su
The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1–10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy–Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni’s correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species. PMID:22837688
Kang, Jung-Ha; Park, Jung-Youn; Jo, Hyun-Su
The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.
Full Text Available The melon ( L. fruit is an important crop and model system for the genomic study of both fleshy fruit development and the Cucurbitaceae family. To obtain an accurate representation of the melon fruit transcriptome based on expressed sequence tag (EST abundance in 454-pyrosequencing data, we prepared double-stranded complementary DNA (cDNA of melon without the usual amplification and normalization steps. A purification step was also included to eliminate small fragments. Complementary DNAs were obtained from 14 individual fruit libraries derived from two genotypes, separated into flesh and peel tissues, and sampled throughout fruit development. Pyrosequencing was performed using Genome Sequencer FLX (GS FLX technology, resulting in 1,215,359 reads, with mean length of >200 nucleotides. The global digital expression data was validated by comparative reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR of 40 selected genes and expression patterns were similar for the two methods. The results indicate that high-quality, nonbiased cDNA for next-generation sequencing can be prepared from mature, fleshy fruit, which are notorious for difficulties in ribonucleic acid (RNA preparation.
Ribeiro, José M. C.; Genta, Fernando A.; Sorgine, Marcos H. F.; Logullo, Raquel; Mesquita, Rafael D.; Paiva-Silva, Gabriela O.; Majerowicz, David; Medeiros, Marcelo; Koerich, Leonardo; Terra, Walter R.; Ferreira, Clélia; Pimentel, André C.; Bisch, Paulo M.; Leite, Daniel C.; Diniz, Michelle M. P.; Junior, João Lídio da S. G. V.; Da Silva, Manuela L.; Araujo, Ricardo N.; Gandara, Ana Caroline P.; Brosson, Sébastien; Salmon, Didier; Bousbata, Sabrina; González-Caballero, Natalia; Silber, Ariel Mariano; Alves-Bezerra, Michele; Gondim, Katia C.; Silva-Neto, Mário Alberto C.; Atella, Georgia C.; Araujo, Helena; Dias, Felipe A.; Polycarpo, Carla; Vionette-Amaral, Raquel J.; Fampa, Patrícia; Melo, Ana Claudia A.; Tanaka, Aparecida S.; Balczun, Carsten; Oliveira, José Henrique M.; Gonçalves, Renata L. S.; Lazoski, Cristiano; Rivera-Pomar, Rolando; Diambra, Luis; Schaub, Günter A.; Garcia, Elói S.; Azambuja, Patrícia; Braz, Glória R. C.; Oliveira, Pedro L.
The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7–8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for
José M C Ribeiro
Full Text Available The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM, protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM. As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated
Schramm, Katharina; Adamski, Jerzy; Gieger, Christian; Herder, Christian; Carstensen, Maren; Peters, Annette; Rathmann, Wolfgang; Roden, Michael; Strauch, Konstantin; Suhre, Karsten; Kastenmüller, Gabi; Prokisch, Holger; Theis, Fabian J.
Biological systems consist of multiple organizational levels all densely interacting with each other to ensure function and flexibility of the system. Simultaneous analysis of cross-sectional multi-omics data from large population studies is a powerful tool to comprehensively characterize the underlying molecular mechanisms on a physiological scale. In this study, we systematically analyzed the relationship between fasting serum metabolomics and whole blood transcriptomics data from 712 individuals of the German KORA F4 cohort. Correlation-based analysis identified 1,109 significant associations between 522 transcripts and 114 metabolites summarized in an integrated network, the ‘human blood metabolome-transcriptome interface’ (BMTI). Bidirectional causality analysis using Mendelian randomization did not yield any statistically significant causal associations between transcripts and metabolites. A knowledge-based interpretation and integration with a genome-scale human metabolic reconstruction revealed systematic signatures of signaling, transport and metabolic processes, i.e. metabolic reactions mainly belonging to lipid, energy and amino acid metabolism. Moreover, the construction of a network based on functional categories illustrated the cross-talk between the biological layers at a pathway level. Using a transcription factor binding site enrichment analysis, this pathway cross-talk was further confirmed at a regulatory level. Finally, we demonstrated how the constructed networks can be used to gain novel insights into molecular mechanisms associated to intermediate clinical traits. Overall, our results demonstrate the utility of a multi-omics integrative approach to understand the molecular mechanisms underlying both normal physiology and disease. PMID:26086077
Full Text Available Biological systems consist of multiple organizational levels all densely interacting with each other to ensure function and flexibility of the system. Simultaneous analysis of cross-sectional multi-omics data from large population studies is a powerful tool to comprehensively characterize the underlying molecular mechanisms on a physiological scale. In this study, we systematically analyzed the relationship between fasting serum metabolomics and whole blood transcriptomics data from 712 individuals of the German KORA F4 cohort. Correlation-based analysis identified 1,109 significant associations between 522 transcripts and 114 metabolites summarized in an integrated network, the 'human blood metabolome-transcriptome interface' (BMTI. Bidirectional causality analysis using Mendelian randomization did not yield any statistically significant causal associations between transcripts and metabolites. A knowledge-based interpretation and integration with a genome-scale human metabolic reconstruction revealed systematic signatures of signaling, transport and metabolic processes, i.e. metabolic reactions mainly belonging to lipid, energy and amino acid metabolism. Moreover, the construction of a network based on functional categories illustrated the cross-talk between the biological layers at a pathway level. Using a transcription factor binding site enrichment analysis, this pathway cross-talk was further confirmed at a regulatory level. Finally, we demonstrated how the constructed networks can be used to gain novel insights into molecular mechanisms associated to intermediate clinical traits. Overall, our results demonstrate the utility of a multi-omics integrative approach to understand the molecular mechanisms underlying both normal physiology and disease.
He, Liming; Liu, Fang; Karuppiah, Valliappan; Ren, Yi; Li, Zhiyong
To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.
Ye, Lin; Zhang, Tong
This study applied 454 high-throughput pyrosequencing to analyze potentially pathogenic bacteria in activated sludge from 14 municipal wastewater treatment plants (WWTPs) across four countries (China, U.S., Canada, and Singapore), plus the influent and effluent of one of the 14 WWTPs. A total of 370,870 16S rRNA gene sequences with average length of 207 bps were obtained and all of them were assigned to corresponding taxonomic ranks by using RDP classifier and MEGAN. It was found that the most abundant potentially pathogenic bacteria in the WWTPs were affiliated with the genera of Aeromonas and Clostridium. Aeromonas veronii, Aeromonas hydrophila, and Clostridium perfringens were species most similar to the potentially pathogenic bacteria found in this study. Some sequences highly similar (>99%) to Corynebacterium diphtheriae were found in the influent and activated sludge samples from a saline WWTP. Overall, the percentage of the sequences closely related (>99%) to known pathogenic bacteria sequences was about 0.16% of the total sequences. Additionally, a platform-independent Java application (BAND) was developed for graphical visualization of the data of microbial abundance generated by high-throughput pyrosequencing. The approach demonstrated in this study could examine most of the potentially pathogenic bacteria simultaneously instead of one-by-one detection by other methods.
Sundberg, Carina; Al-Soud, Waleed A; Larsson, Madeleine; Alm, Erik; Yekta, Sepehr S; Svensson, Bo H; Sørensen, Søren J; Karlsson, Anna
The microbial community of 21 full-scale biogas reactors was examined using 454 pyrosequencing of 16S rRNA gene sequences. These reactors included seven (six mesophilic and one thermophilic) digesting sewage sludge (SS) and 14 (ten mesophilic and four thermophilic) codigesting (CD) various combinations of wastes from slaughterhouses, restaurants, households, etc. The pyrosequencing generated more than 160,000 sequences representing 11 phyla, 23 classes, and 95 genera of Bacteria and Archaea. The bacterial community was always both more abundant and more diverse than the archaeal community. At the phylum level, the foremost populations in the SS reactors included Actinobacteria, Proteobacteria, Chloroflexi, Spirochetes, and Euryarchaeota, while Firmicutes was the most prevalent in the CD reactors. The main bacterial class in all reactors was Clostridia. Acetoclastic methanogens were detected in the SS, but not in the CD reactors. Their absence suggests that methane formation from acetate takes place mainly via syntrophic acetate oxidation in the CD reactors. A principal component analysis of the communities at genus level revealed three clusters: SS reactors, mesophilic CD reactors (including one thermophilic CD and one SS), and thermophilic CD reactors. Thus, the microbial composition was mainly governed by the substrate differences and the process temperature. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Mark, Kristiina; Cornejo, Carolina; Keller, Christine; Flück, Daniela; Scheidegger, Christoph
Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).
Sun, Wei; Zhang, Fengli; He, Liming; Li, Zhiyong
Diverse sessile organisms inhabit the coral reef ecosystems, including corals, sponges, and sea anemones. In the past decades, scleractinian corals (Cnidaria, Anthozoa, Scleractinia) and their associated microorganisms have attracted much attention. Zoanthids (Cnidaria, Anthozoa, Zoanthidea) are commonly found in coral reefs. However, little is known about the community structure of zoanthid-associated microbiota. In this study, the microbial community associated with the zoanthid Palythoa australiae in the South China Sea was investigated by 454 pyrosequencing. As a result, 2,353 bacterial, 583 archaeal, and 36 eukaryotic microbial ribotypes were detected, respectively. A total of 22 bacterial phyla (16 formally described phyla and six candidate phyla) were recovered. Proteobacteria was the most abundant group, followed by Chloroflexi and Actinobacteria. High-abundance Rhizobiales and diverse Chloroflexi were observed in the bacterial community. The archaeal population was composed of Crenarchaeota and Euryarchaeota, with Marine Group I as the dominant lineage. In particular, Candidatus Nitrosopumilus dominated the archaeal community. Besides bacteria and archaea, the zoanthid harbored eukaryotic microorganisms including fungi and algae though their diversity was very low. This study provided the first insights into the microbial community associated with P. australiae by 454 pyrosequencing, consequently laid a basis for the understanding of the association of P. australiae-microbes symbioses.
Gallagher Margaret L
Full Text Available Abstract Background Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1–2 are derived from CYP2A7, and exons 3–9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. Methods A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow. Results Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics. Conclusion This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.
Prest, Emmanuelle I E C
The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.
Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S
The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.
Cabrera, Ana R; Donohue, Kevin V; Khalil, Sayed M S; Scholl, Elizabeth; Opperman, Charles; Sonenshine, Daniel E; Roe, R Michael
Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function. 2010 Elsevier Ltd. All rights reserved.
Full Text Available Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn's disease and ulcerative colitis (UC. Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS and 13 Fusobacterium adhesion (FadA paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.
Guo, Qi; Wang, Jin-Xing; Su, Li-Zhuo; Lv, Wei; Sun, Yu-Han; Li, Yun
Black locust ( Robinia pseudoacacia L. of the family Fabaceae) is an ecologically and economically important deciduous tree. However, few genomic resources are available for this forest species, and few effective expressed sequence tag-derived simple sequence repeat (EST-SSR) markers have been developed to date. In this study, paired-end sequencing was used to sequence transcriptomes of R. pseudoacacia by the Illumina HiSeq TM2000 platform, and EST-SSR loci were identified by de novo assembly. Furthermore, a total of 1697 primer pairs were successfully designed, from which 286 primers met the selection screening criteria; 94 pairs were randomly selected and tested for validation using polymerase chain reaction amplification. Forty-five primers were verified as polymorphic, with clear bands. The polymorphism information content values were 0.033-0.765, the number of alleles per locus ranged from 2 to 10, and the observed and expected heterozygosities were 0.000-0.931 and 0.035-0.810, respectively, indicating a high level of informativeness. Subsequently, 45 polymorphic EST-SSR loci were tested for amplification efficiency, using the verified primers, in an additional nine species of Leguminosae, 23 loci were amplified in more than three species, of which two loci were amplified successfully in all species. These EST-SSR markers provide a valuable tool for investigating the genetic diversity and population structure of R . pseudoacacia , constructing a DNA fingerprint database, performing quantitative trait locus mapping, and preserving genetic information.
Cárdenas, Leyla; Sánchez, Roland; Gomez, Daniela; Fuenzalida, Gonzalo; Gallardo-Escárate, Cristián; Tanguy, Arnaud
The marine gastropod Concholepas concholepas, locally known as the "loco", is the main target species of the benthonic Chilean fisheries. Genetic and genomic tools are necessary to study the genome of this species in order to understand the molecular basis of its development, growth, and other key traits to improve the management strategies and to identify local adaptation to prevent loss of biodiversity. Here, we use pyrosequencing technologies to generate the first transcriptomic database from adult specimens of the loco. After trimming, a total of 140,756 Expressed Sequence Tag sequences were achieved. Clustering and assembly analysis identified 19,219 contigs and 105,435 singleton sequences. BlastN analysis showed a significant identity with Expressed Sequence Tags of different gastropod species available in public databases. Similarly, BlastX results showed that only 895 out of the total 124,654 had significant hits and may represent novel genes for marine gastropods. From this database, simple sequence repeat motifs were also identified and a total of 38 primer pairs were designed and tested to assess their potential as informative markers and to investigate their cross-species amplification in different related gastropod species. This dataset represents the first publicly available 454 data for a marine gastropod endemic to the southeastern Pacific coast, providing a valuable transcriptomic resource for future efforts of gene discovery and development of functional markers in other marine gastropods. Copyright © 2011 Elsevier B.V. All rights reserved.
Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist
ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus, which were encountered in some cheese samples at low percentages compared with the total bacterial populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient method...
Full Text Available Next generation sequencing platforms have recently been used to rapidly characterize transcriptome sequences from a number of non-model organisms. The present study compares two of the most frequently used platforms, the Roche 454-pyrosequencing and the Illumina sequencing-by-synthesis (SBS, on the same RNA sample obtained from an intertidal gastropod mollusc species, Haliotis midae. All the sequencing reads were deposited in the Short Read Archive (SRA database are retrievable under the accession number [SRR071314 (Illumina Genome Analyzer II] and [SRR1737738, SRR1737737, SRR1737735, SRR1737734 (454 GS FLX] in the SRA database of NCBI. Three transcriptomes, composed of either pure 454 or Illumina reads or a mixture of read types (Hybrid, were assembled using CLC Genomics Workbench software. Illumina assemblies performed the best de novo transcriptome characterization in terms of contig length, whereas the 454 assemblies tended to improve the complete assembly of gene transcripts. Both the Hybrid and Illumina assemblies produced longer contigs covering more of the transcriptome than 454 assemblies. However, the addition of 454 significantly increased the number of genes annotated.
Greene Jonathan R
Full Text Available Abstract Background Genome wide transcriptome maps can provide tools to identify candidate genes that are over-expressed or silenced in certain disease tissue and increase our understanding of the structure and organization of the genome. Expressed Sequence Tags (ESTs from the public dbEST and proprietary Incyte LifeSeq databases were used to derive a transcript map in conjunction with the working draft assembly of the human genome sequence. Results Examination of ESTs derived from brain tissues (excluding brain tumor tissues suggests that these genes are distributed on chromosomes in a non-random fashion. Some regions on the genome are dense with brain-enriched genes while some regions lack brain-enriched genes, suggesting a significant correlation between distribution of genes along the chromosome and tissue type. ESTs from brain tumor tissues have also been mapped to the human genome working draft. We reveal that some regions enriched in brain genes show a significant decrease in gene expression in brain tumors, and, conversely that some regions lacking in brain genes show an increased level of gene expression in brain tumors. Conclusions This report demonstrates a novel approach for tissue specific transcriptome mapping using EST-based quantitative assessment.
University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.; Carlson, Joseph W.; Duff, Michael O.; Landolin, Jane M.; Yang, Li; Artieri, Carlo G.; van Baren, Marijke J.; Boley, Nathan; Booth, Benjamin W.; Brown, James B.; Cherbas, Lucy; Davis, Carrie A.; Dobin, Alex; Li, Renhua; Lin, Wei; Malone, John H.; Mattiuzzo, Nicolas R.; Miller, David; Sturgill, David; Tuch, Brian B.; Zaleski, Chris; Zhang, Dayu; Blanchette, Marco; Dudoit, Sandrine; Eads, Brian; Green, Richard E.; Hammonds, Ann; Jiang, Lichun; Kapranov, Phil; Langton, Laura; Perrimon, Norbert; Sandler, Jeremy E.; Wan, Kenneth H.; Willingham, Aarron; Zhang, Yu; Zou, Yi; Andrews, Justen; Bicke, Peter J.; Brenner, Steven E.; Brent, Michael R.; Cherbas, Peter; Gingeras, Thomas R.; Hoskins, Roger A.; Kaufman, Thomas C.; Oliver, Brian; Celniker, Susan E.
Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development. Drosophila melanogaster is an important non-mammalian model system that has had a critical role in basic biological discoveries, such as identifying chromosomes as the carriers of genetic information and uncovering the role of genes in development. Because it shares a substantial genic content with humans, Drosophila is increasingly used as a translational model for human development, homeostasis and disease. High-quality maps are needed for all functional genomic elements. Previous studies demonstrated that a rich collection of genes is deployed during the life cycle of the fly. Although expression profiling using microarrays has revealed the expression of, 13,000 annotated genes, it is difficult to map splice junctions and individual base modifications generated by RNA editing using such approaches. Single-base resolution is essential to define precisely the elements that comprise the Drosophila transcriptome. Estimates of the number of transcript isoforms are less accurate than estimates of the number of genes
Wang, Bin; Lv, Yangyong; Li, Xuejie; Lin, Yiying; Deng, Hai; Pan, Li
The global regulator LaeA controls the production of many fungal secondary metabolites, possibly via chromatin remodeling. Here we aimed to survey the secondary metabolite profile regulated by LaeA in Aspergillus niger FGSC A1279 by genome sequencing and comparative transcriptomics between the laeA deletion (ΔlaeA) and overexpressing (OE-laeA) mutants. Genome sequencing revealed four putative polyketide synthase genes specific to FGSC A1279, suggesting that the corresponding polyketide compounds might be unique to FGSC A1279. RNA-seq data revealed 281 putative secondary metabolite genes upregulated in the OE-laeA mutants, including 22 secondary metabolite backbone genes. LC-MS chemical profiling illustrated that many secondary metabolites were produced in OE-laeA mutants compared to wild type and ΔlaeA mutants, providing potential resources for drug discovery. KEGG analysis annotated 16 secondary metabolite clusters putatively linked to metabolic pathways. Furthermore, 34 of 61 Zn 2 Cys 6 transcription factors located in secondary metabolite clusters were differentially expressed between ΔlaeA and OE-laeA mutants. Three secondary metabolite clusters (cluster 18, 30 and 33) containing Zn 2 Cys 6 transcription factors that were upregulated in OE-laeA mutants were putatively linked to KEGG pathways, suggesting that Zn 2 Cys 6 transcription factors might play an important role in synthesizing secondary metabolites regulated by LaeA. Taken together, LaeA dramatically influences the secondary metabolite profile in FGSC A1279. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Nuruzzaman, Mohammed; Cao, Hongzhe; Xiu, Hao; Luo, Tiao; Li, Jijia; Chen, Xianghui; Luo, Junli; Luo, Zhiyong
WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Scott A. Ochsner
Full Text Available Public transcriptomic assets in the nuclear receptor (NR signaling field hold considerable collective potential for exposing underappreciated aspects of NR regulation of gene expression. This potential is undermined however by a series of enduring informatic pain points that retard the routine re-use of these datasets. Here we describe a coordinated biocuration and web development approach to redress this situation that is closely aligned with ideals articulated in the FAIR (findable, accessible, interoperable, re-usable principles on data stewardship. To improve findability, biocurators engage authors of studies in collaborating journals to secure datasets for deposition in public archives. Annotated derivatives of the archived datasets are assigned digital object identifiers and regulatory molecule identifiers that support persistent linkages between datasets and their associated research articles, integration in relevant records in gene and small molecule knowledgebases, and indexing by dataset search engines. To enhance their accessibility and interoperability, datasets are visualizable in responsively designed web pages, retrievable in machine-readable spreadsheets, or through an application programming interface. Re-use of the datasets is supported by their interrogation as a universe of data points through the Transcriptomine search engine, highlighting transcriptional intersections between NR signaling pathways, physiological processes and disease states. We illustrate the value of our approach in connecting disparate research communities using a use case of persistent interoperability between the Nuclear Receptor Signaling Atlas and the Pharmacogenomics Knowledgebase. Our FAIR-aligned model demonstrates the enduring value of discovery-scale datasets that accrues from their systematic compilation, biocuration and distribution across the digital biomedical research enterprise.
Full Text Available Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs. From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1-3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51% under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.
Chen, Jingfang; Li, Ronghua; Xia, Yanshi; Bai, Guihua; Guo, Peiguo; Wang, Zhiliang; Zhang, Hua; Siddique, Kadambot H M
Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1-3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.
Větrovský, Tomáš; Baldrian, Petr
Roč. 49, č. 8 (2013), s. 1027-1037 ISSN 0178-2762 R&D Projects: GA MŠk LD12050; GA MŠk LD12048; GA ČR GAP504/12/0709 Institutional support: RVO:61388971 Keywords : Fungal community * Internal transcribed spacer * Pyrosequencing pipeline Subject RIV: EE - Microbiology, Virology Impact factor: 3.396, year: 2013
Pu, Jian; Kazama, Shinobu; Miura, Takayuki; Azraini, Nabila Dhyan; Konta, Yoshimitsu; Ito, Hiroaki; Ueki, You; Cahyaningrum, Ermaya Eka; Omura, Tatsuo; Watanabe, Toru
Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.
Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia
There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336
Vettraino, A M; Bonants, P; Tomassini, A; Bruni, N; Vannini, A
To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%) Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.
Reyes, Carlen; Dellwig, Olaf; Dähnke, K.
To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5-1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing....
Chen, Shuangyan [Chinese Academy of Sciences (CAS), Institute of Botany (IB), Beijing; Huang, Xin [Chinese Academy of Sciences (CAS), Institute of Botany (IB), Beijing; Yang, Xiaohan [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Liu, Gongshe [Chinese Academy of Sciences (CAS), Institute of Botany (IB), Beijing
BACKGROUND: Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. RESULTS: The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. CONCLUSIONS: This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.
Chen, Shuangyan; Huang, Xin; Yan, Xueqing; Liang, Ye; Wang, Yuezhu; Li, Xiaofeng; Peng, Xianjun; Ma, Xingyong; Zhang, Lexin; Cai, Yueyue; Ma, Tian; Cheng, Liqin; Qi, Dongmei; Zheng, Huajun; Yang, Xiaohan; Li, Xiaoxia; Liu, Gongshe
Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.
Man Kit Cheung
Full Text Available BACKGROUND: Tuberculosis (TB remains a global threat in the 21st century. Traditional studies of the disease are focused on the single pathogen Mycobacterium tuberculosis. Recent studies have revealed associations of some diseases with an imbalance in the microbial community. Characterization of the TB microbiota could allow a better understanding of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Here, the sputum microbiota in TB infection was examined by using 16S rRNA pyrosequencing. A total of 829,873 high-quality sequencing reads were generated from 22 TB and 14 control sputum samples. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were the five major bacterial phyla recovered, which together composed over 98% of the microbial community. Proteobacteria and Bacteroidetes were more represented in the TB samples and Firmicutes was more predominant in the controls. Sixteen major bacterial genera were recovered. Streptococcus, Neisseria and Prevotella were the most predominant genera, which were dominated by several operational taxonomic units grouped at a 97% similarity level. Actinomyces, Fusobacterium, Leptotrichia, Prevotella, Streptococcus, and Veillonella were found in all TB samples, possibly representing the core genera in TB sputum microbiota. The less represented genera Mogibacterium, Moryella and Oribacterium were enriched statistically in the TB samples, while a genus belonging to the unclassified Lactobacillales was enriched in the controls. The diversity of microbiota was similar in the TB and control samples. CONCLUSIONS/SIGNIFICANCE: The composition and diversity of sputum microbiota in TB infection was characterized for the first time by using high-throughput pyrosequencing. It lays the framework for examination of potential roles played by the diverse microbiota in TB pathogenesis and progression, and could ultimately facilitate advances in TB treatment.
Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W
Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.
Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Lieberman, Isador H; Krebs, Viktor; Togawa, Daisuke; Fujishiro, Takaaki; Procop, Gary W
We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.
Full Text Available Objective The gastrointestinal tract of sheep contain complex microbial communities that influence numerous aspects of the sheep’s health and development. The objective of this study was to analyze the composition and diversity of the microbiota in the gastrointestinal tract sections (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum of sheep. Methods This analysis was performed by 454 pyrosequencing using the V3-V6 region of the 16S rRNA genes. Samples were collected from five healthy, small tailed Han sheep aged 10 months, obtained at market. The bacterial composition of sheep gastrointestinal microbiota was investigated at the phylum, class, order, family, genus, and species levels. Results The dominant bacterial phyla in the entire gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. In the stomach, the three most dominant genera in the sheep were Prevotella, unclassified Lachnospiraceae, and Butyrivibrio. In the small intestine, the three most dominant genera in the sheep were Escherichia, unclassified Lachnospiraceae, and Ruminococcus. In the large intestine, the three most dominant genera in the sheep were Ruminococcus, unclassified Ruminococcaceae, and Prevotella. R. flavefaciens, B. fibrisolvens, and S. ruminantium were three most dominant species in the sheep gastrointestinal tract. Principal Coordinates Analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: stomach (rumen, reticulum, omasum, and abomasum, small intestine (duodenum, jejunum, and ileum, and large intestine (cecum, colon, and rectum. Conclusion This is the first study to characterize the entire gastrointestinal microbiota in sheep by use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the gastrointestinal bacterial community of sheep.
Lank Simon M
Full Text Available Abstract Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.
Subha, Bakthavachallam; Song, Young Chae; Woo, Jung Hui
The present study aims to optimize the slow release biostimulant ball (BSB) for bioremediation of contaminated coastal sediment using response surface methodology (RSM). Different bacterial communities were evaluated using a pyrosequencing-based approach in contaminated coastal sediments. The effects of BSB size (1-5cm), distance (1-10cm) and time (1-4months) on changes in chemical oxygen demand (COD) and volatile solid (VS) reduction were determined. Maximum reductions of COD and VS, 89.7% and 78.8%, respectively, were observed at a 3cm ball size, 5.5cm distance and 4months; these values are the optimum conditions for effective treatment of contaminated coastal sediment. Most of the variance in COD and VS (0.9291 and 0.9369, respectively) was explained in our chosen models. BSB is a promising method for COD and VS reduction and enhancement of SRB diversity. Copyright © 2015 Elsevier Ltd. All rights reserved.
Liu, Yun; Song, Shuqun; Chen, Tiantian; Li, Caiwen
Pyrosequencing of the 18S rRNA gene has been widely adopted to study the eukaryotic diversity in various types of environments, and has an advantage over traditional morphology methods in exploring unknown microbial communities. To comprehensively assess the diversity and community composition of marine protists in the coastal waters of China, we applied both morphological observations and high-throughput sequencing of the V2 and V3 regions of 18S rDNA simultaneously to analyze samples collected from the surface layer of the Yellow and East China Seas. Dinoflagellates, diatoms and ciliates were the three dominant protistan groups as revealed by the two methods. Diatoms were the first dominant protistan group in the microscopic observations, with Skeletonema mainly distributed in the nearshore eutrophic waters and Chaetoceros in higher temperature and higher pH waters. The mixotrophic dinoflagellates, Gymnodinium and Gyrodinium, were more competitive in the oligotrophic waters. The pyrosequencing method revealed an extensive diversity of dinoflagellates. Chaetoceros was the only dominant diatom group in the pyrosequencing dataset. Gyrodinium represented the most abundant reads and dominated the offshore oligotrophic protistan community as they were in the microscopic observations. The dominance of parasitic dinoflagellates in the pyrosequencing dataset, which were overlooked in the morphological observations, indicates more attention should be paid to explore the potential role of this group. Both methods provide coherent clustering of samples. Nutrient levels, salinity and pH were the main factors influencing the distribution of protists. This study demonstrates that different primer pairs used in the pyrosequencing will indicate different protistan community structures. A suitable marker may reveal more comprehensive composition of protists and provide valuable information on environmental drivers.
Vukmirovic, Milica; Kaminski, Naftali
Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease characterized by aberrant remodeling of the lung parenchyma with extensive changes to the phenotypes of all lung resident cells. The introduction of transcriptomics, genome scale profiling of thousands of RNA transcripts, caused a significant inversion in IPF research. Instead of generating hypotheses based on animal models of disease, or biological plausibility, with limited validation in humans, investigators were able to generate hypotheses based on unbiased molecular analysis of human samples and then use animal models of disease to test their hypotheses. In this review, we describe the insights made from transcriptomic analysis of human IPF samples. We describe how transcriptomic studies led to identification of novel genes and pathways involved in the human IPF lung such as: matrix metalloproteinases, WNT pathway, epithelial genes, role of microRNAs among others, as well as conceptual insights such as the involvement of developmental pathways and deep shifts in epithelial and fibroblast phenotypes. The impact of lung and transcriptomic studies on disease classification, endotype discovery, and reproducible biomarkers is also described in detail. Despite these impressive achievements, the impact of transcriptomic studies has been limited because they analyzed bulk tissue and did not address the cellular and spatial heterogeneity of the IPF lung. We discuss new emerging technologies and applications, such as single-cell RNAseq and microenvironment analysis that may address cellular and spatial heterogeneity. We end by making the point that most current tissue collections and resources are not amenable to analysis using the novel technologies. To take advantage of the new opportunities, we need new efforts of sample collections, this time focused on access to all the microenvironments and cells in the IPF lung. PMID:29670881
Identification and Comparison of Candidate Olfactory Genes in the Olfactory and Non-Olfactory Organs of Elm Pest Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) Based on Transcriptome Analysis.
Wang, Yinliang; Chen, Qi; Zhao, Hanbo; Ren, Bingzhong
The leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) is a predominant forest pest that causes substantial damage to the lumber industry and city management. However, no effective and environmentally friendly chemical method has been discovered to control this pest. Until recently, the molecular basis of the olfactory system in A. quadriimpressum was completely unknown. In this study, antennae and leg transcriptomes were analyzed and compared using deep sequencing data to identify the olfactory genes in A. quadriimpressum. Moreover, the expression profiles of both male and female candidate olfactory genes were analyzed and validated by bioinformatics, motif analysis, homology analysis, semi-quantitative RT-PCR and RT-qPCR experiments in antennal and non-olfactory organs to explore the candidate olfactory genes that might play key roles in the life cycle of A. quadriimpressum. As a result, approximately 102.9 million and 97.3 million clean reads were obtained from the libraries created from the antennas and legs, respectively. Annotation led to 34344 Unigenes, which were matched to known proteins. Annotation data revealed that the number of genes in antenna with binding functions and receptor activity was greater than that of legs. Furthermore, many pathway genes were differentially expressed in the two organs. Sixteen candidate odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 34 odorant receptors (ORs), 20 inotropic receptors  and 2 sensory neuron membrane proteins (SNMPs) and their isoforms were identified. Additionally, 15 OBPs, 9 CSPs, 18 ORs, 6 IRs and 2 SNMPs were predicted to be complete ORFs. Using RT-PCR, RT-qPCR and homology analysis, AquaOBP1/2/4/7/C1/C6, AquaCSP3/9, AquaOR8/9/10/14/15/18/20/26/29/33, AquaIR8a/13/25a showed olfactory-specific expression, indicating that these genes might play a key role in olfaction-related behaviors in A. quadriimpressum such as foraging and seeking. AquaOBP4/C5, AquaOBP4/C5, AquaCSP7
Full Text Available The leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae is a predominant forest pest that causes substantial damage to the lumber industry and city management. However, no effective and environmentally friendly chemical method has been discovered to control this pest. Until recently, the molecular basis of the olfactory system in A. quadriimpressum was completely unknown. In this study, antennae and leg transcriptomes were analyzed and compared using deep sequencing data to identify the olfactory genes in A. quadriimpressum. Moreover, the expression profiles of both male and female candidate olfactory genes were analyzed and validated by bioinformatics, motif analysis, homology analysis, semi-quantitative RT-PCR and RT-qPCR experiments in antennal and non-olfactory organs to explore the candidate olfactory genes that might play key roles in the life cycle of A. quadriimpressum. As a result, approximately 102.9 million and 97.3 million clean reads were obtained from the libraries created from the antennas and legs, respectively. Annotation led to 34344 Unigenes, which were matched to known proteins. Annotation data revealed that the number of genes in antenna with binding functions and receptor activity was greater than that of legs. Furthermore, many pathway genes were differentially expressed in the two organs. Sixteen candidate odorant binding proteins (OBPs, 10 chemosensory proteins (CSPs, 34 odorant receptors (ORs, 20 inotropic receptors  and 2 sensory neuron membrane proteins (SNMPs and their isoforms were identified. Additionally, 15 OBPs, 9 CSPs, 18 ORs, 6 IRs and 2 SNMPs were predicted to be complete ORFs. Using RT-PCR, RT-qPCR and homology analysis, AquaOBP1/2/4/7/C1/C6, AquaCSP3/9, AquaOR8/9/10/14/15/18/20/26/29/33, AquaIR8a/13/25a showed olfactory-specific expression, indicating that these genes might play a key role in olfaction-related behaviors in A. quadriimpressum such as foraging and seeking. AquaOBP4/C5, Aqua
Liu, Xin; Guan, Huirui; Song, Min; Fu, Yanping; Han, Xiaomin; Lei, Meng; Ren, Jingyu; Guo, Bin; He, Wei; Wei, Yahui
Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. In this study, 10 candidate RGs namely, 18S , 60S , CYP , GAPCP1 , GAPDH2 , EF1B , MDH , SAND , TUA1 , and TUA6 , were singled out from the transcriptome database of S. chamaejasme , and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND , TUA1 and CYP , GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes ( P5CS2 and GI ) further verified that the RGs that we selected were suitable for gene expression normalization. This work is the first attempt to
Full Text Available Background Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. Method In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH were estimated with the programs geNorm, NormFinder, and BestKeeper. Result Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI further verified that the RGs that we selected were suitable for gene expression normalization. Discussion
Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S
Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Full Text Available Abstract Background Although the gene expression profile of several tissues in humans and in rodent animal models has been explored, analysis of the complete choroid plexus (CP transcriptome is still lacking. A better characterization of the CP transcriptome can provide key insights into its functions as one of the barriers that separate the brain from the periphery and in the production of cerebrospinal fluid. Methods This work extends further what is known about the mouse CP transcriptome through a microarray analysis of CP tissue from normal mice under physiological conditions. Results We found that the genes most highly expressed are those implicated in energy metabolism (oxidative phosphorylation, glycolysis/gluconeogenesis and in ribosomal function, which is in agreement with the secretory nature of the CP. On the other hand, genes encoding for immune mediators are among those with lower expression in basal conditions. In addition, we found genes known to be relevant during brain development, and not previously identified to be expressed in the CP, including those encoding for various axonal guidance and angiogenesis molecules and for growth factors. Some of these are known to influence the neural stem cell niche in the subventricular zone, highlighting the involvement of the CP as a likely modulator of neurogenesis. Interestingly, our observations confirm that the CP transcriptome is unique, displaying low homology with that of other tissues. Of note, we describe here that the closest similarity is with the transcriptome of the endothelial cells of the blood-brain barrier. Conclusions Based on the data presented here, it will now be possible to further explore the function of particular proteins of the CP secretome in health and in disease.
Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong
We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.
Plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT; the details are in the attached slide presentation presentation on plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT, given at the OECD meeting on June 23, 2016
Full Text Available Soybean is the most important legume crop in the world. Several diseases in soybean lead to serious yield losses in major soybean-producing countries. Moreover, soybean can be infected by diverse viruses. Recently, we carried out a large-scale screening to identify viruses infecting soybean using available soybean transcriptome data. Of the screened transcriptomes, a soybean transcriptome for soybean seed development analysis contains several virus-associated sequences. In this study, we identified five viruses, including soybean mosaic virus (SMV, infecting soybean by de novo transcriptome assembly followed by blast search. We assembled a nearly complete consensus genome sequence of SMV China using transcriptome data. Based on phylogenetic analysis, the consensus genome sequence of SMV China was closely related to SMV isolates from South Korea. We examined single nucleotide variations (SNVs for SMVs in the soybean seed transcriptome revealing 780 SNVs, which were evenly distributed on the SMV genome. Four SNVs, C-U, U-C, A-G, and G-A, were frequently identified. This result demonstrated the quasispecies variation of the SMV genome. Taken together, this study carried out bioinformatics analyses to identify viruses using soybean transcriptome data. In addition, we demonstrated the application of soybean transcriptome data for virus genome assembly and SNV analysis.
Lü, Bingjian; Xu, Jing; Lai, Maode; Zhang, Hao; Chen, Jian
Accumulating databases in human genome research have enabled integrated genome-wide study on complicated diseases such as cancers. A practical approach is to mine a global transcriptome profile of disease from public database. New concepts of these diseases might emerge by landscaping this profile. In this study, we clustered human colorectal normal mucosa (N), inflammatory bowel disease (IBD), adenoma (A) and cancer (T) related expression sequence tags (EST) into UniGenes via an in-house GetUni software package and analyzed the transcriptome overview of these libraries by GOTree Machine (GOTM). Additionally, we downloaded UniGene based cDNA libraries of colon and analyzed them by Xprofiler to cross validate the efficiency of GetUni. Semi-quantitative RT-PCR was used to validate the expression of β-catenin and. 7 novel genes in colorectal cancers. The efficiency of GetUni was successfully validated by Xprofiler and RT-PCR. Genes in library N, IBD and A were all found in library T. A total of 14,879 genes were identified with 2,355 of them having at least 2 transcripts. Differences in gene enrichment among these libraries were statistically significant in 50 signal transduction pathways and Pfam protein domains by GOTM analysis P < 0.01 Hypergeometric Test). Genes in two metabolic pathways, ribosome and glycolysis, were more enriched in the expression profiles of A and IBD than in N and T. Seven transmembrane receptor superfamily genes were typically abundant in cancers. Colorectal cancers are genetically heterogeneous. Transcription variants are common in them. Aberrations of ribosome and glycolysis pathway might be early indicators of precursor lesions in colon cancers. The electronic gene expression profile could be used to highlight the integral molecular events in colorectal cancers
Peng, Qian; Yang, Yanping; Guo, Yanyun; Han, Ye
The vinegar pei harbors complex bacterial communities. Prior studies revealing the bacterial diversity involved were mainly conducted by culture-dependent methods and PCR-DGGE. In this study, 454 pyrosequencing was used to investigate the bacterial communities in vinegar pei during the acetic acid fermentation (AAF) of Tianjin Duliu aged vinegar (TDAV). The results showed that there were 7 phyla and 24 families existing in the vinegar pei, with 2 phyla (Firmicutes, Protebacteria) and 4 families (Lactobacillaceae, Acetobacteracae, Enterobacteriaceae, Chloroplast) predominating. The genus-level identification revealed that 9 genera were the relatively stable, consistent components in different stages of AAF, including the most abundant genus Lactobacillus followed by Acetobacter and Serratia. Additionally, the bacterial community in the early fermentation stage was more complex than those in the later stages, indicating that the accumulation of organic acids provided an appropriate environment to filter unwanted bacteria and to accelerate the growth of required ones. This study provided basic information of bacterial patterns in vinegar pei and relevant changes during AAF of TDAV, and could be used as references in the following study on the implementation of starter culture as well as the improvement of AAF process.
Juan M. Arroyo
Full Text Available Premise of the study: Microsatellite primers were developed for the parasitic mistletoe Phoradendron californicum to investigate to what extent population genetic structure depends on host tree distribution within a highly fragmented landscape. Methods and Results: Fourteen unlinked polymorphic and four monomorphic nuclear microsatellite markers were developed using a genomic shotgun pyrosequencing method. A total of 187 alleles plus four monomorphic loci alleles were found in 98 individuals sampled in three populations from the Sonoran Desert in the Baja California peninsula (Mexico. Loci averaged 13.3 alleles per locus (range 4–28, and observed and expected heterozygosities within populations varied from 0.167–0.879 and 0.364–0.932, respectively. Conclusions: Levels of polymorphism of the reported markers are adequate for studies of diversity and fragmentation in natural populations of this parasitic plant. Cross-species amplifications in P. juniperinum and P. diguetianum only showed four markers that could be useful in P. diguetianum.
Dai, Mulan; Hamel, Chantal; St Arnaud, Marc; He, Yong; Grant, Cynthia; Lupwayi, Newton; Janzen, Henry; Malhi, Sukhdev S; Yang, Xiaohong; Zhou, Zhiqin
The arbuscular mycorrhizal (AM) fungal resources present in wheat fields of the Canadian Prairie were explored using 454 pyrosequencing. Of the 33 dominant AM fungal operational taxonomic units (OTUs) found in the 76 wheat fields surveyed at anthesis in 2009, 14 clustered as Funneliformis - Rhizophagus, 16 as Claroideoglomus, and 3 as Diversisporales. An OTU of Funneliformis mosseae and one OTU of Diversisporales each accounted for approximately 16% of all AM fungal OTUs. The former was ubiquitous, and the latter was mainly restricted to the Black and Dark Brown Chernozems. AM fungal OTU community composition was better explained by the Chernozem great groups (P = 0.044) than by measured soil properties. Fifty-two percent of the AM fungal OTUs were unrelated to measured soil properties. Black Chernozems hosted the largest AM fungal OTU diversity and almost twice the number of AM fungal sequences seen in Dark Brown Chernozems, the great group ranking second for AM fungal sequence abundance. Brown Chernozems hosted the lowest AM fungal abundance and an AM fungal diversity as low as that seen in Gray soils. We concluded that Black Chernozems are most conducive to AM fungal proliferation. AM fungi are generally distributed according to Chernozem great groups in the Canadian Prairie, although some taxa are evenly distributed in all soil groups.
Gomar-Vercher, S; Cabrera-Rubio, R; Mira, A; Montiel-Company, J M; Almerich-Silla, J M
Different dental caries status could be related with alterations in oral microbiota. Previous studies have collected saliva as a representative medium of the oral ecosystem. The purpose of this study was to assess the composition of oral microbiota and its relation to the presence of dental caries at different degrees of severity. One hundred ten saliva samples from 12-year-old children were taken and divided into six groups defined in strict accordance with their dental caries prevalence according to the International Caries Detection and Assessment System II criteria. These samples were studied by pyrosequencing PCR products of the 16S ribosomal RNA gene. The results showed statistically significant intergroup differences at the class and genus taxonomic levels. Streptococcus is the most frequent genus in all groups; although it did not show intergroup statistical differences. In patients with cavities, Porphyromonas and Prevotella showed an increasing percentage compared to healthy individuals. Bacterial diversity diminished as the severity of the disease increased, so those patients with more advanced stages of caries presented less bacterial diversity than healthy subjects. Although microbial composition tended to be different, the intragroup variation is large, as evidenced by the lack of clear intragroup clustering in principal component analyses. Thus, no clear differences were found, indicating that using bacterial composition as the sole source of biomarkers for dental caries may not be reliable in the unstimulated saliva samples used in the current study.
Yousaf, Sohail; Bulgari, Daniela; Bergna, Alessandro; Pancher, Michael; Quaglino, Fabio; Casati, Paola; Campisano, Andrea
Generally, plants are not considered as hosts for human and animal pathogens (HAP). The recent produce-associated outbreaks of food-borne diseases have drawn attention toward significant deficiencies in our understanding of the ecology of HAP, and their potential for interkingdom transfer. To examine the association of microorganisms classified as HAP with plants, we surveyed the presence and distribution of HAP bacterial taxa (henceforth HAPT, for brevity's sake) in the endosphere of grapevine (Vitis vinifera L.) both in the plant stems and leaves. An enrichment protocol was used on leaves to detect taxa with very low abundance in undisturbed tissues. We used pyrosequencing and phylogenetic analyses of the 16S rDNA gene. We identified several HAPT, and focused on four genera (Propionibacterium, Staphylococcus, Clostridium, and Burkholderia). The majority of the bacterial sequences in the genus Propionibacterium, from grapevine leaf and stem, were identified as P. acnes. Clostridia were detected in leaves and stems, but their number was much higher in leaves after enrichment. HAPT were indentified both in leaves and wood of grapevines. This depicts the ability of these taxa to be internalized within plant tissues and maintain their population levels in a variety of environments. Our analysis highlighted the presence of HAPT in the grapevine endosphere and unexpected occurrence of these bacterial taxa in this atypical environment.
Full Text Available Despite recent successes in the control of dental caries, the mechanism of caries development remains unclear. To investigate the causes of dental decay, especially in early childhood caries, the supragingival microflora composition of 20 twins with discordant caries phenotypes were analyzed using high-throughput pyrosequencing. In addition, the parents completed a lifestyle questionnaire. A total of 228,789 sequencing reads revealed 10 phyla, 84 genera, and 155 species of microflora, the relative abundances of these strains varied dramatically among the children, Comparative analysis between groups revealed that Veillonella, Corynebacterium and Actinomyces were presumed to be caries-related genera, Fusobacterium, Kingella and Leptotrichia were presumed to be healthy-related genus, yet this six genera were not statistically significant (P>0.05. Moreover, a cluster analysis revealed that the microbial composition of samples in the same group was often dissimilar but that the microbial composition observed in twins was usually similar. Although the genetic and environmental factors that strongly influence the microbial composition of dental caries remains unknown, we speculate that genetic factors primarily influence the individual's susceptibility to dental caries and that environmental factors primarily regulate the microbial composition of the dental plaque and the progression to caries. By using improved twins models and increased sample sizes, our study can be extended to analyze the specific genetic and environmental factors that affect the development of caries.
Jing, H.; Rocke, E.; Kong, L.; Xia, X.; Liu, H.; Landry, M. R.
Marine planktonic protists, including microalgae and protistan grazers, are an important contributor to global primary production and carbon and mineral cycles, however, little is known about their population shifts along the oxic-anoxic gradient in the water column. We used 454 pyrosequencing of the 18S rRNA gene and gene transcripts to study the community composition of whole and active protists throughout a water column in the Costa Rica Dome, where a stable oxygen minimum zone (OMZ) exists at a depth of 400~700 m. A clear shift of protist composition from photosynthetic Dinoflagellates in the surface to potential parasitic Dinoflagellates and Ciliates in the deeper water was revealed along the vertical profile at both rRNA and rDNA levels. Those protist groups recovered only at the rDNA level represent either lysed aggregates sinking from the upper waters or potential hosts for parasitic groups. UPGMA clustering demonstrated that total and active protists in the anoxic core of OMZ (550 m) were distinct from those in other water depths. The reduced community diversity and presence of a parasitic/symbiotic trophic lifestyle in the OMZ, especially the anoxic core, suggests that OMZs can exert a selective pressure on protist communities. Such changes in community structure and a shift in trophic lifestyle could result in a modulation of the microbial loop and associated biogeochemical cycling.
Garcia, A; Polonio, J C; Polli, A D; Santos, C M; Rhoden, S A; Quecine, M C; Azevedo, J L; Pamphile, J A
Sapindus saponaria L. of Sapindaceae family is popularly known as soldier soap and is found in Central and South America. A study of such medicinal plants might reveal a more complex diversity of microorganisms as compared to non-medicinal plants, considering their metabolic potential and the chemical communication between their natural microbiota. Rhizosphere is a highly diverse microbial habitat with respect to both the diversity of species and the size of the community. Rhizosphere bacteriome associated with medicinal plant S. saponaria is still poorly known. The objective of this study was to assess the rhizosphere microbiome of the medicinal plant S. saponaria using pyrosequencing, a culture-independent approach that is increasingly being used to estimate the number of bacterial species present in different environments. In their rhizosphere microbiome, 26 phyla were identified from 5089 sequences of 16S rRNA gene, with a predominance of Actinobacteria (33.54%), Acidobacteria (22.62%), and Proteobacteria (24.72%). The rarefaction curve showed a linear increase, with 2660 operational taxonomic units at 3% distance sequence dissimilarity, indicating that the rhizosphere microbiome associated with S. saponaria was highly diverse with groups of bacteria important for soil management, which could be further exploited for agricultural and biotechnological purposes.
Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin; Zhou, Zhi; Wu, Chen; Nagarajan, Sanjay
Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere.
Harris, Stephen; Croft, Julie; O'Flynn, Ciaran; Deusch, Oliver; Colyer, Alison; Allsopp, Judi; Milella, Lisa; Davis, Ian J
Periodontitis is the most frequently diagnosed health problem in cats yet little is known about the bacterial species important for the disease. The objective of this study was to identify bacterial species associated with health, gingivitis or mild periodontitis (gingivitis or mild periodontitis. Pyrosequencing of the V1-V3 region of the 16S rDNA from these plaque samples generated more than one million reads and identified a total of 267 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all gingival health categories, particularly in health along with Moraxella and Fusobacteria. The Peptostreptococcaceae were the most abundant family in gingivitis and mild periodontitis. Logistic regression analysis identified species from various genera that were significantly associated with health, gingivitis or mild periodontitis. The species identified were very similar to those observed in canine plaque in the corresponding health and disease states. Such similarities were not observed between cat and human at the bacterial species level but with disease progression similarities did emerge at the phylum level. This suggests that interventions targeted at human pathogenic species will not be effective for use in cats but there is more potential for commonalities in interventions for cats and dogs.
Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A
The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.
Frank, Renee; Baloch, Zubair W; Gentile, Caren; Watt, Christopher D; LiVolsi, Virginia A
Multifocal fibrosing thyroiditis (MFT) is characterized by numerous foci of fibrosis in a stellate configuration with fibroelastotic and fibroblastic centers entrapping epithelial structures. MFT has been proposed as a risk factor for papillary thyroid carcinoma (PTC) development. We attempted to identify whether MFT showed such molecular changes and could possibly be related to PTC. We identified seven cases of PTC with MFT in our institutional pathology database and personal consult service of one of the authors (VAL) for the years 1999 to 2012. Areas of PTC, MFT, and normal tissue were selected for BRAF analysis. Macro-dissection, DNA extraction and PCR amplification, and pyrosequencing were performed to detect BRAF mutations in codon 600. All of the MFT lesions and normal thyroid tissue were negative for BRAF mutations. Of the seven PTCs analyzed, five (71 %) were negative for BRAF mutations, while two cases were positive. In our study, none of the MFT lesions harbored BRAF mutations, whereas 29 % (two of seven) PTCs in the same gland were positive. Hence, in this small study, we found no evidence that the MFT lesion is a direct precursor to PTC. It is likely an incidental bystander in the process and a reflection of the background thyroiditis.
Full Text Available O6-methylguanine-DNA-methyltransferase (MGMT has emerged as a relevant predictor of therapeutic response and good prognosis in patients with glioblastoma (GBM. Transcriptionally active MGMT rapidly removes the alkyl adducts, preventing the formation of cross-links and thereby causing resistance to alkylating drugs. Studies with pyrosequencing (PSQ showed that this technique has a higher reproducibility and sensitivity than other techniques. However, the definition of a prognostically relevant threshold for the percentage of MGMT methylation remains one of the most critical issues in the use of PSQ analysis. The aim of this study was to define the cut-off value correlated with good favourable prognostic outcomes. We retrospectively analyzed 51 patients (33 males, 18 females with GBM who underwent surgery or biopsy. The Receiver Operating Characteristics analysis showed that the best possible criteria for PSQ-detected percentage of MGMT methylation that predicted progression-free survival (PFS and overall survival (OS were 19% and 13%, respectively. Patients with ≤19% of PSQ-detected MGMT had a shorter PFS (HR: 0.24, p<0.01; those ones with ≤13% had a shorter OS (HR: 0.33, p<0.05. Our study reinforces the importance of MGMT in the management of GBM patients, but future studies with larger sample sizes are warranted to confirm our findings.
Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd
The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.
Full Text Available BACKGROUND: Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC, have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom. METHODOLOGY AND PRINCIPAL FINDINGS: The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae. CONCLUSIONS: Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the
Oikonomou, Georgios; Machado, Vinicius Silva; Santisteban, Carlos; Schukken, Ynte Hein; Bicalho, Rodrigo Carvalho
Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk sam...
OKI, Kaihei; DUGERSUREN, Jamyan; DEMBEREL, Shirchin; WATANABE, Koichi
Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare’s milk), 5 Khoormog (fermented camel’s milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 ...
Introduction Age is an important factor in the development of osteoarthritis. Microarray studies provide insight into cartilage aging but do not reveal the full transcriptomic phenotype of chondrocytes such as small noncoding RNAs, pseudogenes, and microRNAs. RNA-Seq is a powerful technique for the interrogation of large numbers of transcripts including nonprotein coding RNAs. The aim of the study was to characterise molecular mechanisms associated with age-related changes in gene signatures. Methods RNA for gene expression analysis using RNA-Seq and real-time PCR analysis was isolated from macroscopically normal cartilage of the metacarpophalangeal joints of eight horses; four young donors (4 years old) and four old donors (>15 years old). RNA sequence libraries were prepared following ribosomal RNA depletion and sequencing was undertaken using the Illumina HiSeq 2000 platform. Differentially expressed genes were defined using Benjamini-Hochberg false discovery rate correction with a generalised linear model likelihood ratio test (P ageing cartilage. Conclusion There was an age-related dysregulation of matrix, anabolic and catabolic cartilage factors. This study has increased our knowledge of transcriptional networks in cartilage ageing by providing a global view of the transcriptome. PMID:23971731
Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms. © 2010 Wiley-Liss, Inc., A Wiley Company.
Full Text Available The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA, an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data.
Trama, Jason P; Adelson, Martin E; Mordechai, Eli
Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. To develop a rapid method for identifying patients infected with MCV via swab sampling. Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.
Pradhan, Seema; Bandhiwal, Nitesh; Shah, Niraj; Kant, Chandra; Gaur, Rashmi; Bhatia, Sabhyata
Understanding developmental processes, especially in non-model crop plants, is extremely important in order to unravel unique mechanisms regulating development. Chickpea (C. arietinum L.) seeds are especially valued for their high carbohydrate and protein content. Therefore, in order to elucidate the mechanisms underlying seed development in chickpea, deep sequencing of transcriptomes from four developmental stages was undertaken. In this study, next generation sequencing platform was utilized to sequence the transcriptome of four distinct stages of seed development in chickpea. About 1.3 million reads were generated which were assembled into 51,099 unigenes by merging the de novo and reference assemblies. Functional annotation of the unigenes was carried out using the Uniprot, COG and KEGG databases. RPKM based digital expression analysis revealed specific gene activities at different stages of development which was validated using Real time PCR analysis. More than 90% of the unigenes were found to be expressed in at least one of the four seed tissues. DEGseq was used to determine differentially expressing genes which revealed that only 6.75% of the unigenes were differentially expressed at various stages. Homology based comparison revealed 17.5% of the unigenes to be putatively seed specific. Transcription factors were predicted based on HMM profiles built using TF sequences from five legume plants and analyzed for their differential expression during progression of seed development. Expression analysis of genes involved in biosynthesis of important secondary metabolites suggested that chickpea seeds can serve as a good source of antioxidants. Since transcriptomes are a valuable source of molecular markers like simple sequence repeats (SSRs), about 12,000 SSRs were mined in chickpea seed transcriptome and few of them were validated. In conclusion, this study will serve as a valuable resource for improved chickpea breeding.
Full Text Available Understanding developmental processes, especially in non-model crop plants, is extremely important in order to unravel unique mechanisms regulating development. Chickpea (C. arietinum L. seeds are especially valued for their high carbohydrate and protein content. Therefore, in order to elucidate the mechanisms underlying seed development in chickpea, deep sequencing of transcriptomes from four developmental stages was undertaken. In this study, next generation sequencing platform was utilised to sequence the transcriptome of four distinct stages of seed development in chickpea. About 1.3 million reads were generated which were assembled into 51,099 unigenes by merging the de novo and reference assemblies. Functional annotation of the unigenes was carried out using the Uniprot, COG and KEGG databases. RPKM based digital expression analysis revealed specific gene activities at different stages of development which was validated using Real time PCR analysis. More than 90% of the unigenes were found to be expressed in at least one of the four seed tissues. DEGseq was used to determine differentially expressing genes which revealed that only 6.75% of the unigenes were differentially expressed at various stages. Homology based comparison revealed 17.5% of the unigenes to be putatively seed specific. Transcription factors were predicted based on HMM profiles built using TF sequences from five legume plants and analysed for their differential expression during progression of seed development. Expression analysis of genes involved in biosynthesis of important secondary metabolites suggested that chickpea seeds can serve as a good source of antioxidants. Since transcriptomes are a valuable source of molecular markers like simple sequence repeats (SSRs, about 12,000 SSRs were mined in chickpea seed transcriptome and few of them were validated. In conclusion, this study will serve as a valuable resource for improved chickpea breeding.
Full Text Available Abstract Background Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of Gossypium herbaceum that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation. Results The variations in root length and growth were found among four genotypes of G.herbaceum when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols were also highly expressed in Vagad and Gujcot-21. Conclusion Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these
Moon Young Lee
Full Text Available Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC, which serve as slow-wave electrical pacemakers for gastrointestinal (GI smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.
Rebecca M. Davidson
Full Text Available Transcriptome sequencing is a powerful method for studying global expression patterns in large, complex genomes. Evaluation of sequence-based expression profiles during reproductive development would provide functional annotation to genes underlying agronomic traits. We generated transcriptome profiles for 12 diverse maize ( L. reproductive tissues representing male, female, developing seed, and leaf tissues using high throughput transcriptome sequencing. Overall, ∼80% of annotated genes were expressed. Comparative analysis between sequence and hybridization-based methods demonstrated the utility of ribonucleic acid sequencing (RNA-seq for expression determination and differentiation of paralagous genes (∼85% of maize genes. Analysis of 4975 gene families across reproductive tissues revealed expression divergence is proportional to family size. In all pairwise comparisons between tissues, 7 (pre- vs. postemergence cobs to 48% (pollen vs. ovule of genes were differentially expressed. Genes with expression restricted to a single tissue within this study were identified with the highest numbers observed in leaves, endosperm, and pollen. Coexpression network analysis identified 17 gene modules with complex and shared expression patterns containing many previously described maize genes. The data and analyses in this study provide valuable tools through improved gene annotation, gene family characterization, and a core set of candidate genes to further characterize maize reproductive development and improve grain yield potential.
Wu, Xuhang; Fu, Yan; Yang, Deying; Zhang, Runhui; Zheng, Wanpeng; Nie, Huaming; Xie, Yue; Yan, Ning; Hao, Guiying; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou
The larval stage of Taenia multiceps, a global cestode, encysts in the central nervous system (CNS) of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome of T. multiceps. We obtained a total of 31,282 unigenes (mean length 920 bp) using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases (Nr, UniprotKB/Swiss-Prot, COG, KEGG, UniProtKB/TrEMBL, InterPro and Pfam). We identified 26,110 (83.47%) unigenes and inferred 20,896 (66.8%) coding sequences (CDS). Further comparative transcripts analysis with other cestodes (Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis) and intestinal parasites (Trichinella spiralis, Ancylostoma caninum and Ascaris suum) showed that 5,100 common genes were shared among three Taenia tapeworms, 261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR. This study provides an extensive transcriptome of the adult stage of T. multiceps, and demonstrates that comparative transcriptomic investigations deserve to be further studied. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of the biology of T. multiceps, and helps in the identification of drug targets and
Full Text Available BACKGROUND: The larval stage of Taenia multiceps, a global cestode, encysts in the central nervous system (CNS of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome of T. multiceps. METHODOLOGY/PRINCIPAL FINDINGS: We obtained a total of 31,282 unigenes (mean length 920 bp using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases (Nr, UniprotKB/Swiss-Prot, COG, KEGG, UniProtKB/TrEMBL, InterPro and Pfam. We identified 26,110 (83.47% unigenes and inferred 20,896 (66.8% coding sequences (CDS. Further comparative transcripts analysis with other cestodes (Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis and intestinal parasites (Trichinella spiralis, Ancylostoma caninum and Ascaris suum showed that 5,100 common genes were shared among three Taenia tapeworms, 261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR. CONCLUSIONS/SIGNIFICANCE: This study provides an extensive transcriptome of the adult stage of T. multiceps, and demonstrates that comparative transcriptomic investigations deserve to be further studied. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of
Zi Long Wang
Full Text Available BACKGROUND: The Eastern hive honey bee, Apis cerana cerana is a native and widely bred honey bee species in China. Molecular biology research about this honey bee species is scarce, and genomic information for A. c. cerana is not currently available. Transcriptome and expression profiling data for this species are therefore important resources needed to better understand the biological mechanisms of A. c. cerana. In this study, we obtained the transcriptome information of A. c. cerana by RNA-sequencing and compared gene expression differences between queens and workers of A. c. cerana by digital gene expression (DGE analysis. RESULTS: Using high-throughput Illumina RNA sequencing we obtained 51,581,510 clean reads corresponding to 4.64 Gb total nucleotides from a single run. These reads were assembled into 46,999 unigenes with a mean length of 676 bp. Based on a sequence similarity search against the five public databases (NR, Swissport, GO, COG, KEGG with a cut-off E-value of 10(-5 using BLASTX, a total of 24,630 unigenes were annotated with gene descriptions, gene ontology terms, or metabolic pathways. Using these transcriptome data as references we analyzed the gene expression differences between the queens and workers of A. c. cerana using a tag-based digital gene expression method. We obtained 5.96 and 5.66 million clean tags from the queen and worker samples, respectively. A total of 414 genes were differentially expressed between them, with 189 up-regulated and 225 down-regulated in queens. CONCLUSIONS: Our transcriptome data provide a comprehensive sequence resource for future A. c. cerana study, establishing an important public information platform for functional genomic studies in A. c. cerana. Furthermore, the DGE data provide comprehensive gene expression information for the queens and workers, which will facilitate our understanding of the molecular mechanisms of the different physiological aspects of the two castes.
Full Text Available Abstract Background Barley and particularly wheat are two grass species of immense agricultural importance. In spite of polyploidization events within the latter, studies have shown that genotypically and phenotypically these species are very closely related and, indeed, fertile hybrids can be created by interbreeding. The advent of two genome-scale Affymetrix GeneChips now allows studies of the comparison of their transcriptomes. Results We have used the Wheat GeneChip to create a "gene expression atlas" for the wheat transcriptome (cv. Chinese Spring. For this, we chose mRNA from a range of tissues and developmental stages closely mirroring a comparable study carried out for barley (cv. Morex using the Barley1 GeneChip. This, together with large-scale clustering of the probesets from the two GeneChips into "homologous groups", has allowed us to perform a genomic-scale comparative study of expression patterns in these two species. We explore the influence of the polyploidy of wheat on the results obtained with the Wheat GeneChip and quantify the correlation between conservation in gene sequence and gene expression in wheat and barley. In addition, we show how the conservation of expression patterns can be used to elucidate, probeset by probeset, the reliability of the Wheat GeneChip. Conclusion While there are many differences in expression on the level of individual genes and tissues, we demonstrate that the wheat and barley transcriptomes appear highly correlated. This finding is significant not only because given small evolutionary distance between the two species it is widely expected, but also because it demonstrates that it is possible to use the two GeneChips for comparative studies. This is the case even though their probeset composition reflects rather different design principles as well as, of course, the present incomplete knowledge of the gene content of the two species. We also show that, in general, the Wheat GeneChip is not able
Marcela S Montecchia
Full Text Available The Southern Andean Yungas in Northwest Argentina constitute one of the main biodiversity hotspots in the world. Considerable changes in land use have taken place in this ecoregion, predominantly related to forest conversion to croplands, inducing losses in above-ground biodiversity and with potential impact on soil microbial communities. In this study, we used high-throughput pyrosequencing of the 16S ribosomal RNA gene to assess whether land-use change and time under agriculture affect the composition and diversity of soil bacterial communities. We selected two areas dedicated to sugarcane and soybean production, comprising both short- and long-term agricultural sites, and used the adjacent native forest soils as a reference. Land-use change altered the composition of bacterial communities, with differences between productive areas despite the similarities between both forests. At the phylum level, only Verrucomicrobia and Firmicutes changed in abundance after deforestation for sugarcane and soybean cropping, respectively. In cultivated soils, Verrucomicrobia decreased sharply (~80%, while Firmicutes were more abundant. Despite the fact that local diversity was increased in sugarcane systems and was not altered by soybean cropping, phylogenetic beta diversity declined along both chronosequences, evidencing a homogenization of soil bacterial communities over time. In spite of the detected alteration in composition and diversity, we found a core microbiome resistant to the disturbances caused by the conversion of forests to cultivated lands and few or none exclusive OTUs for each land-use type. The overall changes in the relative abundance of copiotrophic and oligotrophic taxa may have an impact in soil ecosystem functionality. However, communities with many taxa in common may also share many functional attributes, allowing to maintain at least some soil ecosystem services after forest conversion to croplands.
Li, Tongtong; Long, Meng; Gatesoupe, François-Joël; Zhang, Qianqian; Li, Aihua; Gong, Xiaoning
Gut microbiota is increasingly regarded as an integral component of the host, due to important roles in the modulation of the immune system, the proliferation of the intestinal epithelium and the regulation of the dietary energy intake. Understanding the factors that influence the composition of these microbial communities is essential to health management, and the application to aquatic animals still requires basic investigation. In this study, we compared the bacterial communities harboured in the intestines and in the rearing water of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius cuvieri), and bighead carp (Hypophthalmichthys nobilis), by using 454-pyrosequencing with barcoded primers targeting the V4 to V5 regions of the bacterial 16S rRNA gene. The specimens of the three species were cohabiting in the same pond. Between 6,218 and 10,220 effective sequences were read from each sample, resulting in a total of 110,398 sequences for 13 samples from gut microbiota and pond water. In general, the microbial communities of the three carps were dominated by Fusobacteria, Firmicutes, Proteobacteria and Bacteroidetes, but the abundance of each phylum was significantly different between species. At the genus level, the overwhelming group was Cetobacterium (97.29 ± 0.46 %) in crucian carp, while its abundance averaged c. 40 and 60 % of the sequences read in the other two species. There was higher microbial diversity in the gut of filter-feeding bighead carp than the gut of the two other species, with grazing feeding habits. The composition of intestine microbiota of grass carp and crucian carp shared higher similarity when compared with bighead carp. The principal coordinates analysis (PCoA) with the weighted UniFrac distance and the heatmap analysis suggested that gut microbiota was not a simple reflection of the microbial community in the local habitat but resulted from species-specific selective pressures, possibly dependent on behavioural, immune
James R White
Full Text Available BACKGROUND: Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge "holobiont" system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs. Approximately 65,550 rRNA sequences (24% could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa, and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising >34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. CONCLUSIONS/SIGNIFICANCE: Slight shifts in several bacterial taxa
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.
Estelle Silvia Kilias
Full Text Available Protists in the central Arctic Ocean are adapted to the harsh environmental conditions of its various habitats. During the Polarstern cruise ARK-XXVI/3 in 2011, at one sea-ice station, large aggregates accumulated at the bottom of the melt ponds. In this study, the protist assemblages of the bottom layer of the sea-ice and melt-pond aggregate were investigated using flow cytometry and 454-pyrosequencing. The objective is to provide a first molecular overview of protist biodiversity in these habitats and to consider the overlaps and/or differences in the community compositions. Results of flow cytometry pointed to a cell size distribution that was dominated by 3–10 µm nanoflagellates. The phylogenetic classification of all sequences was conducted at a high taxonomic level, while a selection of abundant (≥1% of total reads sequences was further classified at a lower level. At a high taxonomic level, both habitats showed very similar community structures, dominated by chrysophytes and chlorophytes. At a lower taxonomic level, dissimilarities in the diversity of both groups were encountered in the abundant biosphere. While sea-ice chlorophytes and chrysophytes were dominated by Chlamydomonas/Chloromonas spp. and Ochromonas spp., the melt-pond aggregate was dominated by Carteria sp., Ochromonas spp. and Dinobryon faculiferum. We suppose that the similarities in richness and community structure are a consequence of melt-pond freshwater seeping through porous sea ice in late summer. Differences in the abundant biosphere nevertheless indicate that environmental conditions in both habitats vary enough to select for different dominant species.
Détrée, Camille; López-Landavery, Edgar; Gallardo-Escárate, Cristian; Lafarga-De la Cruz, Fabiola
The population of the Chilean endemic marine gastropod Concholepas concholepas locally called "loco" has dramatically decreased in the past 50 years as a result of intense activity of local fisheries and high environmental variability observed along the Chilean coast, including episodes of hypoxia, changes in sea surface temperature, ocean acidification and diseases. In this study, we set out to explore the molecular basis of C. concholepas to cope with biotic stressors such as exposure to the pathogenic bacterium Vibrio anguillarum. Here, 454pyrosequencing was conducted and 61 transcripts related to the immune response in this muricid species were identified. Among these, the expression of six genes (CcNFκβ, CcIκβ, CcLITAF, CcTLR, CcCas8 and CcCath) involved in the regulation of inflammatory, apoptotic and immune processes upon stimuli, were evaluated during the first 33 h post challenge (hpc). The results showed that CcTLR, CcCas8 and CcCath have an initial response at 4 hpc, evidencing an up-regulation from 4 to 24 hpc. Notably, the response of CcNFKB occurred 2 h later with a statistically significant up-regulation at 6 hpc and 10 hpc. Furthermore, the challenge with V. anguillarum induced a statistically significant down-regulation of CcIKB between 2 and 10 hpc as well as a down-regulation of CcLITAF between 2 and 4 hpc followed in both cases by an up-regulation between 24 and 33 hpc. This work describes the first transcriptomic effort to characterize the immune response of C. concholepas and constitutes a valuable transcriptomic resource for future efforts to develop sustainable aquaculture and conservations tools for this endemic marine snail species. Copyright © 2017 Elsevier Ltd. All rights reserved.
Full Text Available As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages...
Sumedha S Gunewardena
Full Text Available During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth to maturity (60-days after birth. Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2 RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome.
The unarmored dinoflagellate Karenia brevis is among the most prominent harmful, bloom-forming phytoplankton species in the Gulf of Mexico. During blooms, the polyketides PbTx-1 and PbTx-2 (brevetoxins) are produced by K. brevis. Brevetoxins negatively impact human health and the Gulf shellfish harvest. However, the genes underlying brevetoxin synthesis are currently unknown. Because the K. brevis genome is extremely large ( 1 × 1011 base pairs long), and with a high proportion of repetitive, non-coding DNA, it has not been sequenced. In fact, large, repetitive genomes are common among the dinoflagellate group. High-throughput RNA sequencing technology enabled us to assemble Karenia transcriptomes de novo and investigate potential genes in the brevetoxin pathway through comparative transcriptomics. The brevetoxin profile varies among K. brevis clonal cultures. For example, well-documented Wilson-CCFWC268 typically produces 8-10 pg PbTx per cell, whereas SP1 produces differences in gene expression. Of the 85,000 transcripts in the K. brevis transcriptome, 4,600 transcripts, including novel unannotated orthologs and putative polyketide synthases (PKSs), were only expressed by brevetoxin-producing K. brevis and K. papilionacea, not K. mikimotoi. Examination of gene expression between the typical- and low-toxin Wilson clones identified about 3,500 genes with significantly different expression levels, including 2 putative PKSs. One of the 2 PKSs was only found in the brevetoxin-producing Karenia species. These transcriptomes could not have been characterized without high-throughput RNA sequencing.
Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin
Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.
Duan, Jinjie; Sanggaard, Kristian Wejse; Schauser, Leif; Lauridsen, Sanne Enok; Enghild, Jan J; Schierup, Mikkel Heide; Wang, Tobias
Exceptional and extreme feeding behaviour makes the Burmese python (Python bivittatus) an interesting model to study physiological remodelling and metabolic adaptation in response to refeeding after prolonged starvation. In this study, we used transcriptome sequencing of 5 visceral organs during fasting as well as 24 hours and 48 hours after ingestion of a large meal to unravel the postprandial changes in Burmese pythons. We first used the pooled data to perform a de novo assembly of the transcriptome and supplemented this with a proteomic survey of enzymes in the plasma and gastric fluid. We constructed a high-quality transcriptome with 34 423 transcripts, of which 19 713 (57%) were annotated. Among highly expressed genes (fragments per kilo base per million sequenced reads > 100 in 1 tissue), we found that the transition from fasting to digestion was associated with differential expression of 43 genes in the heart, 206 genes in the liver, 114 genes in the stomach, 89 genes in the pancreas, and 158 genes in the intestine. We interrogated the function of these genes to test previous hypotheses on the response to feeding. We also used the transcriptome to identify 314 secreted proteins in the gastric fluid of the python. Digestion was associated with an upregulation of genes related to metabolic processes, and translational changes therefore appear to support the postprandial rise in metabolism. We identify stomach-related proteins from a digesting individual and demonstrate that the sensitivity of modern liquid chromatography/tandem mass spectrometry equipment allows the identification of gastric juice proteins that are present during digestion. © The Authors 2017. Published by Oxford University Press.
Weitkamp, Jörn-Hendrik; Guthrie, Scott O; Wong, Hector R; Moldawer, Lyle L; Baker, Henry V; Wynn, James L
Histologic chorioamnionitis (HCA) is commonly associated with preterm birth and deleterious post-natal outcomes including sepsis and necrotizing enterocolitis. Transcriptomic analysis has been used to uncover gene signatures that permit diagnosis and prognostication, show new therapeutic targets, and reveal mechanisms that underlie differential outcomes with other complex disease states in neonates such as sepsis. To define the transcriptomic and inflammatory protein response in peripheral blood among infants with exposure to histologic chorioamnionitis. Prospective, observational study. Uninfected preterm neonates retrospectively categorized based on placental pathology with no HCA exposure (n=18) or HCA exposure (n=15). We measured the transcriptomic and inflammatory mediator response in prospectively collected whole blood. We found 488 significant (p<0.001), differentially expressed genes in whole blood samples among uninfected neonates with HCA exposure that collectively represented activated innate and adaptive immune cellular pathways and revealed a potential regulatory role for the pleotropic microRNA molecule miR-155. Differentially secreted plasma cytokines in patients with HCA exposure compared to patients without HCA included MCP-1, MPO, and MMP-9 (p<0.05). Exposure to HCA distinctively activates the neonatal immune system in utero with potentially long-term health consequences. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Full Text Available Aerial bulbils are an important propagative organ, playing an important role in population expansion. However, the detailed gene regulatory patterns and molecular mechanism underlying bulbil formation remain unclear. Triploid Lilium lancifolium, which develops many aerial bulbils on the leaf axils of middle-upper stem, is a useful species for investigating bulbil formation. To investigate the mechanism of bulbil formation in triploid L. lancifolium, we performed histological and transcriptomic analyses using samples of leaf axils located in the upper and lower stem of triploid L. lancifolium during bulbil formation. Histological results indicated that the bulbils of triploid L. lancifolium are derived from axillary meristems that initiate de novo from cells on the adaxial side of the petiole base. Transcriptomic analysis generated ~650 million high-quality reads and 11,871 differentially expressed genes (DEGs. Functional analysis showed that the DEGs were significantly enriched in starch and sucrose metabolism and plant hormone signal transduction. Starch synthesis and accumulation likely promoted the initiation of upper bulbils in triploid L. lancifolium. Hormone-associated pathways exhibited distinct patterns of change in each sample. Auxin likely promoted the initiation of bulbils and then inhibited further bulbil formation. High biosynthesis and low degradation of cytokinin might have led to bulbil formation in the upper leaf axil. The present study achieved a global transcriptomic analysis focused on gene expression changes and pathways' enrichment during upper bulbil formation in triploid L. lancifolium, laying a solid foundation for future molecular studies on bulbil formation.
Full Text Available Cucurbita pepo (squash, pumpkin, gourd, a worldwide-cultivated vegetable of American origin, is extremely variable in fruit characteristics. However, the information associated with genes and genetic markers for pumpkin is very limited. In order to identify new genes and to develop genetic markers, we performed a transcriptome analysis (RNA-Seq of two contrasting pumpkin cultivars. Leaves and female flowers of cultivars, ‘Big Moose’ with large round fruits and ‘Munchkin’ with small round fruits, were harvested for total RNA extraction. We obtained a total of 6 GB (Big Moose; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056882 and 5 GB (Munchkin; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056883 sequence data (NCBI SRA database SRX1502732 and SRX1502735, respectively, which correspond to 18,055,786 and 14,824,292 150-base reads. After quality assessment, the clean sequences where 17,995,932 and 14,774,486 respectively. The numbers of total transcripts for ‘Big Moose’ and ‘Munchkin’ were 84,727 and 68,051, respectively. TransDecoder identified possible coding regions in assembled transcripts. This study provides transcriptome data for two contrasting pumpkin cultivars, which might be useful for genetic marker development and comparative transcriptome analyses. Keywords: RNA-Seq, Pumpkin, Contrasting cultivars, Cucurbita pepo
Full Text Available Sour cherry (Prunus cerasus in the genus Prunus in the family Rosaceae is one of the most popular stone fruit trees worldwide. Of known sour cherry cultivars, the Schattenmorelle is a famous old sour cherry with a high amount of fruit production. The Schattenmorelle was selected before 1650 and described in the 1800s. This cultivar was named after gardens of the Chateau de Moreille in which the cultivar was initially found. In order to identify new genes and to develop genetic markers for sour cherry, we performed a transcriptome analysis of a sour cherry. We selected the cultivar Schattenmorelle, which is among commercially important cultivars in Europe and North America. We obtained 2.05 GB raw data from the Schattenmorelle (NCBI accession number: SRX1187170. De novo transcriptome assembly using Trinity identified 61,053 transcripts in which N50 was 611 bp. Next, we identified 25,585 protein coding sequences using TransDecoder. The identified proteins were blasted against NCBI's non-redundant database for annotation. Based on blast search, we taxonomically classified the obtained sequences. As a result, we provide the transcriptome of sour cherry cultivar Schattenmorelle using next generation sequencing.
Full Text Available Abstract Background There has been much evidence recently for a link between transcriptional regulation and chromosomal gene order, but the relationship between genomic organization, regulation and gene function in higher eukaryotes remains to be precisely defined. Results Here, we present evidence for organization of a large proportion of a human transcriptome into gene clusters throughout the genome, which are partly regulated by the same transcription factors, share biological functions and are characterized by non-housekeeping genes. This analysis was based on the cardiac transcriptome identified by our genome-wide array analysis of 55 human heart samples. We found 37% of these genes to be arranged mainly in adjacent pairs or triplets. A significant number of pairs of adjacent genes are putatively regulated by common transcription factors (p = 0.02. Furthermore, these gene pairs share a significant number of GO functional classification terms. We show that the human cardiac transcriptome is organized into many small clusters across the whole genome, rather than being concentrated in a few larger clusters. Conclusion Our findings suggest that genes expressed in concert are organized in a linear arrangement for coordinated regulation. Determining the relationship between gene arrangement, regulation and nuclear organization as well as gene function will have broad biological implications.
John A Daniel
Conference Series has been instrumental in conducting international Biochemistry meetings for seven years, and very excited to expand Europe, America and Asia Pacific continents. Previous meetings were held in major cities like Philadelphia, Orlando with success the meetings again scheduled in three continents. 3rd International Conference on Transcriptomics to be held during October 30 - November 01, 2017 at Bangkok, Thailand The Global Transcriptomics business sector to develop at a C...
Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends.
Marzorati, Massimo; Maignien, Lois; Verhelst, An; Luta, Gabriela; Sinnott, Robert; Kerckhof, Frederiek Maarten; Boon, Nico; Van de Wiele, Tom; Possemiers, Sam
The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. -36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species-of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)-conventional molecular ecological approaches, such as PCR-DGGE and qPCR, still remain a very useful complementary tool.
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
Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Kongklieng, Amornmas; Tantrawatpan, Chairat; Boonmars, Thidarut; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai
Canine babesiosis, hepatozoonosis, ehrlichiosis, and anaplasmosis are tick-borne diseases caused by different hemopathogens. These diseases are causes of morbidity and mortality in dogs. The classic method for parasite detection and differentiation is based on microscopic observation of blood smears. The limitations of the microscopic method are that its performance requires a specially qualified person with professional competence, and it is ineffective in differentiating closely related species. This study applied PCR amplification with high throughput pyrosequencing for molecular differential detection of the following 4 hemoparasites common to tropical areas in dog blood samples: Babesia vogeli, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. PCR was initially used to amplify specific target regions of the ribosomal RNA genes of each parasite using 2 primer pairs that included 18S rRNA for protozoa (B. vogeli and H. canis) and 16S rRNA for rickettsia (E. canis and A. platys). Babesia vogeli and H. canis were discriminated using 9 nucleotide positions out of 30 base pairs, whereas E. canis and A. platys were differentiated using 15 nucleotide positions out of 34 base pairs that were determined from regions adjacent to 3' ends of the sequencing primers. This method provides a challenging alternative for a rapid diagnosis and surveillance of these tick-borne diseases in canines. Copyright © 2014 Elsevier GmbH. All rights reserved.
Önsan Z İlsen
Full Text Available Abstract Background New analysis methods are being developed to integrate data from transcriptome, proteome, interactome, metabolome, and other investigative approaches. At the same time, existing methods are being modified to serve the objectives of systems biology and permit the interpretation of the huge datasets currently being generated by high-throughput methods. Results Transcriptomic and metabolic data from chemostat fermentors were collected with the aim of investigating the relationship between these two data sets. The variation in transcriptome data in response to three physiological or genetic perturbations (medium composition, growth rate, and specific gene deletions was investigated using linear modelling, and open reading-frames (ORFs whose expression changed significantly in response to these perturbations were identified. Assuming that the metabolic profile is a function of the transcriptome profile, expression levels of the different ORFs were used to model the metabolic variables via Partial Least Squares (Projection to Latent Structures – PLS using PLS toolbox in Matlab. Conclusion The experimental design allowed the analyses to discriminate between the effects which the growth medium, dilution rate, and the deletion of specific genes had on the transcriptome and metabolite profiles. Metabolite data were modelled as a function of the transcriptome to determine their congruence. The genes that are involved in central carbon metabolism of yeast cells were found to be the ORFs with the most significant contribution to the model.
Background Common carp (Cyprinus carpio) is thought to have undergone one extra round of genome duplication compared to zebrafish. Transcriptome analysis has been used to study the existence and timing of genome duplication in species for which genome sequences are incomplete. Large-scale transcriptome data for the common carp genome should help reveal the timing of the additional duplication event. Results We have sequenced the transcriptome of common carp using 454 pyrosequencing. After assembling the 454 contigs and the published common carp sequences together, we obtained 49,669 contigs and identified genes using homology searches and an ab initio method. We identified 4,651 orthologous pairs between common carp and zebrafish and found 129,984 paralogous pairs within the common carp. An estimation of the synonymous substitution rate in the orthologous pairs indicated that common carp and zebrafish diverged 120 million years ago (MYA). We identified one round of genome duplication in common carp and estimated that it had occurred 5.6 to 11.3 MYA. In zebrafish, no genome duplication event after speciation was observed, suggesting that, compared to zebrafish, common carp had undergone an additional genome duplication event. We annotated the common carp contigs with Gene Ontology terms and KEGG pathways. Compared with zebrafish gene annotations, we found that a set of biological processes and pathways were enriched in common carp. Conclusions The assembled contigs helped us to estimate the time of the fourth-round of genome duplication in common carp. The resource that we have built as part of this study will help advance functional genomics and genome annotation studies in the future. PMID:22424280
Xiomara V Thomas
Full Text Available BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103 were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.
Ingmar S Middelbos
Full Text Available BACKGROUND: Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. PRINCIPAL FINDINGS: Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5% diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23-40% of reads, Firmicutes (14-28% of reads and Bacteroidetes (31-34% of reads being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. CONCLUSIONS: Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases.
Abdelrahman, Mostafa; El-Sayed, Magdi; Sato, Shusei; Hirakawa, Hideki; Ito, Shin-ichi; Tanaka, Keisuke; Mine, Yoko; Sugiyama, Nobuo; Suzuki, Minoru; Yamauchi, Naoki
The genus Allium is a rich source of steroidal saponins, and its medicinal properties have been attributed to these bioactive compounds. The saponin compounds with diverse structures play a pivotal role in Allium’s defense mechanism. Despite numerous studies on the occurrence and chemical structure of steroidal saponins, their biosynthetic pathway in Allium species is poorly understood. The monosomic addition lines (MALs) of the Japanese bunching onion (A. fistulosum, FF) with an extra chromosome from the shallot (A. cepa Aggregatum group, AA) are powerful genetic resources that enable us to understand many physiological traits of Allium. In the present study, we were able to isolate and identify Alliospiroside A saponin compound in A. fistulosum with extra chromosome 2A from shallot (FF2A) and its role in the defense mechanism against Fusarium pathogens. Furthermore, to gain molecular insight into the Allium saponin biosynthesis pathway, high-throughput RNA-Seq of the root, bulb, and leaf of AA, MALs, and FF was carried out using Illumina's HiSeq 2500 platform. An open access Allium Transcript Database (Allium TDB, http://alliumtdb.kazusa.or.jp) was generated based on RNA-Seq data. The resulting assembled transcripts were functionally annotated, revealing 50 unigenes involved in saponin biosynthesis. Differential gene expression (DGE) analyses of AA and MALs as compared with FF (as a control) revealed a strong up-regulation of the saponin downstream pathway, including cytochrome P450, glycosyltransferase, and beta-glucosidase in chromosome 2A. An understanding of the saponin compounds and biosynthesis-related genes would facilitate the development of plants with unique saponin content and, subsequently, improved disease resistance. PMID:28800607
Abdelrahman, Mostafa; El-Sayed, Magdi; Sato, Shusei; Hirakawa, Hideki; Ito, Shin-Ichi; Tanaka, Keisuke; Mine, Yoko; Sugiyama, Nobuo; Suzuki, Yutaka; Yamauchi, Naoki; Shigyo, Masayoshi
The genus Allium is a rich source of steroidal saponins, and its medicinal properties have been attributed to these bioactive compounds. The saponin compounds with diverse structures play a pivotal role in Allium's defense mechanism. Despite numerous studies on the occurrence and chemical structure of steroidal saponins, their biosynthetic pathway in Allium species is poorly understood. The monosomic addition lines (MALs) of the Japanese bunching onion (A. fistulosum, FF) with an extra chromosome from the shallot (A. cepa Aggregatum group, AA) are powerful genetic resources that enable us to understand many physiological traits of Allium. In the present study, we were able to isolate and identify Alliospiroside A saponin compound in A. fistulosum with extra chromosome 2A from shallot (FF2A) and its role in the defense mechanism against Fusarium pathogens. Furthermore, to gain molecular insight into the Allium saponin biosynthesis pathway, high-throughput RNA-Seq of the root, bulb, and leaf of AA, MALs, and FF was carried out using Illumina's HiSeq 2500 platform. An open access Allium Transcript Database (Allium TDB, http://alliumtdb.kazusa.or.jp) was generated based on RNA-Seq data. The resulting assembled transcripts were functionally annotated, revealing 50 unigenes involved in saponin biosynthesis. Differential gene expression (DGE) analyses of AA and MALs as compared with FF (as a control) revealed a strong up-regulation of the saponin downstream pathway, including cytochrome P450, glycosyltransferase, and beta-glucosidase in chromosome 2A. An understanding of the saponin compounds and biosynthesis-related genes would facilitate the development of plants with unique saponin content and, subsequently, improved disease resistance.
Jacqueline D Farrell
Full Text Available Perennial ryegrass is a highly heterozygous outbreeding grass species used for turf and forage production. Heterozygosity can affect de-Bruijn graph assembly making de novo transcriptome assembly of species such as perennial ryegrass challenging. Creating a reference transcriptome from a homozygous perennial ryegrass genotype can circumvent the challenge of heterozygosity. The goals of this study were to perform RNA-sequencing on multiple tissues from a highly inbred genotype to develop a reference transcriptome. This was complemented with RNA-sequencing of a highly heterozygous genotype for SNP calling.De novo transcriptome assembly of the inbred genotype created 185,833 transcripts with an average length of 830 base pairs. Within the inbred reference transcriptome 78,560 predicted open reading frames were found of which 24,434 were predicted as complete. Functional annotation found 50,890 transcripts with a BLASTp hit from the Swiss-Prot non-redundant database, 58,941 transcripts with a Pfam protein domain and 1,151 transcripts encoding putative secreted peptides. To evaluate the reference transcriptome we targeted the high-affinity K+ transporter gene family and found multiple orthologs. Using the longest unique open reading frames as the reference sequence, 64,242 single nucleotide polymorphisms were found. One thousand sixty one open reading frames from the inbred genotype contained heterozygous sites, confirming the high degree of homozygosity.Our study has developed an annotated, comprehensive transcriptome reference for perennial ryegrass that can aid in determining genetic variation, expression analysis, genome annotation, and gene mapping.
Full Text Available Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs were obtained at the >97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall
Full Text Available We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn, the mutation frequency (Mf, and the nucleotidic diversity (Pi were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each, in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.
Full Text Available Maize (Zea mays L. is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere
Rong, Yan; Wang, Yi; Guan, Yina; Ma, Jiangtao; Cai, Zhiqiang; Yang, Guanghua; Zhao, Xiyue
Graphene (GN) and graphene oxides (GOs) are novel carbon nanomaterial; they have been attracting much attention because of their excellent properties and are widely applied in many areas, including energy, electronics, biomedicine, environmental science, etc. With industrial production and consumption of GN/GO, they will inevitably enter the soil and water environments. GN/GO may directly cause certain harm to microorganisms and lead to ecological and environmental risks. GOs are GN derivatives with abundant oxygen-containing functional groups in their graphitic backbone. The structure and chemistry of GN show obvious differences compared to those of GO, which lead to the different environmental behaviors. In this study, four different types of soil (S1-S4) were employed to investigate the effect of GN and GO on soil enzymatic activity, microbial population, and bacterial community through pyrosequencing of 16S rRNA gene amplicons. The results showed that soil enzyme activity (invertase, protease, catalase, and urease) and microbial population (bacteria, actinomycetes, and fungi) changed after GN/GO release into soils. Soil microbial community species are more rich, and the diversity also increases after GO/GN application. The phylum of Proteobacteria increased at 90 days after treatment (DAT) after GN/GO application. The phylum of Chloroflexi occurred after GN application at 90 DAT in S1 soil and reached 4.6%. Proteobacteria was the most abundant phylum in S2, S3, and S4 soils; it ranged from 43.6 to 71.4% in S2 soil, from 45.6 to 73.7% in S3 soil, and from 38.1 to 56.7% in S4 soil. The most abundant genera were Bacillus (37.5-47.0%) and Lactococcus (28.0-39.0%) in S1 soil, Lysobacter and Flavobacterium in S2 soil, Pedobacter in S3 soil, and Massilia in S4 soil. The effect of GN and GO on the soil microbial community is time-dependent, and there are no significant differences between the samples at 10 and 90 DAT.
Full Text Available Abstract Background Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion
Full Text Available Abstract Background Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans. Results The main result was the identification of DNA barcodes targeting the Firmicutes and Bacteroidetes phyla. These barcodes were validated using previously published 16S rRNA gut microbiota clone libraries. In addition, an accurate F/B ratio was found when the DNA barcodes were applied to short pyrosequencing reads of published gut metagenomes. Finally, the barcodes were utilized to define the F/B ratio of 16S rRNA pyrosequencing data generated from brain abscess pus and cystic fibrosis sputum. Conclusion Using DNA barcodes of Bacteroidetes and Firmicutes 16S rRNA genes combined with pyrosequencing is a cost-effective method for monitoring relevant changes in the relative abundance of Firmicutes and Bacteroidetes bacterial communities in microbial ecosystems.
Carrion, S.; Torres, L.; Castañer, O.
This review highlights the human studies that explore the benefits of the Mediterranean diet and olive oil, based on gene expression analysis. We summarized consistent human transcriptomic studies on cardiovascular risk, based on TMD and olive oil interventions, with real life doses and conditions. A literature review was carried out leading up to February 2016. The results show that the TMD, specially supplemented with virgin olive oil, produces beneficial changes in the transcriptomic response of relevant genes in cardiovascular risk such as CAT, GPX1 and SIRT2. p65 and MCP-1, IL1B, IL6, CXCL1, INF-γ, ARHGAP15 and IL7R, which are involved in inflammation; and ABCA1, SR-B1, PPARBP, PPARα, PPARγ, PPARδ, CD-36 and COX-1, which play an important role in cholesterol efflux. The available data illustrate a transcriptomic effect on atherosclerosis, inflammation and oxidative stress pathways as well as the mentioned genes. [es
Full Text Available This review highlights the human studies that explore the benefits of the Mediterranean diet and olive oil, based on gene expression analysis. We summarized consistent human transcriptomic studies on cardiovascular risk, based on TMD and olive oil interventions, with real life doses and conditions. A literature review was carried out leading up to February 2016. The results show that the TMD, specially supplemented with virgin olive oil, produces beneficial changes in the transcriptomic response of relevant genes in cardiovascular risk such as CAT, GPX1 and SIRT2. p65 and MCP-1, IL1B, IL6, CXCL1, INF-γ, ARHGAP15 and IL7R, which are involved in inflammation; and ABCA1, SR-B1, PPARBP, PPARα, PPARγ, PPARδ, CD-36 and COX-1, which play an important role in cholesterol efflux. The available data illustrate a transcriptomic effect on atherosclerosis, inflammation and oxidative stress pathways as well as the mentioned genes.
De novo transcriptome assembly was performed using CLC Genome Workbench software and a total of 54,584 unique contigs were generated, with an N50 of 1343 base pair (bp and a mean length of 829 bp. This work contributed with a specific Japanese plum skin transcriptome, providing two libraries of contrasting fruit skin color phenotype (yellow and red and increasing substantially the GB of raw data available until now for this specie.
3Centre for Environmental Research, Near East University, 33010, Lefkosha, Turkish Republic of the Northern Cyprus. 4Department of ...... Transcriptomic analysis of sense and antisense strands of .... 2008 Stem cell transcriptome profiling via.
Joseph, Pius; Umbright, Christina; Sellamuthu, Rajendran
The number of new chemicals that are being synthesized each year has been steadily increasing. While chemicals are of immense benefit to mankind, many of them have a significant negative impact, primarily owing to their inherent chemistry and toxicity, on the environment as well as human health. In addition to chemical exposures, human exposures to numerous non-chemical toxic agents take place in the environment and workplace. Given that human exposure to toxic agents is often unavoidable and many of these agents are found to have detrimental human health effects, it is important to develop strategies to prevent the adverse health effects associated with toxic exposures. Early detection of adverse health effects as well as a clear understanding of the mechanisms, especially at the molecular level, underlying these effects are key elements in preventing the adverse health effects associated with human exposure to toxic agents. Recent developments in genomics, especially transcriptomics, have prompted investigations into this important area of toxicology. Previous studies conducted in our laboratory and elsewhere have demonstrated the potential application of blood gene expression profiling as a sensitive, mechanistically relevant and practical surrogate approach for the early detection of adverse health effects associated with exposure to toxic agents. The advantages of blood gene expression profiling as a surrogate approach to detect early target organ toxicity and the molecular mechanisms underlying the toxicity are illustrated and discussed using recent studies on hepatotoxicity and pulmonary toxicity. Furthermore, the important challenges this emerging field in toxicology faces are presented in this review article. PMID:23456664
Full Text Available As a field within the energy research sector, bioenergy is continuously expanding. Although much has been achieved and the yields of both ethanol and butanol have been improved, many avenues of research to further increase these yields still remain. This review covers current research related with transcriptomics and the application of this high-throughput analytical tool to engineer both microbes and plants with the penultimate goal being better biofuel production and yields. The initial focus is given to the responses of fermentative microbes during the fermentative production of acids, such as butyric acid, and solvents, including ethanol and butanol. As plants offer the greatest natural renewable source of fermentable sugars within the form of lignocellulose, the second focus area is the transcriptional responses of microbes when exposed to plant hydrolysates and lignin-related compounds. This is of particular importance as the acid/base hydrolysis methods commonly employed to make the plant-based cellulose available for enzymatic hydrolysis to sugars also generates significant amounts of lignin-derivatives that are inhibitory to fermentative bacteria and microbes. The article then transitions to transcriptional analyses of lignin-degrading organisms, such as Phanerochaete chrysosporium, as an alternative to acid/base hydrolysis. The final portion of this article will discuss recent transcriptome analyses of plants and, in particular, the genes involved in lignin production. The rationale behind these studies is to eventually reduce the lignin content present within these plants and, consequently, the amount of inhibitors generated during the acid/base hydrolysis of the lignocelluloses. All four of these topics represent key areas where transcriptomic research is currently being conducted to identify microbial genes and their responses to products and inhibitors as well as those related with lignin degradation/formation.
Huth, Troy J; Place, Sean P
The notothenioids comprise a diverse group of fishes that rapidly radiated after isolation by the Antarctic Circumpolar Current approximately 14-25 million years ago. Given that evolutionary adaptation has led to finely tuned traits with narrow physiological limits in these organisms, this system provides a unique opportunity to examine physiological trade-offs and limits of adaptive responses to environmental perturbation. As such, notothenioids have a rich history with respect to studies attempting to understand the vulnerability of polar ecosystems to the negative impacts associated with global climate change. Unfortunately, despite being a model system for understanding physiological adaptations to extreme environments, we still lack fundamental molecular tools for much of the Nototheniidae family. Specimens of the emerald notothen, Trematomus bernacchii, were acclimated for 28 days in flow-through seawater tanks maintained near ambient seawater temperatures (-1.5°C) or at +4°C. Following acclimation, tissue specific cDNA libraries for liver, gill and brain were created by pooling RNA from n = 5 individuals per temperature treatment. The tissue specific libraries were bar-coded and used for 454 pyrosequencing, which yielded over 700 thousand sequencing reads. A de novo assembly and annotation of these reads produced a functional transcriptome library of T. bernacchii containing 30,107 unigenes, 13,003 of which possessed significant homology to a known protein product. Digital gene expression analysis of these extremely cold adapted fish reinforced the loss of an inducible heat shock response and allowed the preliminary exploration into other elements of the cellular stress response. Preliminary exploration of the transcriptome of T. bernacchii under elevated temperatures enabled a semi-quantitative comparison to prior studies aimed at characterizing the thermal response of this endemic fish whose size, abundance and distribution has established it as a
Xing, Zhilin; Zhao, Tiantao; Gao, Yanhui; He, Zhi; Zhang, Lijie; Peng, Xuya; Song, Liyan
Real-time CH 4 oxidation in a landfill cover soil was studied using automated gas sampling that determined biogas (CH 4 and CO 2 ) and O 2 concentrations at various depths in a simulated landfill cover soil (SLCS) column reactor. The real-time monitoring system obtained more than 10,000 biogas (CH 4 and CO 2 ) and O 2 data points covering 32 steady states of CH 4 oxidation with 32 different CH 4 fluxes (0.2-125mol·m -2 ·d -1 ). The kinetics of CH 4 oxidation at different depths (0-20cm, 20-40cm, and 40-60cm) of SLCS were well fit by a CH 4 -O 2 dual-substrate model based on 32 values (averaged, n=5-15) of equilibrated CH 4 concentrations. The quality of the fit (R 2 ranged from 0.90 to 0.96) was higher than those reported in previous studies, which suggests that real time monitoring is beneficial for CH 4 oxidation simulations. MiSeq pyrosequencing indicated that CH 4 flux events changed the bacterial community structure (e.g., increased the abundance of Bacteroidetes and Methanotrophs) and resulted in a relative increase in the amount of type I methanotrophs (Methylobacter and Methylococcales) and a decrease in the amount of type II methanotrophs (Methylocystis). Copyright © 2017 Elsevier Ltd. All rights reserved.
Pyrosequencing of the bacteria associated with Platygyra carnosus corals with skeletal growth anomalies reveals differences in bacterial community composition in apparently healthy and diseased tissues
Jenny Chun-Yee Ng
Full Text Available Corals are rapidly declining globally due to coral diseases. Skeletal growth anomalies (SGA or coral tumors are a group of coral diseases that affect coral reefs worldwide, including Hong Kong waters in the Indo-Pacific region. To better understand how bacterial communities may vary in corals with SGA, for the first time, we examined the bacterial composition associated with the apparently healthy and the diseased tissues of SGA-affected Platgyra carnosus using 16S ribosomal rRNA gene pyrosequencing. Taxonomic analysis revealed Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria as the main phyla in both the apparently healthy and the diseased tissues. A significant difference in the bacterial community composition was observed between the two conditions at the OTU level. Diseased tissues were associated with higher abundances of Acidobacteria and Gemmatimonadetes, and a lower abundance of Spirochaetes. Several OTUs belonging to Rhodobacteraceae, Rhizobiales, Gammaproteobacteria, and Cytophaga-Flavobacterium-Bacteroidetes (CFB were strongly associated with the diseased tissues. These groups of bacteria may contain potential pathogens involved with the development of SGA or opportunistic secondary or tertiary colonizers that proliferated upon the health-compromised coral host. We suggest that these bacterial groups to be further studied based on inoculation experiments and testing of Koch’s postulates in efforts to understand the etiology and progression of SGA.
Pagaling, E; Yang, K; Yan, T
To investigate the acidophilic bacterial communities involved in microbially induced concrete corrosion (MICC). Our study sites located downstream from a forced main provided a unique opportunity to study the microbial communities involved in MICC under different environmental conditions (gradients of atmospheric H(2)S, sulphate concentration and pH) and under pipe modifications (coated vs uncoated). Bacterial cell density estimated by both cultivation- and DNA-based methods was low in the corroded sewer samples. Pyrosequencing and cloning showed that Mycobacterium and Acidithiobacillus dominated the acidophilic microbial communities. Methylacidiphilum was also dominant in samples where methane was detected. Correlation analysis indicated that Mycobacterium and Acidithiobacillus were significantly affected by pH and that Mycobacterium could better withstand highly acidic conditions compared to Acidithiobacillus. Communities dominated by Mycobacterium favoured conditions in the lined sewer pipes, while communities with a higher relative abundance of Acidithiobacillus favoured the unlined sewer pipes. Identifying the key micro-organisms involved in MICC and knowing how they interact with their environment are essential aspects for identifying steps towards concrete corrosion management. © 2014 The Society for Applied Microbiology.
Full Text Available The geothermal energy of groundwater has aroused increasing interest as a solution to climate change. The groundwater heat pumps (GWHP system using groundwater is the most environmentally friendly system to date and has been examined in several studies. However, biological clogging by microorganisms negatively affects the thermal efficiency of the GWHP system. In this study, we employed air surging, the most popular among well management methods, and pyrosequencing to analyze the genetic diversity in bacteria before and after air surging in a geothermal well. Furthermore, the diversity of dominant bacterial genera and those related to clogging were evaluated. The bacterial diversity of the groundwater well increased after air surging. Nevertheless, the proportion of bacterial genera thought to be related to microbiological clogging decreased. In cooling and heating systems based on the geothermal energy of groundwater, the wells should be maintained regularly by air surging to reduce efficiency problems caused by microbiological clogging and to prevent secondary damage to human health, e.g., pneumonia due to human pathogenic bacteria including Pseudomonas aeruginosa and Acinetobacter.
Connon, Richard E; Jeffries, Ken M; Komoroske, Lisa M; Todgham, Anne E; Fangue, Nann A
There is growing recognition of the need to understand the mechanisms underlying organismal resilience (i.e. tolerance, acclimatization) to environmental change to support the conservation management of sensitive and economically important species. Here, we discuss how functional genomics can be used in conservation biology to provide a cellular-level understanding of organismal responses to environmental conditions. In particular, the integration of transcriptomics with physiological and ecological research is increasingly playing an important role in identifying functional physiological thresholds predictive of compensatory responses and detrimental outcomes, transforming the way we can study issues in conservation biology. Notably, with technological advances in RNA sequencing, transcriptome-wide approaches can now be applied to species where no prior genomic sequence information is available to develop species-specific tools and investigate sublethal impacts that can contribute to population declines over generations and undermine prospects for long-term conservation success. Here, we examine the use of transcriptomics as a means of determining organismal responses to environmental stressors and use key study examples of conservation concern in fishes to highlight the added value of transcriptome-wide data to the identification of functional response pathways. Finally, we discuss the gaps between the core science and policy frameworks and how thresholds identified through transcriptomic evaluations provide evidence that can be more readily used by resource managers. © 2018. Published by The Company of Biologists Ltd.
Jones, Beryl M.; Wcislo, William T.; Robinson, Gene E.
Transcriptomes provide excellent foundational resources for mechanistic and evolutionary analyses of complex traits. We present a developmental transcriptome for the facultatively eusocial bee Megalopta genalis, which represents a potential transition point in the evolution of eusociality. A de novo transcriptome assembly of Megalopta genalis was generated using paired-end Illumina sequencing and the Trinity assembler. Males and females of all life stages were aligned to this transcriptome fo...
Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Rodelas, Belén; Abbas, Ben A; Martinez-Toledo, Maria Victoria; van Loosdrecht, Mark C M; Osorio, F; Gonzalez-Lopez, Jesus
Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44-49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.
Ontiveros-Valencia, Aura; Tang, Youneng; Zhao, He-Ping; Friese, David; Overstreet, Ryan; Smith, Jennifer; Evans, Patrick; Rittmann, Bruce E; Krajmalnik-Brown, Rosa
We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO3(-)) and perchlorate (ClO4(-)) in contaminated groundwater. The groundwater also contained oxygen (O2) and sulfate (SO4(2-)), which became important electron sinks that affected the NO3(-) and ClO4(-) removal rates. Using pyrosequencing, we elucidated how important phylotypes of each "primary" microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO4(2-) reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the "primary" groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.
Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M
Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.
Full Text Available Identification of anaerobic ammonium oxidizing (anammox bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON and three full-scale bioreactors (anammox, CANON, and DEMON, was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature. The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.
Li, Shuzhao; Todor, Andrei; Luo, Ruiyan
Molecular analysis of blood samples is pivotal to clinical diagnosis and has been intensively investigated since the rise of systems biology. Recent developments have opened new opportunities to utilize transcriptomics and metabolomics for personalized and precision medicine. Efforts from human immunology have infused into this area exquisite characterizations of subpopulations of blood cells. It is now possible to infer from blood transcriptomics, with fine accuracy, the contribution of immune activation and of cell subpopulations. In parallel, high-resolution mass spectrometry has brought revolutionary analytical capability, detecting > 10,000 metabolites, together with environmental exposure, dietary intake, microbial activity, and pharmaceutical drugs. Thus, the re-examination of blood chemicals by metabolomics is in order. Transcriptomics and metabolomics can be integrated to provide a more comprehensive understanding of the human biological states. We will review these new data and methods and discuss how they can contribute to personalized medicine.
Full Text Available Despite recent technological advances, the study of the human transcriptome is still in its early stages. Here we provide an overview of the complex human transcriptomic landscape, present the bioinformatics challenges posed by the vast quantities of transcriptomic data, and discuss some of the studies that have tried to determine how much of the human genome is transcribed. Recent evidence has suggested that more than 90% of the human genome is transcribed into RNA. However, this view has been strongly contested by groups of scientists who argued that many of the observed transcripts are simply the result of transcriptional noise. In this review, we conclude that the full extent of transcription remains an open question that will not be fully addressed until we decipher the complete range and biological diversity of the transcribed genomic sequences.
Roel C. Rabara
Full Text Available Drought is one of the limiting environmental factors that affect crop production. Understanding the molecular basis of how plants respond to this water deficit stress is key to developing drought tolerant crops. In this study we generated time course-based transcriptome profiles of tobacco plants under water deficit conditions using microarray technology. In this paper, we describe in detail the experimental procedures and analyses performed in our study. The data set we generated (available in the NCBI/GEO database under GSE67434 has been analysed to identify genes that are involved in the regulation of tobacco's responses to drought.
van Leeuwen, D. M.; Pedersen, Marie; Knudsen, Lisbeth E.
checkpoint and aneuploidy. The MN-related gene network was tested against a transcriptomics case study associated with MN measurements. In this case study, transcriptomic data from children and adults differentially exposed to ambient air pollution in the Czech Republic were analysed and visualised......Mechanistically relevant information on responses of humans to xenobiotic exposure in relation to chemically induced biological effects, such as micronuclei (MN) formation can be obtained through large-scale transcriptomics studies. Network analysis may enhance the analysis and visualisation...... of such data. Therefore, this study aimed to develop a 'MN formation' network based on a priori knowledge, by using the pathway tool MetaCore. The gene network contained 27 genes and three gene complexes that are related to processes involved in MN formation, e.g. spindle assembly checkpoint, cell cycle...
Aramillo Irizar, Peer; Schäuble, Sascha; Esser, Daniela; Groth, Marco; Frahm, Christiane; Priebe, Steffen; Baumgart, Mario; Hartmann, Nils; Marthandan, Shiva; Menzel, Uwe; Müller, Julia; Schmidt, Silvio; Ast, Volker; Caliebe, Amke; König, Rainer; Krawczak, Michael; Ristow, Michael; Schuster, Stefan; Cellerino, Alessandro; Diekmann, Stephan; Englert, Christoph; Hemmerich, Peter; Sühnel, Jürgen; Guthke, Reinhard; Witte, Otto W; Platzer, Matthias; Ruppin, Eytan; Kaleta, Christoph
Disease epidemiology during ageing shows a transition from cancer to degenerative chronic disorders as dominant contributors to mortality in the old. Nevertheless, it has remained unclear to what extent molecular signatures of ageing reflect this phenomenon. Here we report on the identification of a conserved transcriptomic signature of ageing based on gene expression data from four vertebrate species across four tissues. We find that ageing-associated transcriptomic changes follow trajectories similar to the transcriptional alterations observed in degenerative ageing diseases but are in opposite direction to the transcriptomic alterations observed in cancer. We confirm the existence of a similar antagonism on the genomic level, where a majority of shared risk alleles which increase the risk of cancer decrease the risk of chronic degenerative disorders and vice versa. These results reveal a fundamental trade-off between cancer and degenerative ageing diseases that sheds light on the pronounced shift in their epidemiology during ageing.
Liu, Daofeng; Sui, Shunzhao; Ma, Jing; Li, Zhineng; Guo, Yulong; Luo, Dengpan; Yang, Jianfeng; Li, Mingyang
Wintersweet (Chimonanthus praecox) is familiar as a garden plant and woody ornamental flower. On account of its unique flowering time and strong fragrance, it has a high ornamental and economic value. Despite a long history of human cultivation, our understanding of wintersweet genetics and molecular biology remains scant, reflecting a lack of basic genomic and transcriptomic data. In this study, we assembled three cDNA libraries, from three successive stages in flower development, designated as the flower bud with displayed petal, open flower and senescing flower stages. Using the Illumina RNA-Seq method, we obtained 21,412,928, 26,950,404, 24,912,954 qualified Illumina reads, respectively, for the three successive stages. The pooled reads from all three libraries were then assembled into 106,995 transcripts, 51,793 of which were annotated in the NCBI non-redundant protein database. Of these annotated sequences, 32,649 and 21,893 transcripts were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 15,587 transcripts onto 312 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at the open flower and senescing flower stages. An analysis of differentially expressed genes involved in plant hormone signal transduction pathways indicated that although flower opening and senescence may be independent of the ethylene signaling pathway in wintersweet, salicylic acid may be involved in the regulation of flower senescence. We also succeeded in isolating key genes of floral scent biosynthesis and proposed a biosynthetic pathway for monoterpenes and sesquiterpenes in wintersweet flowers, based on the annotated sequences. This comprehensive transcriptomic analysis presents fundamental information on the genes and pathways which are involved in flower development in wintersweet. And our data
Full Text Available Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.Using a pool of life stages (pupae, larvae, adults: females and males for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp. De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.
H5N1 influenza A virus is a serious threat to human population. With a considerable mortality rate, strategies for coping with the infection are being developed. Our research group and some others investigated the potential therapeutic and preventive measures for tackling H5N1 infections. Protein based and transcriptomics ...
Roos David S
Full Text Available Abstract Background Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown. Results We have used serial analysis of gene expression (SAGE to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (~300,000 SAGE tags representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity. Conclusion This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1–5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmissi