The first insight into the salvia (lamiaceae) genome via bac library construction and high-throughput sequencing of target bac clones

International Nuclear Information System (INIS)

Hao, D.C.; Vautrin, S.; Berges, H.; Chen, S.L.

2015-01-01

Salvia is a representative genus of Lamiaceae, a eudicot family with significant species diversity and population adaptibility. One of the key goals of Salvia genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of medicinal plants to increase their health and productivity. Large-insert genomic libraries are a fundamental tool for achieving this purpose. We report herein the construction, characterization and screening of a gridded BAC library for Salvia officinalis (sage). The S. officinalis BAC library consists of 17,764 clones and the average insert size is 107 Kb, corresponding to 3 haploid genome equivalents. Seventeen positive clones (average insert size 115 Kb) containing five terpene synthase (TPS) genes were screened out by PCR and 12 of them were subject to Illumina HiSeq 2000 sequencing, which yielded 28,097,480 90-bp raw reads (2.53 Gb). Scaffolds containing sabinene synthase (Sab), a Sab homolog, TPS3 (kaurene synthase-like 2), copalyl diphosphate synthase 2 and one cytochrome P450 gene were retrieved via de novo assembly and annotation, which also have flanking noncoding sequences, including predicted promoters and repeat sequences. Among 2,638 repeat sequences, there are 330 amplifiable microsatellites. This BAC library provides a new resource for Lamiaceae genomic studies, including microsatellite marker development, physical mapping, comparative genomics and genome sequencing. Characterization of positive clones provided insights into the structure of the Salvia genome. These sequences will be used in the assembly of a future genome sequence for S. officinalis. (author)

A set of BAC clones spanning the human genome.

NARCIS (Netherlands)

Krzywinski, M.; Bosdet, I.; Smailus, D.; Chiu, R.; Mathewson, C.; Wye, N.; Barber, S.; Brown-John, M.; Chan, S.; Chand, S.; Cloutier, A.; Girn, N.; Lee, D.; Masson, A.; Mayo, M.; Olson, T.; Pandoh, P.; Prabhu, A.L.; Schoenmakers, E.F.P.M.; Tsai, M.Y.; Albertson, D.; Lam, W.W.; Choy, C.O.; Osoegawa, K.; Zhao, S.; Jong, P.J. de; Schein, J.; Jones, S.; Marra, M.A.

2004-01-01

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human

Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L. genome

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Cloutier Sylvie

2011-05-01

Full Text Available Abstract Background Flax (Linum usitatissimum L. is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES from 43,776 clones, providing initial insights into the genome. Results The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb. The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%, followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding. Conclusion The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

Science.gov (United States)

M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

2009-01-01

The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

A Blumeria graminis f.sp. hordei BAC library - contig building and microsynteny studies

DEFF Research Database (Denmark)

Pedersen, C.; Wu, B.; Giese, H.

2002-01-01

A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making...... contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa...

Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

Science.gov (United States)

Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

2016-01-01

Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

454 sequencing of pooled BAC clones on chromosome 3H of barley

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Yamaji Nami

2011-05-01

Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

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Blackmon Barbara P

2011-07-01

Full Text Available Abstract Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

A Comparative BAC Map for the Gilthead Sea Bream (Sparus aurata L.

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Heiner Kuhl

2011-01-01

Full Text Available This study presents the first comparative BAC map of the gilthead sea bream (Sparus aurata, a highly valuated marine aquaculture fish species in the Mediterranean. High-throughput end sequencing of a BAC library yielded 92,468 reads (60.6 Mbp. Comparative mapping was achieved by anchoring BAC end sequences to the three-spined stickleback (Gasterosteus aculeatus genome. BACs that were consistently ordered along the stickleback chromosomes accounted for 14,265 clones. A fraction of 5,249 BACs constituted a minimal tiling path that covers 73.5% of the stickleback chromosomes and 70.2% of the genes that have been annotated. The N50 size of 1,485 “BACtigs” consisting of redundant BACs is 337,253 bp. The largest BACtig covers 2.15 Mbp in the stickleback genome. According to the insert size distribution of mapped BACs the sea bream genome is 1.71-fold larger than the stickleback genome. These results represent a valuable tool to researchers in the field and may support future projects to elucidate the whole sea bream genome.

A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

DEFF Research Database (Denmark)

She, Qunxin; Confalonieri, F.; Zivanovic, Y.

2000-01-01

The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

Creation of BAC genomic resources for cocoa ( Theobroma cacao L.) for physical mapping of RGA containing BAC clones.

Science.gov (United States)

Clément, D; Lanaud, C; Sabau, X; Fouet, O; Le Cunff, L; Ruiz, E; Risterucci, A M; Glaszmann, J C; Piffanelli, P

2004-05-01

We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.

GenMapDB: a database of mapped human BAC clones

OpenAIRE

Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

2001-01-01

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

The complexity of Rhipicephalus (Boophilus microplus genome characterised through detailed analysis of two BAC clones

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Valle Manuel

2011-07-01

Full Text Available Abstract Background Rhipicephalus (Boophilus microplus (Rmi a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the de-novo assembly of two R. microplus BAC sequences from the understudied R microplus genome. Based on available R. microplus sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction. Results In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs. Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA encoding gene sequence (rDNA, related internal transcribed spacer and complex intergenic region. In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb papilin gene was a helicase gene. This helicase overlapped in two exonic regions with the papilin. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits

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Feltus Frank A

2011-07-01

Full Text Available Abstract Background Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18 to duodecaploid (12X = 108. Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. Results A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective. Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. Conclusions The construction of the first switchgrass BAC library and comparative analysis of

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

Science.gov (United States)

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a

Recombining overlapping BACs into a single larger BAC

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Huxley Clare

2004-01-01

Full Text Available Abstract Background BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. Results The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. Conclusion The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones.

Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement.

Science.gov (United States)

Zhang, Tianzhen; Hu, Yan; Jiang, Wenkai; Fang, Lei; Guan, Xueying; Chen, Jiedan; Zhang, Jinbo; Saski, Christopher A; Scheffler, Brian E; Stelly, David M; Hulse-Kemp, Amanda M; Wan, Qun; Liu, Bingliang; Liu, Chunxiao; Wang, Sen; Pan, Mengqiao; Wang, Yangkun; Wang, Dawei; Ye, Wenxue; Chang, Lijing; Zhang, Wenpan; Song, Qingxin; Kirkbride, Ryan C; Chen, Xiaoya; Dennis, Elizabeth; Llewellyn, Danny J; Peterson, Daniel G; Thaxton, Peggy; Jones, Don C; Wang, Qiong; Xu, Xiaoyang; Zhang, Hua; Wu, Huaitong; Zhou, Lei; Mei, Gaofu; Chen, Shuqi; Tian, Yue; Xiang, Dan; Li, Xinghe; Ding, Jian; Zuo, Qiyang; Tao, Linna; Liu, Yunchao; Li, Ji; Lin, Yu; Hui, Yuanyuan; Cao, Zhisheng; Cai, Caiping; Zhu, Xiefei; Jiang, Zhi; Zhou, Baoliang; Guo, Wangzhen; Li, Ruiqiang; Chen, Z Jeffrey

2015-05-01

Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

A first generation BAC-based physical map of the rainbow trout genome

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Thorgaard Gary H

2009-10-01

Full Text Available Abstract Background Rainbow trout (Oncorhynchus mykiss are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL have been identified for production and life-history traits in rainbow trout. A bacterial artificial chromosome (BAC physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. Results The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF method. The clones were assembled into physical map contigs using the finger-printing contig (FPC program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1 comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2 anchoring large contigs to the microsatellite-based genetic linkage map. Conclusion The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome

The relationship of normal body temperature, end-expired breath temperature, and BAC/BrAC ratio in 98 physically fit human test subjects.

Science.gov (United States)

Cowan, J Mack; Burris, James M; Hughes, James R; Cunningham, Margaret P

2010-06-01

The relationship between normal body temperature, end-expired breath temperature, and blood alcohol concentration (BAC)/breath alcohol concentration (BrAC) ratio was studied in 98 subjects (84 men, 14 women). Subjects consumed alcohol sufficient to produce a BrAC of at least 0.06 g/210 L 45-75 min after drinking. Breath samples were analyzed using an Intoxilyzer 8000 specially equipped to measure breath temperature. Venous blood samples and body temperatures were then taken. The mean body temperature of the men (36.6 degrees C) was lower than the women (37.0 degrees C); however, their mean breath temperatures were virtually identical (men: 34.5 degrees C; women: 34.6 degrees C). The BAC exceeded the BrAC for every subject. BAC/BrAC ratios were calculated from the BAC and BrAC analytical results. There was no difference in the BAC/BrAC ratios for men (1:2379) and women (1:2385). The correlation between BAC and BrAC was high (r = 0.938, p body temperature and end-expired breath temperature, body temperature and BAC/BrAC ratio, and breath temperature and BAC/BrAC ratio were much lower. Neither normal body temperature nor end-expired breath temperature was strongly associated with BAC/BrAC ratio.

Identification and Mapping of Simple Sequence Repeat Markers from Common Bean (Phaseolus vulgaris L. Bacterial Artificial Chromosome End Sequences for Genome Characterization and Genetic–Physical Map Integration

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Juana M. Córdoba

2010-11-01

Full Text Available Microsatellite markers or simple sequence repeat (SSR loci are useful for diversity characterization and genetic–physical mapping. Different in silico microsatellite search methods have been developed for mining bacterial artificial chromosome (BAC end sequences for SSRs. The overall goal of this study was genome characterization based on SSRs in 89,017 BAC end sequences (BESs from the G19833 common bean ( L. library. Another objective was to identify new SSR taking into account three tandem motif identification programs (Automated Microsatellite Marker Development [AMMD], Tandem Repeats Finder [TRF], and SSRLocator [SSRL]. Among the microsatellite search engines, SSRL identified the highest number of SSRs; however, when primer design was attempted, the number dropped due to poor primer design regions. Automated Microsatellite Marker Development software identified many SSRs with valuable AT/TA or AG/TC motifs, while TRF found fewer SSRs and produced no primers. A subgroup of 323 AT-rich, di-, and trinucleotide SSRs were selected from the AMMD results and used in a parental survey with DOR364 and G19833, of which 75 could be mapped in the corresponding population; these represented 4052 BAC clones. Together with 92 previously mapped BES- and 114 non-BES-derived markers, a total of 280 SSRs were included in the polymerase chain reaction (PCR-based map, integrating a total of 8232 BAC clones in 162 contigs from the physical map.

Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

Science.gov (United States)

Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

2016-02-01

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.).

Science.gov (United States)

Cloutier, Sylvie; Miranda, Evelyn; Ward, Kerry; Radovanovic, Natasa; Reimer, Elsa; Walichnowski, Andrzej; Datla, Raju; Rowland, Gordon; Duguid, Scott; Ragupathy, Raja

2012-08-01

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

Science.gov (United States)

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

Directory of Open Access Journals (Sweden)

Stefano Lonardi

2013-04-01

Full Text Available For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

Combinatorial pooling enables selective sequencing of the barley gene space.

Science.gov (United States)

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Science.gov (United States)

Stabej, P; Leegwater, P A J; Imholz, S; Versteeg, S A; Zijlstra, C; Stokhof, A A; Domanjko-Petriè, A; van Oost, B A

2005-01-01

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

An efficient approach to BAC based assembly of complex genomes.

Science.gov (United States)

Visendi, Paul; Berkman, Paul J; Hayashi, Satomi; Golicz, Agnieszka A; Bayer, Philipp E; Ruperao, Pradeep; Hurgobin, Bhavna; Montenegro, Juan; Chan, Chon-Kit Kenneth; Staňková, Helena; Batley, Jacqueline; Šimková, Hana; Doležel, Jaroslav; Edwards, David

2016-01-01

There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

DEFF Research Database (Denmark)

Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael

2011-01-01

BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each...... consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

The first generation of a BAC-based physical map of Brassica rapa

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Lee Soo

2008-06-01

Full Text Available Abstract Background The genus Brassica includes the most extensively cultivated vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. A physical map of the B. rapa genome is a fundamental tool for analysis of Brassica "A" genome structure. Integration of a physical map with an existing genetic map by linking genetic markers and BAC clones in the sequencing pipeline provides a crucial resource for the ongoing genome sequencing effort and assembly of whole genome sequences. Results A genome-wide physical map of the B. rapa genome was constructed by the capillary electrophoresis-based fingerprinting of 67,468 Bacterial Artificial Chromosome (BAC clones using the five restriction enzyme SNaPshot technique. The clones were assembled into contigs by means of FPC v8.5.3. After contig validation and manual editing, the resulting contig assembly consists of 1,428 contigs and is estimated to span 717 Mb in physical length. This map provides 242 anchored contigs on 10 linkage groups to be served as seed points from which to continue bidirectional chromosome extension for genome sequencing. Conclusion The map reported here is the first physical map for Brassica "A" genome based on the High Information Content Fingerprinting (HICF technique. This physical map will serve as a fundamental genomic resource for accelerating genome sequencing, assembly of BAC sequences, and comparative genomics between Brassica genomes. The current build of the B. rapa physical map is available at the B. rapa Genome Project website for the user community.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

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Ritland Carol

2009-08-01

Full Text Available Abstract Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs and full-length (FLcDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR and a cytochrome P450 (CYP720B4 from a non-arrayed genomic BAC library of white spruce (Picea glauca. Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR and 94 kbp (CYP720B4 long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs, high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

Science.gov (United States)

Hamberger, Björn; Hall, Dawn; Yuen, Mack; Oddy, Claire; Hamberger, Britta; Keeling, Christopher I; Ritland, Carol; Ritland, Kermit; Bohlmann, Jörg

2009-08-06

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The

Fight Bac! | Partnership for Food Safety Education

Science.gov (United States)

Fight Bac! Fight Bac! Fight Bac! Partnership for Food Safety Education Supporting consumers to & Symptoms Food Safety Glossary Food Safety Education Food Safety Education Month 2017 Don't Wing Spanish Resources Food Safety Education Food Safety Education Month 2017 Don't Wing It The Story of Your

Generation of a BAC-based physical map of the melon genome

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Puigdomènech Pere

2010-05-01

Full Text Available Abstract Background Cucumis melo (melon belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has high intra-specific genetic variation, morphologic diversity and a small genome size (450 Mb, which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. A number of genetic and genomic resources have already been developed, such as several genetic maps and BAC genomic libraries. These tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. However, no physical map of any Cucurbitaceae has yet been developed. A project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. A BAC-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced. Results A melon physical map was constructed using a 5.7 × BAC library and a genetic map previously developed in our laboratories. High-information-content fingerprinting (HICF was carried out on 23,040 BAC clones, digesting with five restriction enzymes and SNaPshot labeling, followed by contig assembly with FPC software. The physical map has 1,355 contigs and 441 singletons, with an estimated physical length of 407 Mb (0.9 × coverage of the genome and the longest contig being 3.2 Mb. The anchoring of 845 BAC clones to 178 genetic markers (100 RFLPs, 76 SNPs and 2 SSRs also allowed the genetic positioning of 183 physical map contigs/singletons, representing 55 Mb (12% of the melon genome, to individual chromosomal loci. The melon FPC database is available for download at http://melonomics.upv.es/static/files/public/physical_map/. Conclusions Here we report the construction

Sequencing of 15622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

Czech Academy of Sciences Publication Activity Database

Munoz-Amatriain, M.; Lonardi, S.; Luo, M.C.; Madishetty, K.; Svensson, J.T.; Moscou, M. J.; Wanamaker, S.; Kudrna, D.; Zheng, J.; Šimková, Hana; Doležel, Jaroslav; Grimwood, J.; Mammadov, J.; Close, T.J.

2015-01-01

Roč. 84, č. 1 (2015), s. 216-227 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Barley * Hordeum vulgare L * BAC sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.468, year: 2015

De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

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Scholz Uwe

2009-11-01

Full Text Available Abstract Background De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L. is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable. Results To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes. By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies. Conclusion The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.

Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region

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Pan Hui-Juan

2007-09-01

Full Text Available Abstract Background Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. Results A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1 PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2 DNA sequencing validation of positive clones, and (3 restriction digest fingerprinting verification of inter-clone overlapping. Conclusion The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Science.gov (United States)

Morellet, Nelly; Li, Xianghong; Wieninger, Silke A; Taylor, Jennifer L; Bischerour, Julien; Moriau, Séverine; Lescop, Ewen; Bardiaux, Benjamin; Mathy, Nathalie; Assrir, Nadine; Bétermier, Mireille; Nilges, Michael; Hickman, Alison B; Dyda, Fred; Craig, Nancy L; Guittet, Eric

2018-01-01

Abstract The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity. PMID:29385532

Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

Science.gov (United States)

Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

2013-01-01

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

International Nuclear Information System (INIS)

Jang Songhun; Zhou Fang; Xia Laixin; Zhao Wei; Cheng Hanhua; Zhou Rongjia

2006-01-01

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes

A BAC/BIBAC-based physical map of chickpea, Cicer arietinum L

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Abbo Shahal

2010-09-01

Full Text Available Abstract Background Chickpea (Cicer arietinum L. is the third most important pulse crop worldwide. Despite its importance, relatively little is known about its genome. The availability of a genome-wide physical map allows rapid fine mapping of QTL, development of high-density genome maps, and sequencing of the entire genome. However, no such a physical map has been developed in chickpea. Results We present a genome-wide, BAC/BIBAC-based physical map of chickpea developed by fingerprint analysis. Four chickpea BAC and BIBAC libraries, two of which were constructed in this study, were used. A total of 67,584 clones were fingerprinted, and 64,211 (~11.7 × of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBAC contigs, with each containing an average of 28.3 clones and having an average physical length of 559 kb. The contigs collectively span approximately 1,088 Mb. By using the physical map, we identified the BAC/BIBAC contigs containing or closely linked to QTL4.1 for resistance to Didymella rabiei (RDR and QTL8 for days to first flower (DTF, thus further verifying the physical map and confirming its utility in fine mapping and cloning of QTL. Conclusion The physical map represents the first genome-wide, BAC/BIBAC-based physical map of chickpea. This map, along with other genomic resources previously developed in the species and the genome sequences of related species (soybean, Medicago and Lotus, will provide a foundation necessary for many areas of advanced genomics research in chickpea and other legume species. The inclusion of transformation-ready BIBACs in the map greatly facilitates its utility in functional analysis of the legume genomes.

Transposon-mediated BAC transgenesis in zebrafish and mice

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Sumiyama Kenta

2009-10-01

Full Text Available Abstract Background Bacterial artificial chromosomes (BACs are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. Results We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. Conclusion The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes

DEFF Research Database (Denmark)

Silahtaroglu, Asli; Poulsen, Tim; Giselø, Claus

2001-01-01

in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH. We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV...

Construction of BAC Libraries from Flow-Sorted Chromosomes.

Science.gov (United States)

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

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Inoko Hidetoshi

2006-05-01

Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

Science.gov (United States)

Luo, Meizhong; Kim, Hyeran; Kudrna, Dave; Sisneros, Nicholas B; Lee, So-Jeong; Mueller, Christopher; Collura, Kristi; Zuccolo, Andrea; Buckingham, E Bryan; Grim, Suzanne M; Yanagiya, Kazuyo; Inoko, Hidetoshi; Shiina, Takashi; Flajnik, Martin F; Wing, Rod A; Ohta, Yuko

2006-05-03

Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Sequence finishing and mapping of Drosophila melanogasterheterochromatin

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Hoskins, Roger A.; Carlson, Joseph W.; Kennedy, Cameron; Acevedo,David; Evans-Holm, Martha; Frise, Erwin; Wan, Kenneth H.; Park, Soo; Mendez-Lago, Maria; Rossi, Fabrizio; Villasante, Alfredo; Dimitri,Patrizio; Karpen, Gary H.; Celniker, Susan E.

2007-06-15

Genome sequences for most metazoans are incomplete due tothe presence of repeated DNA in the pericentromeric heterochromatin. Theheterochromatic regions of D. melanogaster contain 20 Mb of sequenceamenable to mapping, sequence assembly and finishing. Here we describethe generation of 15 Mb of finished or improved heterochromatic sequenceusing available clone resources and assembly and mapping methods. We alsoconstructed a BAC-based physical map that spans approximately 13 Mb ofthe pericentromeric heterochromatin, and a cytogenetic map that positionsapproximately 11 Mb of BAC contigs and sequence scaffolds in specificchromosomal locations. The integrated sequence assembly and maps greatlyimprove our understanding of the structure and composition of this poorlyunderstood fraction of a metazoan genome and provide a framework forfunctional analyses.

BAC-HAPPY mapping (BAP mapping: a new and efficient protocol for physical mapping.

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Giang T H Vu

2010-02-01

Full Text Available Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping, that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

Chromosomal mapping of canine-derived BAC clones to the red fox and American mink genomes.

Science.gov (United States)

Kukekova, Anna V; Vorobieva, Nadegda V; Beklemisheva, Violetta R; Johnson, Jennifer L; Temnykh, Svetlana V; Yudkin, Dmitry V; Trut, Lyudmila N; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D; Acland, Gregory M; Graphodatsky, Alexander S

2009-01-01

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

A BAC-based physical map of the Drosophila buzzatii genome

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Gonzalez, Josefa; Nefedov, Michael; Bosdet, Ian; Casals, Ferran; Calvete, Oriol; Delprat, Alejandra; Shin, Heesun; Chiu, Readman; Mathewson, Carrie; Wye, Natasja; Hoskins, Roger A.; Schein, JacquelineE.; de Jong, Pieter; Ruiz, Alfredo

2005-03-18

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophilabuzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a {approx}18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be {approx}23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using Finger Printed Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670singletons. Finally, we anchored 181 large contigs (containing 7,788clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

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Rodrigues NB

2002-01-01

Full Text Available In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3% sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds. Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8% contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds. The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds. From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

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Bharti Arvind K

2008-12-01

Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

A Snapshot of the Emerging Tomato Genome Sequence

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Lukas A. Mueller

2009-03-01

Full Text Available The genome of tomato ( L. is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy, and the United States as part of the larger “International Solanaceae Genome Project (SOL: Systems Approach to Diversity and Adaptation” initiative. The tomato genome sequencing project uses an ordered bacterial artificial chromosome (BAC approach to generate a high-quality tomato euchromatic genome sequence for use as a reference genome for the Solanaceae and euasterids. Sequence is deposited at GenBank and at the SOL Genomics Network (SGN. Currently, there are around 1000 BACs finished or in progress, representing more than a third of the projected euchromatic portion of the genome. An annotation effort is also underway by the International Tomato Annotation Group. The expected number of genes in the euchromatin is ∼40,000, based on an estimate from a preliminary annotation of 11% of finished sequence. Here, we present this first snapshot of the emerging tomato genome and its annotation, a short comparison with potato ( L. sequence data, and the tools available for the researchers to exploit this new resource are also presented. In the future, whole-genome shotgun techniques will be combined with the BAC-by-BAC approach to cover the entire tomato genome. The high-quality reference euchromatic tomato sequence is expected to be near completion by 2010.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

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Boris Troyanovsky

2016-01-01

Full Text Available Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

A highly redundant BAC library of Atlantic salmon (Salmo salar: an important tool for salmon projects

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Koop Ben F

2005-04-01

library. We have demonstrated the library's ability to identify specific genes and genetic markers using hybridization, PCR and fingerprinting experiments. In addition, multiple fingerprinting contigs indicated a pseudo-tetraploidity of the Atlantic salmon genome. The highly redundant CHORI-214 BAC library is expected to be an important resource for mapping and sequencing of the Atlantic salmon genome.

Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond.

Science.gov (United States)

Ting, Jonathan T; Feng, Guoping

2014-01-01

The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic tools have played a central role in the optogenetic revolution in neuroscience. Indeed, an overwhelming proportion of studies in this field have made use of BAC transgenic Cre driver lines to achieve targeted expression of optogenetic probes in the brain. In addition, several BAC transgenic mouse lines have been established for direct cell-type specific expression of Channelrhodopsin-2 (ChR2). While the benefits of these new tools largely outweigh any accompanying challenges, many available BAC transgenic lines may suffer from confounds due in part to increased gene dosage of one or more "extra" genes contained within the large BAC DNA sequences. Here we discuss this under-appreciated issue and propose strategies for developing the next generation of BAC transgenic lines that are devoid of extra genes. Furthermore, we provide evidence that these strategies are simple, reproducible, and do not disrupt the intended cell-type specific transgene expression patterns for several distinct BAC clones. These strategies may be widely implemented for improved BAC transgenesis across diverse disciplines.

Rhipicephalus (Boophilus) microplus strain Deutsch, 5 BAC clone sequencing, including two encoding Cytochrome P450s and one encoding CzEst9 carboxylesterase

Science.gov (United States)

The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. BAC clones give insight into the genome struct...

A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R

Czech Academy of Sciences Publication Activity Database

Bartoš, Jan; Paux, E.; Kofler, R.; Havránková, Miroslava; Kopecký, David; Suchánková, Pavla; Šafář, Jan; Šimková, Hana; Town, C.D.; Lelley, T.; Feuillet, C.; Doležel, Jaroslav

2008-01-01

Roč. 8, č. 95 (2008), s. 1-12 ISSN 1471-2229 R&D Projects: GA ČR GP521/06/P412; GA ČR GD521/05/H013; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : BAC libraries * flow-sorted chromosomes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.030, year: 2008

Effects of nitrogen ion irradiation on endoglucanase activity and gene mutation of Bacillus subtilis Bac01

International Nuclear Information System (INIS)

Lv Jie; Mao Peihong; Jin Xiang; Yu Long; Ying Hanjie

2009-01-01

Bacillus subtilis Bac01 was mutated by 15 keV N + ions of 1.5xl0 16 cm -2 . The mutant strain Bac11 with high yield of endoglucanase was isolated using carboxymethylcellulose sodium and congo red indicative plates. It exhibited higher endoglucanase activity (381.89IU) than the original strain Bac01 (93.33IU). Two 1,500 bp endoglucanase gene fragments were obtained with PCR amplification from B. subtilis Bac01 and mutant strain Bac11. BLAST comparison result indicated that 10 nucleotides mutated. Bioinformatics methods were used to analyze the two predicted amino acid sequences, and it was found that 5 amino acid residues changed, being all in the cellulose-binding domain of endoglucanase. (authors)

Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

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Mikhail Nefedov

2011-01-01

Full Text Available We have developed a new approach to screen bacterial artificial chromosome (BAC libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380 with temperature inducible homologous recombination (HR capability. We amplified one library segment, induced HR at 42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences

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Yandell Mark

2010-07-01

Full Text Available Abstract Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24. The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity elsewhere in the genome, but only 23% have identical copies (99% identity. The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is

A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs

Science.gov (United States)

2012-01-01

Background The Nile tilapia (Oreochromis niloticus) is the second most farmed fish species worldwide. It is also an important model for studies of fish physiology, particularly because of its broad tolerance to an array of environments. It is a good model to study evolutionary mechanisms in vertebrates, because of its close relationship to haplochromine cichlids, which have undergone rapid speciation in East Africa. The existing genomic resources for Nile tilapia include a genetic map, BAC end sequences and ESTs, but comparative genome analysis and maps of quantitative trait loci (QTL) are still limited. Results We have constructed a high-resolution radiation hybrid (RH) panel for the Nile tilapia and genotyped 1358 markers consisting of 850 genes, 82 markers corresponding to BAC end sequences, 154 microsatellites and 272 single nucleotide polymorphisms (SNPs). From these, 1296 markers could be associated in 81 RH groups, while 62 were not linked. The total size of the RH map is 34,084 cR3500 and 937,310 kb. It covers 88% of the entire genome with an estimated inter-marker distance of 742 Kb. Mapping of microsatellites enabled integration to the genetic map. We have merged LG8 and LG24 into a single linkage group, and confirmed that LG16-LG21 are also merged. The orientation and association of RH groups to each chromosome and LG was confirmed by chromosomal in situ hybridizations (FISH) of 55 BACs. Fifty RH groups were localized on the 22 chromosomes while 31 remained small orphan groups. Synteny relationships were determined between Nile tilapia, stickleback, medaka and pufferfish. Conclusion The RH map and associated FISH map provide a valuable gene-ordered resource for gene mapping and QTL studies. All genetic linkage groups with their corresponding RH groups now have a corresponding chromosome which can be identified in the karyotype. Placement of conserved segments indicated that multiple inter-chromosomal rearrangements have occurred between Nile tilapia

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

Science.gov (United States)

Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

2014-11-01

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

Genome Sequencing

DEFF Research Database (Denmark)

Sato, Shusei; Andersen, Stig Uggerhøj

2014-01-01

The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based on transcr......The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based...

Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening

Institute of Scientific and Technical Information of China (English)

SU Ai-guo; LI Shuang-shuang; LIU Guo-zheng; LEI Bin-bin; KANG Ding-ming; LI Zhao-hu; MA Zhi-ying; HUA Jin-ping

2014-01-01

The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artiifcial chromosome (BAC) library. Thirty-ifve primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and veriifed for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be ampliifed, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

NARCIS (Netherlands)

Choi, H.K.; Kim, D.; Uhm, T.; Limpens, E.H.M.; Lim, H.; Mun, J.H.; Kalo, P.; Penmetsa, R.V.; Seres, A.; Kulikova, O.; Roe, B.A.; Bisseling, T.; Kiss, G.B.; Cook, D.R.

2004-01-01

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an E, population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene

Sequence Ready Characterization of the Pericentromeric Region of 19p12

Energy Technology Data Exchange (ETDEWEB)

Evan E. Eichler

2006-08-31

Current mapping and sequencing strategies have been inadequate within the proximal portion of 19p12 due, in part, to the presence of a recently expanded ZNF (zinc-finger) gene family and the presence of large (25-50 kb) inverted beta-satellite repeat structures which bracket this tandemly duplicated gene family. The virtual of absence of classically defined “unique” sequence within the region has hampered efforts to identify and characterize a suitable minimal tiling path of clones which can be used as templates required for finished sequencing of the region. The goal of this proposal is to develop and implement a novel sequence-anchor strategy to generate a contiguous BAC map of the most proximal portion of chromosome 19p12 for the purpose of complete sequence characterization. The target region will be an estimated 4.5 Mb of DNA extending from STS marker D19S450 (the beginning of the ZNF gene cluster) to the centromeric (alpha-satellite) junction of 19p11. The approach will entail 1) pre-selection of 19p12 BAC and cosmid clones (NIH approved library) utilizing both 19p12 -unique and 19p12-SPECIFIC repeat probes (Eichler et al., 1998); 2) the generation of a BAC/cosmid end-sequence map across the region with a density of one marker every 8kb; 3) the development of a second-generation of STS (sequence tagged sites) which will be used to identify and verify clonal overlap at the level of the sequence; 4) incorporation of these sequence-anchored overlapping clones into existing cosmid/BAC restriction maps developed at Livermore National Laboratory; and 5) validation of the organization of this region utilizing high-resolution FISH techniques (extended chromatin analysis) on monochromosomal 19 somatic cell hybrids and parental cell lines of source material. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence

Comparative BAC-based mapping in the white-throated sparrow, a novel behavioral genomics model, using interspecies overgo hybridization

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Gonser Rusty A

2011-06-01

Full Text Available Abstract Background The genomics era has produced an arsenal of resources from sequenced organisms allowing researchers to target species that do not have comparable mapping and sequence information. These new "non-model" organisms offer unique opportunities to examine environmental effects on genomic patterns and processes. Here we use comparative mapping as a first step in characterizing the genome organization of a novel animal model, the white-throated sparrow (Zonotrichia albicollis, which occurs as white or tan morphs that exhibit alternative behaviors and physiology. Morph is determined by the presence or absence of a complex chromosomal rearrangement. This species is an ideal model for behavioral genomics because the association between genotype and phenotype is absolute, making it possible to identify the genomic bases of phenotypic variation. Findings We initiated a genomic study in this species by characterizing the white-throated sparrow BAC library via filter hybridization with overgo probes designed for the chicken, turkey, and zebra finch. Cross-species hybridization resulted in 640 positive sparrow BACs assigned to 77 chicken loci across almost all macro-and microchromosomes, with a focus on the chromosomes associated with morph. Out of 216 overgos, 36% of the probes hybridized successfully, with an average number of 3.0 positive sparrow BACs per overgo. Conclusions These data will be utilized for determining chromosomal architecture and for fine-scale mapping of candidate genes associated with phenotypic differences. Our research confirms the utility of interspecies hybridization for developing comparative maps in other non-model organisms.

PLE-wu, a new member of piggyBac transposon family from insect, is active in mammalian cells.

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Wu, Chunxiao; Wang, Shu

2014-10-01

piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An efficient approach to BAC based assembly of complex genomes

Czech Academy of Sciences Publication Activity Database

Visendi, P.; Berkman, P.J.; Hayashi, S.; Golicz, A.A.; Bayer, P.E.; Ruperao, P.; Hurgobin, B.; Montenegro, J.; Chan, C.K.K.; Staňková, Helena; Batley, J.; Šimková, Hana; Doležel, Jaroslav; Edwards, D.

2016-01-01

Roč. 12, JAN 20 (2016), s. 2 ISSN 1746-4811 R&D Projects: GA MŠk(CZ) LO1204; GA ČR(CZ) GAP501/12/2554 Institutional support: RVO:61389030 Keywords : Next-generation sequencing * SASSY * BAC Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.510, year: 2016

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.).

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Kim, Hyoun-Joung; Nahm, Seok-Hyeon; Lee, Heung-Ryul; Yoon, Gi-Bo; Kim, Ki-Taek; Kang, Byoung-Cheorl; Choi, Doil; Kweon, Oh Yeol; Cho, Myeong-Cheoul; Kwon, Jin-Kyung; Han, Jung-Heon; Kim, Jeong-Ho; Park, Minkyu; Ahn, Jong Hwa; Choi, Soon Ho; Her, Nam Han; Sung, Joo-Hee; Kim, Byung-Dong

2008-12-01

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.

Partial Diversity Generates Effector Immunity Specificity of the Bac41-Like Bacteriocins of Enterococcus faecalis Clinical Strains.

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Kurushima, Jun; Ike, Yasuyoshi; Tomita, Haruyoshi

2016-09-01

Bacteriocin 41 (Bac41) is the plasmid-encoded bacteriocin produced by the opportunistic pathogen Enterococcus faecalis Its genetic determinant consists of bacL1 (effector), bacL2 (regulator), bacA (effector), and bacI (immunity). The secreted effectors BacL1 and BacA coordinate to induce the lytic cell death of E. faecalis Meanwhile, the immunity factor BacI provides self-resistance to the Bac41 producer, E. faecalis, against the action of BacL1 and BacA. In this study, we demonstrated that more than half of the 327 clinical strains of E. faecalis screened had functional Bac41 genes. Analysis of the genetic structure of the Bac41 genes in the DNA sequences of the E. faecalis strains revealed that the Bac41-like genes consist of a relatively conserved region and a variable region located downstream from bacA Based on similarities in the variable region, the Bac41-like genes could be classified into type I, type IIa, and type IIb. Interestingly, the distinct Bac41 types had specific immunity factors for self-resistance, BacI1 or BacI2, and did not show cross-immunity to the other type of effector. We also demonstrated experimentally that the specificity of the immunity was determined by the combination of the C-terminal region of BacA and the presence of the unique BacI1 or BacI2 factor. These observations suggested that Bac41-like bacteriocin genes are extensively disseminated among E. faecalis strains in the clinical environment and can be grouped into at least three types. It was also indicated that the partial diversity results in specificity of self-resistance which may offer these strains a competitive advantage. Bacteriocins are antibacterial effectors produced by bacteria. In general, a bacteriocin-coding gene is accompanied by a cognate immunity gene that confers self-resistance on the bacteriocin-producing bacterium itself. We demonstrated that one of the bacteriocins, Bac41, is disseminated among E. faecalis clinical strains and the Bac41 subtypes with

Development of Chromosome-Specific BAC Resources for Genomics of Bread Wheat

Czech Academy of Sciences Publication Activity Database

Šafář, Jan; Šimková, Hana; Kubaláková, Marie; Čihalíková, Jarmila; Suchánková, Pavla; Bartoš, Jan; Doležel, Jaroslav

2010-01-01

Roč. 129, 1-3 (2010), s. 211-223 ISSN 1424-8581 R&D Projects: GA ČR GA521/07/1573; GA MŠk(CZ) LC06004 Grant - others:European Community’s Seventh Framework Programme(XE) FP7/2007–2013 Institutional research plan: CEZ:AV0Z50380511 Keywords : BAC library * Chromosome * DNA markers Subject RIV: GE - Plant Breeding Impact factor: 1.783, year: 2010

Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

Science.gov (United States)

2011-01-01

Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. PMID:21447194

Genome resources for climate-resilient cowpea, an essential crop for food security.

Science.gov (United States)

Muñoz-Amatriaín, María; Mirebrahim, Hamid; Xu, Pei; Wanamaker, Steve I; Luo, MingCheng; Alhakami, Hind; Alpert, Matthew; Atokple, Ibrahim; Batieno, Benoit J; Boukar, Ousmane; Bozdag, Serdar; Cisse, Ndiaga; Drabo, Issa; Ehlers, Jeffrey D; Farmer, Andrew; Fatokun, Christian; Gu, Yong Q; Guo, Yi-Ning; Huynh, Bao-Lam; Jackson, Scott A; Kusi, Francis; Lawley, Cynthia T; Lucas, Mitchell R; Ma, Yaqin; Timko, Michael P; Wu, Jiajie; You, Frank; Barkley, Noelle A; Roberts, Philip A; Lonardi, Stefano; Close, Timothy J

2017-03-01

Cowpea (Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought-prone climates, and a primary source of protein in sub-Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K-499-35 include a whole-genome shotgun (WGS) assembly, a bacterial artificial chromosome (BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi-parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited-input small-holder farming and climate stress. © 2016 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC libraries using High Resolution Melt analysis

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Caligari Peter DS

2010-05-01

Full Text Available Abstract Background The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR and High Resolution Melt (HRM analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

Isolation of BAC Clones Containing Conserved Genes from Libraries of Three Distantly Related Moths: A Useful Resource for Comparative Genomics of Lepidoptera

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Yuji Yasukochi

2011-01-01

Full Text Available Lepidoptera, butterflies and moths, is the second largest animal order and includes numerous agricultural pests. To facilitate comparative genomics in Lepidoptera, we isolated BAC clones containing conserved and putative single-copy genes from libraries of three pests, Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella, harboring the haploid chromosome number, =31, which are not closely related with each other or with the silkworm, Bombyx mori, (=28, the sequenced model lepidopteran. A total of 108–184 clones representing 101–182 conserved genes were isolated for each species. For 79 genes, clones were isolated from more than two species, which will be useful as common markers for analysis using fluorescence in situ hybridization (FISH, as well as for comparison of genome sequence among multiple species. The PCR-based clone isolation method presented here is applicable to species which lack a sequenced genome but have a significant collection of cDNA or EST sequences.

Investigation of decolorization of textile wastewater in an anaerobic/aerobic biological activated carbon system (A/A BAC).

Science.gov (United States)

Pasukphun, N; Vinitnantharat, S; Gheewala, S

2010-04-01

The aim of this study is to investigate the decolorization in anaerobic/aerobic biological activated carbon (A/A BAC) system. The experiment was divided into 2 stages; stage I is batch test for preliminary study of dye removal equilibrium time. The preliminary experiment (stage I) provided the optimal data for experimental design of A/A BAC system in SBR (stage II). Stage II is A/A BAC system imitated Sequencing Batch Reactor (SBR) which consist of 5 main periods; fill, react, settle, draw and idle. React period include anaerobic phase followed by aerobic phase. The BAC main media; Granular Activated Carbon (GAC), Mixed Cultures (MC) and Biological Activated Carbon (BAC) were used for dye and organic substances removal in three different solutions; Desizing Agent Solution (DAS), dye Solution (DS) and Synthetic Textile Wastewater (STW). Results indicate that GAC adsorption plays role in dye removal followed by BAC and MC activities, respectively. In the presence desizing agent, decolorization by MC was improved because desizing agent acts as co-substrates for microorganisms. It was found that 50% of dye removal efficiency was achieved in Fill period by MC. GC/MS analysis was used to identify dye intermediate from decolorization. Dye intermediate containing amine group was found in the solution and on BAC surfaces. The results demonstrated that combination of MC and BAC in the system promotes decolorization and dye intermediate removal. In order to improve dye removal efficiency in an A/A BAC system, replacement of virgin GAC, sufficient co-substrates supply and the appropriate anaerobic: aerobic period should be considered.

Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

Science.gov (United States)

Wang, Chao; Shi, Xue; Liu, Lin; Li, Haiyan; Ammiraju, Jetty S S; Kudrna, David A; Xiong, Wentao; Wang, Hao; Dai, Zhaozhao; Zheng, Yonglian; Lai, Jinsheng; Jin, Weiwei; Messing, Joachim; Bennetzen, Jeffrey L; Wing, Rod A; Luo, Meizhong

2013-11-01

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

Comparison of the Equine Reference Sequence with Its Sanger Source Data and New Illumina Reads.

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Jovan Rebolledo-Mendez

Full Text Available The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight's half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects' and Twilight's genome or due to errors in the reference. EquCab2 is regarded as "The Twilight Assembly." The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies

Transposon-mediated BAC transgenesis in human ES cells

Science.gov (United States)

Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J. H.; Anastassiadis, Konstantinos; Stewart, A. Francis

2012-01-01

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs. PMID:22753106

Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

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B. Karsten Tischer

2012-01-01

Full Text Available Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

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Guillaume Manat

Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information

Science.gov (United States)

2012-01-01

Background Cotton is the world’s most important natural textile fiber and a significant oilseed crop. Decoding cotton genomes will provide the ultimate reference and resource for research and utilization of the species. Integration of high-density genetic maps with genomic sequence information will largely accelerate the process of whole-genome assembly in cotton. Results In this paper, we update a high-density interspecific genetic linkage map of allotetraploid cultivated cotton. An additional 1,167 marker loci have been added to our previously published map of 2,247 loci. Three new marker types, InDel (insertion-deletion) and SNP (single nucleotide polymorphism) developed from gene information, and REMAP (retrotransposon-microsatellite amplified polymorphism), were used to increase map density. The updated map consists of 3,414 loci in 26 linkage groups covering 3,667.62 cM with an average inter-locus distance of 1.08 cM. Furthermore, genome-wide sequence analysis was finished using 3,324 informative sequence-based markers and publicly-available Gossypium DNA sequence information. A total of 413,113 EST and 195 BAC sequences were physically anchored and clustered by 3,324 sequence-based markers. Of these, 14,243 ESTs and 188 BACs from different species of Gossypium were clustered and specifically anchored to the high-density genetic map. A total of 2,748 candidate unigenes from 2,111 ESTs clusters and 63 BACs were mined for functional annotation and classification. The 337 ESTs/genes related to fiber quality traits were integrated with 132 previously reported cotton fiber quality quantitative trait loci, which demonstrated the important roles in fiber quality of these genes. Higher-level sequence conservation between different cotton species and between the A- and D-subgenomes in tetraploid cotton was found, indicating a common evolutionary origin for orthologous and paralogous loci in Gossypium. Conclusion This study will serve as a valuable genomic resource

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti.

Science.gov (United States)

Adelman, Zach N; Jasinskiene, Nijole; Vally, K J M; Peek, Corrie; Travanty, Emily A; Olson, Ken E; Brown, Susan E; Stephens, Janice L; Knudson, Dennis L; Coates, Craig J; James, Anthony A

2004-10-01

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15-30% of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.

BAC and RNA sequencing reveal the brown planthopper resistance gene BPH15 in a recombination cold spot that mediates a unique defense mechanism.

Science.gov (United States)

Lv, Wentang; Du, Ba; Shangguan, Xinxin; Zhao, Yan; Pan, Yufang; Zhu, Lili; He, Yuqing; He, Guangcun

2014-08-11

Brown planthopper (BPH, Nilaparvata lugens Stål), is the most destructive phloem-feeding insect pest of rice (Oryza sativa). The BPH-resistance gene BPH15 has been proved to be effective in controlling the pest and widely applied in rice breeding programs. Nevertheless, molecular mechanism of the resistance remain unclear. In this study, we narrowed down the position of BPH15 on chromosome 4 and investigated the transcriptome of BPH15 rice after BPH attacked. We analyzed 13,000 BC2F2 plants of cross between susceptible rice TN1 and the recombinant inbred line RI93 that carrying the BPH15 gene from original resistant donor B5. BPH15 was mapped to a 0.0269 cM region on chromosome 4, which is 210-kb in the reference genome of Nipponbare. Sequencing bacterial artificial chromosome (BAC) clones that span the BPH15 region revealed that the physical size of BPH15 region in resistant rice B5 is 580-kb, much bigger than the corresponding region in the reference genome of Nipponbare. There were 87 predicted genes in the BPH15 region in resistant rice. The expression profiles of predicted genes were analyzed. Four jacalin-related lectin proteins genes and one LRR protein gene were found constitutively expressed in resistant parent and considered the candidate genes of BPH15. The transcriptomes of resistant BPH15 introgression line and the susceptible recipient line were analyzed using high-throughput RNA sequencing. In total, 2,914 differentially expressed genes (DEGs) were identified. BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6 h after infestation, HAI) and late stage (48 HAI). The key defense mechanism was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca(2+) signaling, PR genes, transcription factors, and protein posttranslational modifications. Our work combined BAC and RNA sequencing to identify candidate genes of BPH15 and revealed the resistance mechanism

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

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Canela, Andres; Sridharan, Sriram; Sciascia, Nicholas; Tubbs, Anthony; Meltzer, Paul; Sleckman, Barry P; Nussenzweig, André

2016-09-01

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice. Published by Elsevier Inc.

Genetic stability of pestivirus genomes cloned into BACs

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse

pestivirus genomes to demonstrate the suitability of the BAC vector for harbouring pestivirus genomes. Two BAC clones, comprising the complete genomes of BDV Gifhorn (pBeloGif3) and CSFV Paderborn (pBeloPader10) were passaged 15 times in E.coli representing at least 360 bacteria generations. From 15th...

Genome Improvement at JGI-HAGSC

Energy Technology Data Exchange (ETDEWEB)

Grimwood, Jane; Schmutz, Jeremy J.; Myers, Richard M.

2012-03-03

Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence. For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.

Rapid development of microsatellite markers for Callosobruchus chinensis using Illumina paired-end sequencing.

Directory of Open Access Journals (Sweden)

Can-Xing Duan

Full Text Available BACKGROUND: The adzuki bean weevil, Callosobruchus chinensis L., is one of the most destructive pests of stored legume seeds such as mungbean, cowpea, and adzuki bean, which usually cause considerable loss in the quantity and quality of stored seeds during transportation and storage. However, a lack of genetic information of this pest results in a series of genetic questions remain largely unknown, including population genetic structure, kinship, biotype abundance, and so on. Co-dominant microsatellite markers offer a great resolving power to determine these events. Here, we report rapid microsatellite isolation from C. chinensis via high-throughput sequencing. PRINCIPAL FINDINGS: In this study, 94,560,852 quality-filtered and trimmed reads were obtained for the assembly of genome using Illumina paired-end sequencing technology. In total, the genome with total length of 497,124,785 bp, comprising 403,113 high quality contigs was generated with de novo assembly. More than 6800 SSR loci were detected and a suit of 6303 primer pair sequences were designed and 500 of them were randomly selected for validation. Of these, 196 pair of primers, i.e. 39.2%, produced reproducible amplicons that were polymorphic among 8 C. chinensis genotypes collected from different geographical regions. Twenty out of 196 polymorphic SSR markers were used to analyze the genetic diversity of 18 C. chinensis populations. The results showed the twenty SSR loci were highly polymorphic among these populations. CONCLUSIONS: This study presents a first report of genome sequencing and de novo assembly for C. chinensis and demonstrates the feasibility of generating a large scale of sequence information and SSR loci isolation by Illumina paired-end sequencing. Our results provide a valuable resource for C. chinensis research. These novel markers are valuable for future genetic mapping, trait association, genetic structure and kinship among C. chinensis.

Development of Health Education Learning Module in Bac.TSE-LDPE Programme in TTI: Needs Analysis Study

Science.gov (United States)

Ujang, Alijah; Alias, Norlidah; Siraj, Saedah

2015-01-01

This study is to explore the need to develop learning modules of health education for trainee teachers in the Bachelor Of Teaching (Hons)(Special Education-Learning Disabilities For Primary Education) Programme (Bac.TSE-LDPE) in the Teacher Training Institute (TTI). The questionnaire uses the Likert scale with the close ended questions analysed by…

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Institute of Scientific and Technical Information of China (English)

Qingshu Meng; Kaifu Chen; Lina Ma; Songnian Hu; Jun Yu

2011-01-01

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome

Czech Academy of Sciences Publication Activity Database

Šafář, Jan; Noa-Carrazana, J. C.; Vrána, Jan; Bartoš, Jan; Alkhimova, Olena; Lheureux, F.; Šimková, Hana; Caruana, M. L.; Doležel, Jaroslav; Piffanelli, P.

2004-01-01

Roč. 47, - (2004), 1182ů1191 E-ISSN 1480-3321 R&D Projects: GA AV ČR IAA6038201 Grant - others:research contract IAEA(FR) 12230/RBF Institutional research plan: CEZ:AV0Z5038910 Keywords : bacterial artificial chromosome library * banana * BAC-FISH Subject RIV: EB - Genetics ; Molecular Biology

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

Directory of Open Access Journals (Sweden)

Qing Ye

Full Text Available The bacterial artificial chromosome (BAC system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

Time to Detection with BacT/Alert FA Plus Compared to BacT/Alert FA Blood Culture Media.

Science.gov (United States)

Nutman, A; Fisher Even-Tsur, S; Shapiro, G; Braun, T; Schwartz, D; Carmeli, Y

2016-09-01

Rapid identification of the causative pathogen in patients with bacteremia allows adjustment of antibiotic therapy and improves patient outcomes. We compared in vitro and real-life time to detection (TTD) of two blood culture media, BacT/Alert FA (FA) and BacT/Alert FA Plus (FA Plus), for the nine most common species of bacterial pathogens recovered from blood samples. Experimental data from simulated cultures was compared with microbiology records of TTD for both culture media with growth of the species of interest in clinical blood cultures. In the experimental conditions, median TTD was 3.8 hours (23.9 %) shorter using FA Plus media. The magnitude of reduction differed between species. Similarly, in real life data, FA Plus had shorter TTD than FA media; however, the difference between culture media was smaller, and median TTD was only 1 hour (8.5 %) less. We found shorter TTD with BacT/Alert FA Plus culture media, both experimentally and in real-life conditions and unrelated to antibiotic neutralization, highlighting the importance of appropriate blood culture media selection.

Spatial attributes of the four-helix bundle group of bacteriocins – The high-resolution structure of BacSp222 in solution

KAUST Repository

Nowakowski, Michał

2017-11-01

BacSp222 is a multifunctional bacteriocin produced by Staphylococcus pseudintermedius strain 222, an opportunistic pathogen of domestic animals. At micromolar concentrations, BacSp222 kills Gram-positive bacteria and is cytotoxic toward mammalian cells, while at nanomolar doses, it acts as an immunomodulatory factor, enhancing nitric oxide release in macrophage-like cell lines. The bacteriocin is a cationic, N-terminally formylated, 50-amino-acid-long linear peptide that is rich in tryptophan residues.In this study, the solution structure of BacSp222 was determined and compared to the currently known structures of similar bacteriocins. BacSp222 was isolated from a liquid culture medium in a uniformly 13C- and 15N-labeled form, and NMR data were collected. The structure was calculated based on NMR-derived constraints and consists of a rigid and tightly packed globular bundle of four alpha-helices separated by three short turns.Although the amino acid sequence of BacSp222 has no significant similarity to any known peptide or protein, a 3D structure similarity search indicates a close relation to other four-helix bundle-motif bacteriocins, such as aureocin A53, lacticin Q and enterocins 7A/7B. Assuming similar functions, biology, structure and physicochemical properties, we propose to distinguish the four-helix bundle bacteriocins as a new Type A in subclass IId of bacteriocins, containing linear, non-pediocin-like peptides.

Spatial attributes of the four-helix bundle group of bacteriocins – The high-resolution structure of BacSp222 in solution

KAUST Repository

Nowakowski, Michał; Jaremko, Łukasz; Wladyka, Benedykt; Dubin, Grzegorz; Ejchart, Andrzej; Mak, Paweł

2017-01-01

collected. The structure was calculated based on NMR-derived constraints and consists of a rigid and tightly packed globular bundle of four alpha-helices separated by three short turns.Although the amino acid sequence of BacSp222 has no significant

Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae

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Gonthier Lucy

2010-08-01

Full Text Available Abstract Background The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L. constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Findings Two bacterial artificial chromosome (BAC libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. Conclusions This

Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

Science.gov (United States)

Gonthier, Lucy; Bellec, Arnaud; Blassiau, Christelle; Prat, Elisa; Helmstetter, Nicolas; Rambaud, Caroline; Huss, Brigitte; Hendriks, Theo; Bergès, Hélène; Quillet, Marie-Christine

2010-08-11

The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L.) constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Two bacterial artificial chromosome (BAC) libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. This indicated that both BAC libraries are valuable tools for molecular

Conversion of BAC Clones into Binary BAC (BIBAC) Vectors and Their Delivery into Basidiomycete Fungal Cells Using Agrobacterium tumefaciens

KAUST Repository

Ali, Shawkat

2014-09-19

The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

Science.gov (United States)

Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille

2012-01-01

Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464

Genome sequencing of the sweetpotato whitefly Bemisia tabaci MED/Q.

Science.gov (United States)

Xie, Wen; Chen, Chunhai; Yang, Zezhong; Guo, Litao; Yang, Xin; Wang, Dan; Chen, Ming; Huang, Jinqun; Wen, Yanan; Zeng, Yang; Liu, Yating; Xia, Jixing; Tian, Lixia; Cui, Hongying; Wu, Qingjun; Wang, Shaoli; Xu, Baoyun; Li, Xianchun; Tan, Xinqiu; Ghanim, Murad; Qiu, Baoli; Pan, Huipeng; Chu, Dong; Delatte, Helene; Maruthi, M N; Ge, Feng; Zhou, Xueping; Wang, Xiaowei; Wan, Fanghao; Du, Yuzhou; Luo, Chen; Yan, Fengming; Preisser, Evan L; Jiao, Xiaoguo; Coates, Brad S; Zhao, Jinyang; Gao, Qiang; Xia, Jinquan; Yin, Ye; Liu, Yong; Brown, Judith K; Zhou, Xuguo Joe; Zhang, Youjun

2017-05-01

The sweetpotato whitefly Bemisia tabaci is a highly destructive agricultural and ornamental crop pest. It damages host plants through both phloem feeding and vectoring plant pathogens. Introductions of B. tabaci are difficult to quarantine and eradicate because of its high reproductive rates, broad host plant range, and insecticide resistance. A total of 791 Gb of raw DNA sequence from whole genome shotgun sequencing, and 13 BAC pooling libraries were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 437 kb, and a total length of 658 Mb. Annotation of repetitive elements and coding regions resulted in 265.0 Mb TEs (40.3%) and 20 786 protein-coding genes with putative gene family expansions, respectively. Phylogenetic analysis based on orthologs across 14 arthropod taxa suggested that MED/Q is clustered into a hemipteran clade containing A. pisum and is a sister lineage to a clade containing both R. prolixus and N. lugens. Genome completeness, as estimated using the CEGMA and Benchmarking Universal Single-Copy Orthologs pipelines, reached 96% and 79%. These MED/Q genomic resources lay a foundation for future 'pan-genomic' comparisons of invasive vs. noninvasive, invasive vs. invasive, and native vs. exotic Bemisia, which, in return, will open up new avenues of investigation into whitefly biology, evolution, and management. © The Author 2017. Published by Oxford University Press.

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L.and O.granulata Nees et Am.ex Watt.

Institute of Scientific and Technical Information of China (English)

XIONG Zhiyong; TAN Guangxuan; YOU Aiqing; HE Guangyuan; SHE Chaowen; LI Lijia; SONG Yunchun

2004-01-01

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice, which are the most valuable for study and utilization in China. In this study, the homology and physical locations of three rice resistance genes, Glh,Bph-3 and xa-5 are comparatively analyzed between O. sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH). The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata. By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O. sativa and O. granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O. sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O. sativa and O. granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.

Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector.

Science.gov (United States)

Lobo, N F; Hua-Van, A; Li, X; Nolen, B M; Fraser, M J

2002-04-01

Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes.

Isolation and sequence analysis of the wheat B genome subtelomeric DNA.

Science.gov (United States)

Salina, Elena A; Sergeeva, Ekaterina M; Adonina, Irina G; Shcherban, Andrey B; Afonnikov, Dmitry A; Belcram, Harry; Huneau, Cecile; Chalhoub, Boulos

2009-09-05

Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119,737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11,666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time

SSR_pipeline--computer software for the identification of microsatellite sequences from paired-end Illumina high-throughput DNA sequence data

Science.gov (United States)

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (SSRs; for example, microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains three analysis modules along with a fourth control module that can be used to automate analyses of large volumes of data. The modules are used to (1) identify the subset of paired-end sequences that pass quality standards, (2) align paired-end reads into a single composite DNA sequence, and (3) identify sequences that possess microsatellites conforming to user specified parameters. Each of the three separate analysis modules also can be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc). All modules are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, Windows). The program suite relies on a compiled Python extension module to perform paired-end alignments. Instructions for compiling the extension from source code are provided in the documentation. Users who do not have Python installed on their computers or who do not have the ability to compile software also may choose to download packaged executable files. These files include all Python scripts, a copy of the compiled extension module, and a minimal installation of Python in a single binary executable. See program documentation for more information.

In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

Science.gov (United States)

Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

2011-01-01

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

Bacterial contamination of platelet components not detected by BacT/ALERT®.

Science.gov (United States)

Abela, M A; Fenning, S; Maguire, K A; Morris, K G

2018-02-01

To investigate the possible causes for false negative results in BacT/ALERT ® 3D Signature System despite bacterial contamination of platelet units. The Northern Ireland Blood Transfusion Service (NIBTS) routinely extends platelet component shelf life to 7 days. Components are sampled and screened for bacterial contamination using an automated microbial detection system, the BacT/ALERT ® 3D Signature System. We report on three platelet components with confirmed bacterial contamination, which represent false negative BacT/ALERT ® results and near-miss serious adverse events. NIBTS protocols for risk reduction of bacterial contamination of platelet components are described. The methodology for bacterial detection using BacT/ALERT ® is outlined. Laboratory tests, relevant patient details and relevant follow-up information are analysed. In all three cases, Staphylococcus aureus was isolated from the platelet residue and confirmed on terminal sub-culture using BacT/ALERT ® . In two cases, S. aureus with similar genetic makeup was isolated from the donors. Risk reduction measures for bacterial contamination of platelet components are not always effective. Automated bacterial culture detection does not eliminate the risk of bacterial contamination. Visual inspection of platelet components prior to release, issue and administration remains an important last line of defence. © 2017 British Blood Transfusion Society.

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey

OpenAIRE

Luo, Meizhong; Kim, HyeRan; Kudrna, Dave; Sisneros, Nicholas B; Lee, So-Jeong; Mueller, Christopher; Collura, Kristi; Zuccolo, Andrea; Buckingham, E Bryan; Grim, Suzanne M; Yanagiya, Kazuyo; Inoko, Hidetoshi; Shiina, Takashi; Flajnik, Martin F; Wing, Rod A

2006-01-01

Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) ...

Biomass Resource Allocation among Competing End Uses

Energy Technology Data Exchange (ETDEWEB)

Newes, E.; Bush, B.; Inman, D.; Lin, Y.; Mai, T.; Martinez, A.; Mulcahy, D.; Short, W.; Simpkins, T.; Uriarte, C.; Peck, C.

2012-05-01

The Biomass Scenario Model (BSM) is a system dynamics model developed by the U.S. Department of Energy as a tool to better understand the interaction of complex policies and their potential effects on the biofuels industry in the United States. However, it does not currently have the capability to account for allocation of biomass resources among the various end uses, which limits its utilization in analysis of policies that target biomass uses outside the biofuels industry. This report provides a more holistic understanding of the dynamics surrounding the allocation of biomass among uses that include traditional use, wood pellet exports, bio-based products and bioproducts, biopower, and biofuels by (1) highlighting the methods used in existing models' treatments of competition for biomass resources; (2) identifying coverage and gaps in industry data regarding the competing end uses; and (3) exploring options for developing models of biomass allocation that could be integrated with the BSM to actively exchange and incorporate relevant information.

Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

Directory of Open Access Journals (Sweden)

Gillet Laurent

2011-08-01

Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC, the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA. As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G differs from the other prDNA units (prDNA-inner. Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

CREST--classification resources for environmental sequence tags.

Directory of Open Access Journals (Sweden)

Anders Lanzén

Full Text Available Sequencing of taxonomic or phylogenetic markers is becoming a fast and efficient method for studying environmental microbial communities. This has resulted in a steadily growing collection of marker sequences, most notably of the small-subunit (SSU ribosomal RNA gene, and an increased understanding of microbial phylogeny, diversity and community composition patterns. However, to utilize these large datasets together with new sequencing technologies, a reliable and flexible system for taxonomic classification is critical. We developed CREST (Classification Resources for Environmental Sequence Tags, a set of resources and tools for generating and utilizing custom taxonomies and reference datasets for classification of environmental sequences. CREST uses an alignment-based classification method with the lowest common ancestor algorithm. It also uses explicit rank similarity criteria to reduce false positives and identify novel taxa. We implemented this method in a web server, a command line tool and the graphical user interfaced program MEGAN. Further, we provide the SSU rRNA reference database and taxonomy SilvaMod, derived from the publicly available SILVA SSURef, for classification of sequences from bacteria, archaea and eukaryotes. Using cross-validation and environmental datasets, we compared the performance of CREST and SilvaMod to the RDP Classifier. We also utilized Greengenes as a reference database, both with CREST and the RDP Classifier. These analyses indicate that CREST performs better than alignment-free methods with higher recall rate (sensitivity as well as precision, and with the ability to accurately identify most sequences from novel taxa. Classification using SilvaMod performed better than with Greengenes, particularly when applied to environmental sequences. CREST is freely available under a GNU General Public License (v3 from http://apps.cbu.uib.no/crest and http://lcaclassifier.googlecode.com.

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

Science.gov (United States)

Silar, Philippe; Barreau, Christian; Debuchy, Robert; Kicka, Sébastien; Turcq, Béatrice; Sainsard-Chanet, Annie; Sellem, Carole H; Billault, Alain; Cattolico, Laurence; Duprat, Simone; Weissenbach, Jean

2003-08-01

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

A controlled comparison of the BacT/ALERT® 3D and VIRTUO™ microbial detection systems.

Science.gov (United States)

Totty, H; Ullery, M; Spontak, J; Viray, J; Adamik, M; Katzin, B; Dunne, W M; Deol, P

2017-10-01

The performance of the next-generation BacT/ALERT® VIRTUO™ Microbial Detection System (VIRTUO™, bioMérieux Inc., Hazelwood, MO) was compared to the BacT/ALERT® 3D Microbial Detection System (3D, bioMérieux Inc., Durham, NC) using BacT/ALERT® FA Plus (FA Plus), BacT/ALERT® PF Plus (PF Plus), BacT/ALERT® FN Plus (FN Plus), BacT/ALERT® Standard Aerobic (SA), and BacT/ALERT® Standard Anaerobic (SN) blood culture bottles (bioMérieux Inc., Durham, NC). A seeded limit of detection (LoD) study was performed for each bottle type in both systems. The LoD studies demonstrated that both systems were capable of detecting organisms at nearly identical levels [detection (TTD) between the systems using a panel of clinically relevant microorganisms inoculated at or near the LoD with 0, 4, or 10 mL of healthy human blood. VIRTUO™ exhibited a faster TTD by an average of 3.5 h, as well as demonstrated a significantly improved detection rate of 99.9% compared to 98.8% with 3D (p-value <0.05).

Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System

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Bo Lin

2014-01-01

Full Text Available The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

Isolation and sequence analysis of the wheat B genome subtelomeric DNA

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Huneau Cecile

2009-09-01

Full Text Available Abstract Background Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. Results The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs, 8.2% Spelt52 (namely, the subfamily Spelt52.2 and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0. Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Conclusion Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat

Generating resources for genomics of wheat homoeologous chromosome group 3: 3AS- and 3DS-specific BAC libraries

Czech Academy of Sciences Publication Activity Database

Šafář, Jan; Šimková, Hana; Kubaláková, Marie; Suchánková, Pavla; Čihalíková, Jarmila; Bartoš, Jan; Fiocchetti, F.; Roselli, M.; Gill, B. S.; Doležel, Jaroslav; Lucretti, S.

2009-01-01

Roč. 61, 1-2 (2009), s. 151-160 ISSN 0394-9257 R&D Projects: GA ČR GD521/05/H013; GA ČR GA521/06/1723; GA ČR GA521/07/1573; GA MŠk(CZ) LC06004; GA MŠk OC08025 Institutional research plan: CEZ:AV0Z50380511 Keywords : BAC library * Flow sorting * Homoeologous chromosomes Subject RIV: GE - Plant Breeding

Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

Energy Technology Data Exchange (ETDEWEB)

Korenberg, J.R.

1997-12-31

The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca

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Zhang Ying

2006-11-01

Full Text Available Abstract Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH. Conclusion This is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

Comparison of biological activated carbon (BAC) and membrane bioreactor (MBR) for pollutants removal in drinking water treatment.

Science.gov (United States)

Tian, J Y; Chen, Z L; Liang, H; Li, X; Wang, Z Z; Li, G B

2009-01-01

Biological activated carbon (BAC) and membrane bioreactor (MBR) were systematically compared for the drinking water treatment from slightly polluted raw water under the same hydraulic retention time (HRT) of 0.5 h. MBR exhibited excellent turbidity removal capacity due to the separation of the membrane; while only 60% of influent turbidity was intercepted by BAC. Perfect nitrification was achieved by MBR with the 89% reduction in ammonia; by contrast, BAC only eliminated a moderate amount of influent ammonia (by 54.5%). However, BAC was able to remove more dissolved organic matter (DOM, especially for organic molecules of 3,000 approximately 500 Daltons) and corresponding disinfection by-product formation potential (DBPFP) in raw water than MBR. Unfortunately, particulate organic matter (POM) was detected in the BAC effluent. On the other hand, BAC and MBR displayed essentially the same capacity for biodegradable organic matter (BOM) removal. Fractionation of DOM showed that the removal efficiencies of hydrophobic neutrals, hydrophobic acids, weakly hydrophobic acids and hydrophilic organic matter through BAC treatment were 11.7%, 8.8%, 13.9% and 4.8% higher than that through MBR; while MBR achieved 13.8% higher hydrophobic bases removal as compared with BAC.

When less is more: 'slicing' sequencing data improves read decoding accuracy and de novo assembly quality.

Science.gov (United States)

Lonardi, Stefano; Mirebrahim, Hamid; Wanamaker, Steve; Alpert, Matthew; Ciardo, Gianfranco; Duma, Denisa; Close, Timothy J

2015-09-15

As the invention of DNA sequencing in the 70s, computational biologists have had to deal with the problem of de novo genome assembly with limited (or insufficient) depth of sequencing. In this work, we investigate the opposite problem, that is, the challenge of dealing with excessive depth of sequencing. We explore the effect of ultra-deep sequencing data in two domains: (i) the problem of decoding reads to bacterial artificial chromosome (BAC) clones (in the context of the combinatorial pooling design we have recently proposed), and (ii) the problem of de novo assembly of BAC clones. Using real ultra-deep sequencing data, we show that when the depth of sequencing increases over a certain threshold, sequencing errors make these two problems harder and harder (instead of easier, as one would expect with error-free data), and as a consequence the quality of the solution degrades with more and more data. For the first problem, we propose an effective solution based on 'divide and conquer': we 'slice' a large dataset into smaller samples of optimal size, decode each slice independently, and then merge the results. Experimental results on over 15 000 barley BACs and over 4000 cowpea BACs demonstrate a significant improvement in the quality of the decoding and the final assembly. For the second problem, we show for the first time that modern de novo assemblers cannot take advantage of ultra-deep sequencing data. Python scripts to process slices and resolve decoding conflicts are available from http://goo.gl/YXgdHT; software Hashfilter can be downloaded from http://goo.gl/MIyZHs stelo@cs.ucr.edu or timothy.close@ucr.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Tandemly repeated sequence in 5'end of mtDNA control region of ...

African Journals Online (AJOL)

Extensive length variability was observed in 5' end sequence of the mitochondrial DNA control region of the Japanese Spanish mackerel (Scomberomorus niphonius). This length variability was due to the presence of varying numbers of a 56-bp tandemly repeated sequence and a 46-bp insertion/deletion (indel).

Bac on the Border

OpenAIRE

Umberger, Emily

2007-01-01

Although now on the Tohono O'odom reservation in the modern US Southwest, when the Franciscan church of San Xavier del Bac was built (1780-97), its location was the northern frontier of New Spain. From the outset the church stood out from other northern New Spanish missions in its elaborate decoration, and it still stands out because its contents remain intact, despite changes through time. This essay serves as an introduction to the church as a subject of art historical study. It highlights ...

Rhipicephalus microplus strain Deutsch, 10 BAC clone sequences

Science.gov (United States)

The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. We used labeled DNA probes from the coding reg...

De novo assembly, gene annotation and marker development using Illumina paired-end transcriptome sequences in celery (Apium graveolens L..

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Nan Fu

Full Text Available BACKGROUND: Celery is an increasing popular vegetable species, but limited transcriptome and genomic data hinder the research to it. In addition, a lack of celery molecular markers limits the process of molecular genetic breeding. High-throughput transcriptome sequencing is an efficient method to generate a large transcriptome sequence dataset for gene discovery, molecular marker development and marker-assisted selection breeding. PRINCIPAL FINDINGS: Celery transcriptomes from four tissues were sequenced using Illumina paired-end sequencing technology. De novo assembling was performed to generate a collection of 42,280 unigenes (average length of 502.6 bp that represent the first transcriptome of the species. 78.43% and 48.93% of the unigenes had significant similarity with proteins in the National Center for Biotechnology Information (NCBI non-redundant protein database (Nr and Swiss-Prot database respectively, and 10,473 (24.77% unigenes were assigned to Clusters of Orthologous Groups (COG. 21,126 (49.97% unigenes harboring Interpro domains were annotated, in which 15,409 (36.45% were assigned to Gene Ontology(GO categories. Additionally, 7,478 unigenes were mapped onto 228 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG. Large numbers of simple sequence repeats (SSRs were indentified, and then the rate of successful amplication and polymorphism were investigated among 31 celery accessions. CONCLUSIONS: This study demonstrates the feasibility of generating a large scale of sequence information by Illumina paired-end sequencing and efficient assembling. Our results provide a valuable resource for celery research. The developed molecular markers are the foundation of further genetic linkage analysis and gene localization, and they will be essential to accelerate the process of breeding.

Application of EDTA-functionalized bamboo activated carbon (BAC) for Pb(II) and Cu(II) removal from aqueous solutions

Science.gov (United States)

Lv, Dan; Liu, Yu; Zhou, Jiasheng; Yang, Kunlun; Lou, Zimo; Baig, Shams Ali; Xu, Xinhua

2018-01-01

In this study, a novel bamboo activated carbon (BAC) with ethylene diamine tetraacetic acid (EDTA) functionality was prepared by direct grafting in the presence of tetraethyl orthosilicate (TEOS) as a crosslinking agent. The BAC@SiO2-EDTA was characterized by SEM, TEM, TGA, FTIR, XPS and its adsorption property for removal of Pb(II) and Cu(II) under various experimental conditions was also investigated. The characterization results reflected that EDTA was successfully assembled on the surface of the BAC and average pore size increased from 4.10 to 4.83 nm as BAC grafted with EDTA. Adsorption data fitted very well in Langmuir isotherm model and pseudo-second-order kinetic model. As compared with the raw BAC, the maximum adsorption capacities of BAC@SiO2-EDTA for the Pb(II) and Cu(II) increased from 45.45 to 123.45 mg g-1 and from 6.85 to 42.19 mg g-1, since the existence of EDTA on modified BAC promoted the formation of chemical complex. The removal of heavy metal ions mainly depended on the complexation with EDTA and the electrostatic attractions with negatively charged surface of BAC@SiO2-EDTA. The adsorption of Pb(II)/Cu(II) on the BAC@SiO2-EDTA was pH dependent and pH 5-6 was considered an optimum. However, lower temperature favored the adsorption and the maximum adsorption was recorded at 20 °C. In addition, BAC@SiO2-EDTA had an excellent reusability with about 40% decline in the adsorption capacity for Pb(II) after fifth reuse. Insignificant influences of co-existing cations and natural organic matter (NOM) were found on the adsorption of Pb(II) and Cu(II). All the results demonstrate that BAC@SiO2-EDTA is a potential adsorbent for metal ions in wastewater.

Sensitivity and applicability of the Brazilian version of the Brief Assessment of Cognition in Schizophrenia (BACS

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João Vinícius Salgado

Full Text Available Abstract Cognitive assessment in schizophrenia has traditionally used batteries that are long and complex or differ widely in their content. The Brief Assessment of Cognition in Schizophrenia (BACS has been developed to cover the main cognitive deficits of schizophrenia as well as to be easily and briefly administered, portable, sensitive and reliable. Objectives: To investigate the applicability and sensitivity of the Brazilian Version of the BACS (Brazilian-BACS. Methods: Performance of 20 stable patients with schizophrenia on the Brazilian-BACS was compared to 20 matched healthy controls. Results: Applying the Brazilian-BACS required 43.4±8.4 minutes for patients and 40.5±5.7 minutes for controls (p=0.17. All tests demonstrated significant differences between controls and patients (P<0.01. Pearson's correlation analysis and Cronbach's a evidenced a high internal consistency for patient performance. The cognitive deficit in the patients was approximately 1.5 standard deviations below controls. These results were consistent with those reported in the validation of the original version and in meta-analyses of similar studies. Conclusions: The Brazilian-BACS displayed good applicability and sensitivity in assessing the major cognitive constructs that are impaired in schizophrenia. Thus, the Brazilian-BACS seems to be a promising tool for assessing cognition in patients with schizophrenia in Brazil.

High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.

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Shin-ichi Hashimoto

Full Text Available Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza. More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

A Validation Approach of an End-to-End Whole Genome Sequencing Workflow for Source Tracking of Listeria monocytogenes and Salmonella enterica

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Anne-Catherine Portmann

2018-03-01

Full Text Available Whole genome sequencing (WGS, using high throughput sequencing technology, reveals the complete sequence of the bacterial genome in a few days. WGS is increasingly being used for source tracking, pathogen surveillance and outbreak investigation due to its high discriminatory power. In the food industry, WGS used for source tracking is beneficial to support contamination investigations. Despite its increased use, no standards or guidelines are available today for the use of WGS in outbreak and/or trace-back investigations. Here we present a validation of our complete (end-to-end WGS workflow for Listeria monocytogenes and Salmonella enterica including: subculture of isolates, DNA extraction, sequencing and bioinformatics analysis. This end-to-end WGS workflow was evaluated according to the following performance criteria: stability, repeatability, reproducibility, discriminatory power, and epidemiological concordance. The current study showed that few single nucleotide polymorphism (SNPs were observed for L. monocytogenes and S. enterica when comparing genome sequences from five independent colonies from the first subculture and five independent colonies after the tenth subculture. Consequently, the stability of the WGS workflow for L. monocytogenes and S. enterica was demonstrated despite the few genomic variations that can occur during subculturing steps. Repeatability and reproducibility were also demonstrated. The WGS workflow was shown to have a high discriminatory power and has the ability to show genetic relatedness. Additionally, the WGS workflow was able to reproduce published outbreak investigation results, illustrating its capability of showing epidemiological concordance. The current study proposes a validation approach comprising all steps of a WGS workflow and demonstrates that the workflow can be applied to L. monocytogenes or S. enterica.

A PiggyBac-mediated approach for muscle gene transfer or cell therapy

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Déborah Ley

2014-11-01

Full Text Available An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.

Human pro. cap alpha. 1(III) collagen: cDNA sequence for the 3' end

Energy Technology Data Exchange (ETDEWEB)

Mankoo, B S; Dalgleish, R

1988-03-25

The authors have previously isolated two overlapping cDNA clones, pIII-21 and pIII-33, which encode the C-terminal end of human type III procollagen. They now present the sequence of 2520 bases encoded in these cDNAs which overlaps other previously published sequences for the same gene. The sequence presented differs from previously published sequences at five positions.

Sequence-Based Analysis of Structural Organization and Composition of the Cultivated Sunflower (Helianthus annuus L.) Genome

Science.gov (United States)

Gill, Navdeep; Buti, Matteo; Kane, Nolan; Bellec, Arnaud; Helmstetter, Nicolas; Berges, Hélène; Rieseberg, Loren H.

2014-01-01

Sunflower is an important oilseed crop, as well as a model system for evolutionary studies, but its 3.6 gigabase genome has proven difficult to assemble, in part because of the high repeat content of its genome. Here we report on the sequencing, assembly, and analyses of 96 randomly chosen BACs from sunflower to provide additional information on the repeat content of the sunflower genome, assess how repetitive elements in the sunflower genome are organized relative to genes, and compare the genomic distribution of these repeats to that found in other food crops and model species. We also examine the expression of transposable element-related transcripts in EST databases for sunflower to determine the representation of repeats in the transcriptome and to measure their transcriptional activity. Our data confirm previous reports in suggesting that the sunflower genome is >78% repetitive. Sunflower repeats share very little similarity to other plant repeats such as those of Arabidopsis, rice, maize and wheat; overall 28% of repeats are “novel” to sunflower. The repetitive sequences appear to be randomly distributed within the sequenced BACs. Assuming the 96 BACs are representative of the genome as a whole, then approximately 5.2% of the sunflower genome comprises non TE-related genic sequence, with an average gene density of 18kbp/gene. Expression levels of these transposable elements indicate tissue specificity and differential expression in vegetative and reproductive tissues, suggesting that expressed TEs might contribute to sunflower development. The assembled BACs will also be useful for assessing the quality of several different draft assemblies of the sunflower genome and for annotating the reference sequence. PMID:24833511

Sequence-Based Analysis of Structural Organization and Composition of the Cultivated Sunflower (Helianthus annuus L. Genome

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Navdeep Gill

2014-04-01

Full Text Available Sunflower is an important oilseed crop, as well as a model system for evolutionary studies, but its 3.6 gigabase genome has proven difficult to assemble, in part because of the high repeat content of its genome. Here we report on the sequencing, assembly, and analyses of 96 randomly chosen BACs from sunflower to provide additional information on the repeat content of the sunflower genome, assess how repetitive elements in the sunflower genome are organized relative to genes, and compare the genomic distribution of these repeats to that found in other food crops and model species. We also examine the expression of transposable element-related transcripts in EST databases for sunflower to determine the representation of repeats in the transcriptome and to measure their transcriptional activity. Our data confirm previous reports in suggesting that the sunflower genome is >78% repetitive. Sunflower repeats share very little similarity to other plant repeats such as those of Arabidopsis, rice, maize and wheat; overall 28% of repeats are “novel” to sunflower. The repetitive sequences appear to be randomly distributed within the sequenced BACs. Assuming the 96 BACs are representative of the genome as a whole, then approximately 5.2% of the sunflower genome comprises non TE-related genic sequence, with an average gene density of 18kbp/gene. Expression levels of these transposable elements indicate tissue specificity and differential expression in vegetative and reproductive tissues, suggesting that expressed TEs might contribute to sunflower development. The assembled BACs will also be useful for assessing the quality of several different draft assemblies of the sunflower genome and for annotating the reference sequence.

A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome

Czech Academy of Sciences Publication Activity Database

Parlange, Z.; Oberhaensli, S.; Breen, J.; Platzer, M.; Taudien, S.; Šimková, Hana; Wicker, T.; Doležel, Jaroslav; Keller, B.

2011-01-01

Roč. 11, č. 4 (2011), s. 671-677 ISSN 1438-793X R&D Projects: GA MŠk ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Blumeria graminis * BAC library * BAC-end sequences Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.842, year: 2011

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach

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Delfina Barabaschi

2015-11-01

Full Text Available The huge size, redundancy, and highly repetitive nature of the bread wheat [ (L.] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC. A total of 95,812 bacterial artificial chromosome (BAC clones of short-arm chromosome 5A (5AS and long-arm chromosome 5A (5AL arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC and Linear Topological Contig (LTC tools. Combined anchoring approaches based on polymerase chain reaction (PCR marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs, genome zipper, and chromosome survey sequences allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively of contigs ordered along the chromosome. In the genome of grasses, [ (L. Beauv.], rice ( L., and sorghum [ (L. Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Insect transformation with piggyBac: getting the number of injections just right

Science.gov (United States)

Morrison, N. I.; Shimeld, S. M.

2016-01-01

Abstract The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision‐making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co‐opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov‐Chain Monte‐Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac. PMID:27027400

Differential CT features of infectious pneumonia versus bronchioloalveolar carcinoma (BAC) mimicking pneumonia

International Nuclear Information System (INIS)

Kim, Tae Hoon; Kim, Sang Jin; Ryu, Young Hoon; Chung, Soo Yoon; Seo, Jae Seung; Kim, Young Jin; Choi, Byoung Wook; Lee, Sun Hwa; Cho, Sang Ho

2006-01-01

The purpose of this study was to evaluate retrospectively the differential CT features of bronchioloalveolar carcinoma (BAC) mimicking pneumonia and infectious pneumonia at the lung periphery. CT images were reviewed in 47 patients with focal areas of parenchymal opacification at the lung periphery. We evaluated the presence of ground-glass attenuation, marginal conspicuity of the lesion, CT angiogram sign, air-bronchogram sign, a bubble-like low-attenuation area within the lesion, presence of pleural thickening and retraction associated with the lesion, presence of pleural effusion and extra-pleural fatty hypertrophy, presence of bronchial wall thickening proximal to the lesion, and air-trapping in the normal lung near the lesion. BAC (n=18) depicted the presence of a bubble-like low-attenuation area within the lesion, whereas infectious pneumonia (n=29) represented the pleural thickening associated with the lesion and bronchial wall thickening proximal to the lesion (P 0.05). The focal areas of the parenchymal opacification on the CT images may suggest infectious pneumonia rather than BAC when they show bronchial wall thickening proximal to the lesion and pleural thickening associated with the lesion, whereas BAC is characterized as the presence of a bubble-like low attenuation area within the tumor. (orig.)

Differential CT features of infectious pneumonia versus bronchioloalveolar carcinoma (BAC) mimicking pneumonia

Energy Technology Data Exchange (ETDEWEB)

Kim, Tae Hoon [Yonsei University College of Medicine, Department of Radiology, Seoul (Korea); Yongdong Severance Hospital, Department of Radiology, Seoul (Korea); Kim, Sang Jin; Ryu, Young Hoon; Chung, Soo Yoon; Seo, Jae Seung; Kim, Young Jin; Choi, Byoung Wook [Yonsei University College of Medicine, Department of Radiology, Seoul (Korea); Lee, Sun Hwa [NeoDin Medical Institute, Department of Clinical Pathology, Seoul (Korea); Cho, Sang Ho [Yonsei University College of Medicine, Department of Pathology, Seoul (Korea)

2006-08-15

The purpose of this study was to evaluate retrospectively the differential CT features of bronchioloalveolar carcinoma (BAC) mimicking pneumonia and infectious pneumonia at the lung periphery. CT images were reviewed in 47 patients with focal areas of parenchymal opacification at the lung periphery. We evaluated the presence of ground-glass attenuation, marginal conspicuity of the lesion, CT angiogram sign, air-bronchogram sign, a bubble-like low-attenuation area within the lesion, presence of pleural thickening and retraction associated with the lesion, presence of pleural effusion and extra-pleural fatty hypertrophy, presence of bronchial wall thickening proximal to the lesion, and air-trapping in the normal lung near the lesion. BAC (n=18) depicted the presence of a bubble-like low-attenuation area within the lesion, whereas infectious pneumonia (n=29) represented the pleural thickening associated with the lesion and bronchial wall thickening proximal to the lesion (P<0.05). The other CT findings showed no significant differences (P>0.05). The focal areas of the parenchymal opacification on the CT images may suggest infectious pneumonia rather than BAC when they show bronchial wall thickening proximal to the lesion and pleural thickening associated with the lesion, whereas BAC is characterized as the presence of a bubble-like low attenuation area within the tumor. (orig.)

Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers

Science.gov (United States)

Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B

2015-01-01

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

Insulated piggyBac vectors for insect transgenesis

Directory of Open Access Journals (Sweden)

Horn Carsten

2006-06-01

Full Text Available Abstract Background Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. Results To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. Conclusion The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.

Physical Analysis of the Complex Rye (Secale cereale L.) Alt4 Aluminium (Aluminum) Tolerance Locus Using a Whole-Genome BAC Library of Rye cv. Blanco

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Rye is a diploid crop species with many outstanding qualities, and is also important as a source of new traits for wheat and triticale improvement. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies. The library provides a 6 × genome ...

High-throughput physical map anchoring via BAC-pool sequencing

Czech Academy of Sciences Publication Activity Database

Cviková, Kateřina; Cattonaro, F.; Alaux, M.; Stein, N.; Mayer, K.F.X.; Doležel, Jaroslav; Bartoš, Jan

2015-01-01

Roč. 15, APR 11 (2015) ISSN 1471-2229 R&D Projects: GA ČR GA13-08786S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Physical map * Contig anchoring * Next generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.631, year: 2015

MBBR evaluation for oil refinery wastewater treatment, with post-ozonation and BAC, for wastewater reuse.

Science.gov (United States)

Schneider, E E; Cerqueira, A C F P; Dezotti, M

2011-01-01

This work evaluated the performance of a Moving Bed Biofilm Reactor (MBBR) in the treatment of an oil refinery wastewater. Also, it investigated the possibility of reuse of the MBBR effluent, after ozonation in series with a biological activated carbon (BAC) column. The best performance of the MBBR was achieved with a hydraulic retention time (HRT) of 6 hours, employing a bed to bioreactor volume ratio (V(B)/V(R)) of 0.6. COD and N-NH₄(+) MBBR effluent concentrations ranged from 40 to 75 mg L⁻¹ (removal efficiency of 69-89%) and 2 to 6 mg L⁻¹ (removal efficiency of 45-86%), respectively. Ozonation carried out for 15 min with an ozone concentration of 5 mg L⁻¹ was able to improve the treated wastewater biodegradability. The treatment performance of the BAC columns was practically the same for ozonated and non ozonated MBBR effluents. The dissolved organic carbon (DOC) content of the columns of the activated carbon columns (CAG) was in the range of 2.1-3.8 mg L⁻¹, and the corresponding DOC removal efficiencies were comprised between 52 and 75%. The effluent obtained at the end of the proposed treatment presented a quality, which meet the requirements for water reuse in the oil refinery.

BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

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Yuki Muranishi

2012-01-01

Full Text Available Dickkopf (DKK family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.

Nucleotide sequence of the 3' ends of the double-stranded RNAs of grapevine chrome mosaic nepovirus.

Science.gov (United States)

Le Gall, O; Candresse, T; Dunez, J

1988-02-01

Attempts were made to label the termini of dsRNAs corresponding to the two genomic RNAs of grapevine chrome mosaic nepovirus (GCMV). It was not possible to label the 5' ends of the dsRNAs with [gamma-32P]ATP, which suggests that a genome-linked protein blocks their 5' ends. Both dsRNA species were labelled at their 3' ends with pCp. The 3'-terminal sequences were determined by 'wandering spot' or by partial enzymic cleavage analysis. One strand (presumably positive) ended in a poly(A) 30 to 50 nucleotides long whereas the other (presumably negative) ended in 3'-ACCUUUUAAAAAG (RNA1) or 3'-ACCUUUUAAUAAAG (RNA2). The sequences resemble closely those complementary to the 5' ends of the RNAs of tomato black ring virus (strain S), which is distantly related to GCMV.

Development of Genomic Resources in the Species of Trifolium L. and Its Application in Forage Legume Breeding

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Leif Skøt

2012-05-01

Full Text Available Clovers (genus Trifolium are a large and widespread genus of legumes. A number of clovers are of agricultural importance as forage crops in grassland agriculture, particularly temperate areas. White clover (Trifolium repens L. is used in grazed pasture and red clover (T. pratense L. is widely cut and conserved as a winter feed. For the diploid red clover, genetic and genomic tools and resources have developed rapidly over the last five years including genetic and physical maps, BAC (bacterial artificial chromosome end sequence and transcriptome sequence information. This has paved the way for the use of genome wide selection and high throughput phenotyping in germplasm development. For the allotetraploid white clover progress has been slower although marker assisted selection is in use and relatively robust genetic maps and QTL (quantitative trait locus information now exist. For both species the sequencing of the model legume Medicago truncatula gene space is an important development to aid genomic, biological and evolutionary studies. The first genetic maps of another species, subterranean clover (Trifolium subterraneum L. have also been published and its comparative genomics with red clover and M. truncatula conducted. Next generation sequencing brings the potential to revolutionize clover genomics, but international consortia and effective use of germplasm, novel population structures and phenomics will be required to carry out effective translation into breeding. Another avenue for clover genomic and genetic improvement is interspecific hybridization. This approach has considerable potential with regard to crop improvement but also opens windows of opportunity for studies of biological and evolutionary processes.

Characterization of the Kenaf (Hibiscus cannabinus) Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers

Science.gov (United States)

Li, Hui; Li, Defang; Chen, Anguo; Tang, Huijuan; Li, Jianjun; Huang, Siqi

2016-01-01

Kenaf (Hibiscus cannabinus L.) is an economically important natural fiber crop grown worldwide. However, only 20 expressed tag sequences (ESTs) for kenaf are available in public databases. The aim of this study was to develop large-scale simple sequence repeat (SSR) markers to lay a solid foundation for the construction of genetic linkage maps and marker-assisted breeding in kenaf. We used Illumina paired-end sequencing technology to generate new EST-simple sequences and MISA software to mine SSR markers. We identified 71,318 unigenes with an average length of 1143 nt and annotated these unigenes using four different protein databases. Overall, 9324 complementary pairs were designated as EST-SSR markers, and their quality was validated using 100 randomly selected SSR markers. In total, 72 primer pairs reproducibly amplified target amplicons, and 61 of these primer pairs detected significant polymorphism among 28 kenaf accessions. Thus, in this study, we have developed large-scale SSR markers for kenaf, and this new resource will facilitate construction of genetic linkage maps, investigation of fiber growth and development in kenaf, and also be of value to novel gene discovery and functional genomic studies. PMID:26960153

Characterization of the Kenaf (Hibiscus cannabinus) Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers.

Science.gov (United States)

Li, Hui; Li, Defang; Chen, Anguo; Tang, Huijuan; Li, Jianjun; Huang, Siqi

2016-01-01

Kenaf (Hibiscus cannabinus L.) is an economically important natural fiber crop grown worldwide. However, only 20 expressed tag sequences (ESTs) for kenaf are available in public databases. The aim of this study was to develop large-scale simple sequence repeat (SSR) markers to lay a solid foundation for the construction of genetic linkage maps and marker-assisted breeding in kenaf. We used Illumina paired-end sequencing technology to generate new EST-simple sequences and MISA software to mine SSR markers. We identified 71,318 unigenes with an average length of 1143 nt and annotated these unigenes using four different protein databases. Overall, 9324 complementary pairs were designated as EST-SSR markers, and their quality was validated using 100 randomly selected SSR markers. In total, 72 primer pairs reproducibly amplified target amplicons, and 61 of these primer pairs detected significant polymorphism among 28 kenaf accessions. Thus, in this study, we have developed large-scale SSR markers for kenaf, and this new resource will facilitate construction of genetic linkage maps, investigation of fiber growth and development in kenaf, and also be of value to novel gene discovery and functional genomic studies.

Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

Directory of Open Access Journals (Sweden)

Andrew Jo

Full Text Available Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

Science.gov (United States)

Staňková, Helena; Hastie, Alex R; Chan, Saki; Vrána, Jan; Tulpová, Zuzana; Kubaláková, Marie; Visendi, Paul; Hayashi, Satomi; Luo, Mingcheng; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2016-07-01

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Resource intensities of the front end of the nuclear fuel cycle

International Nuclear Information System (INIS)

Schneider, E.; Phathanapirom, U.; Eggert, R.; Collins, J.

2013-01-01

This paper presents resource intensities, including direct and embodied energy consumption, land and water use, associated with the processes comprising the front end of the nuclear fuel cycle. These processes include uranium extraction, conversion, enrichment, fuel fabrication and depleted uranium de-conversion. To the extent feasible, these impacts are calculated based on data reported by operating facilities, with preference given to more recent data based on current technologies and regulations. All impacts are normalized per GWh of electricity produced. Uranium extraction is seen to be the most resource intensive front end process. Combined, the energy consumed by all front end processes is equal to less than 1% of the electricity produced by the uranium in a nuclear reactor. Land transformation and water withdrawals are calculated at 8.07 m 2 /GWh(e) and 1.37x10 5 l/GWh(e), respectively. Both are dominated by the requirements of uranium extraction, which accounts for over 70% of land use and nearly 90% of water use

Measuring brain activity cycling (BAC) in long term EEG monitoring of preterm babies

International Nuclear Information System (INIS)

Stevenson, Nathan J; Palmu, Kirsi; Wikström, Sverre; Hellström-Westas, Lena; Vanhatalo, Sampsa

2014-01-01

Measuring fluctuation of vigilance states in early preterm infants undergoing long term intensive care holds promise for monitoring their neurological well-being. There is currently, however, neither objective nor quantitative methods available for this purpose in a research or clinical environment. The aim of this proof-of-concept study was, therefore, to develop quantitative measures of the fluctuation in vigilance states or brain activity cycling (BAC) in early preterm infants. The proposed measures of BAC were summary statistics computed on a frequency domain representation of the proportional duration of spontaneous activity transients (SAT%) calculated from electroencephalograph (EEG) recordings. Eighteen combinations of three statistics and six frequency domain representations were compared to a visual interpretation of cycling in the SAT% signal. Three high performing measures (band energy/periodogram: R = 0.809, relative band energy/nonstationary frequency marginal: R = 0.711, g-statistic/nonstationary frequency marginal: R = 0.638) were then compared to a grading of sleep wake cycling based on the visual interpretation of the amplitude-integrated EEG trend. These measures of BAC are conceptually straightforward, correlate well with the visual scores of BAC and sleep wake cycling, are robust enough to cope with the technically compromised monitoring data available in intensive care units, and are recommended for further validation in prospective studies. (paper)

Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

Science.gov (United States)

Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

2017-03-01

Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017. © 2017 American Institute of Chemical Engineers.

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

Science.gov (United States)

Chakraborty, Syandan; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.

2014-01-01

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. PMID:25492703

ASAP: Amplification, sequencing & annotation of plastomes

Directory of Open Access Journals (Sweden)

Folta Kevin M

2005-12-01

Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

A preliminary categorization of end-of-life electrical and electronic equipment as secondary metal resources

International Nuclear Information System (INIS)

Oguchi, Masahiro; Murakami, Shinsuke; Sakanakura, Hirofumi; Kida, Akiko; Kameya, Takashi

2011-01-01

Highlights: → End-of-life electrical and electronic equipment (EEE) as secondary metal resources. → The content and the total amount of metals in specific equipment are both important. → We categorized 21 EEE types from contents and total amounts of various metals. → Important equipment types as secondary resources were listed for each metal kind. → Collectability and possible collection systems of various EEE types were discussed. - Abstract: End-of-life electrical and electronic equipment (EEE) has recently received attention as a secondary source of metals. This study examined characteristics of end-of-life EEE as secondary metal resources to consider efficient collection and metal recovery systems according to the specific metals and types of EEE. We constructed an analogy between natural resource development and metal recovery from end-of-life EEE and found that metal content and total annual amount of metal contained in each type of end-of-life EEE should be considered in secondary resource development, as well as the collectability of the end-of-life products. We then categorized 21 EEE types into five groups and discussed their potential as secondary metal resources. Refrigerators, washing machines, air conditioners, and CRT TVs were evaluated as the most important sources of common metals, and personal computers, mobile phones, and video games were evaluated as the most important sources of precious metals. Several types of small digital equipment were also identified as important sources of precious metals; however, mid-size information and communication technology (ICT) equipment (e.g., printers and fax machines) and audio/video equipment were shown to be more important as a source of a variety of less common metals. The physical collectability of each type of EEE was roughly characterized by unit size and number of end-of-life products generated annually. Current collection systems in Japan were examined and potentially appropriate collection

Validation of the French version of the BACS (the brief assessment of cognition in schizophrenia) among 50 French schizophrenic patients.

Science.gov (United States)

Bralet, Marie-Cécile; Falissard, Bruno; Neveu, Xavier; Lucas-Ross, Margaret; Eskenazi, Anne-Marie; Keefe, Richard S E

2007-09-01

Schizophrenic patients demonstrate impairments in several key dimensions of cognition. These impairments are correlated with important aspects of functional outcome. While assessment of these cognition disorders is increasingly becoming a part of clinical and research practice in schizophrenia, there is no standard and easily administered test battery. The BACS (Brief Assessment of Cognition in Schizophrenia) has been validated in English language [Keefe RSE, Golberg TE, Harvey PD, Gold JM, Poe MP, Coughenour L. The Brief Assessment of Cognition in Schizophrenia: reliability, sensibility, and comparison with a standard neurocognitive battery. Schizophr. Res 2004;68:283-97], and was found to be as sensitive to cognitive dysfunction as a standard battery of tests, with the advantage of requiring less than 35 min to complete. We developed a French adaptation of the BACS and this study tested its ease of administration and concurrent validity. Correlation analyses between the BACS (version A) and a standard battery were performed. A sample of 50 stable schizophrenic patients received the French Version A of the BACS in a first session, and in a second session a standard battery. All the patients completed each of the subtests of the French BACS . The mean duration of completion for the BACS French version was 36 min (S.D.=5.56). A correlation analysis between the BACS (version A) global score and the standard battery global score showed a significant result (r=0.81, p<0.0001). The correlation analysis between the BACS (version A) sub-scores and the standard battery sub-scores showed significant results for verbal memory, working memory, verbal fluency, attention and speed of information processing and executive functions (p<0.001) and for motor speed (p<0.05). The French Version of the BACS is easier to use in French schizophrenic patients compared to a standard battery (administration shorter and completion rate better) and its good psychometric properties suggest

MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

Science.gov (United States)

Gradia, Scott D; Ishida, Justin P; Tsai, Miaw-Sheue; Jeans, Chris; Tainer, John A; Fuss, Jill O

2017-01-01

Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done. © 2017 Elsevier Inc. All rights reserved.

Brassica ASTRA: an integrated database for Brassica genomic research.

Science.gov (United States)

Love, Christopher G; Robinson, Andrew J; Lim, Geraldine A C; Hopkins, Clare J; Batley, Jacqueline; Barker, Gary; Spangenberg, German C; Edwards, David

2005-01-01

Brassica ASTRA is a public database for genomic information on Brassica species. The database incorporates expressed sequences with Swiss-Prot and GenBank comparative sequence annotation as well as secondary Gene Ontology (GO) annotation derived from the comparison with Arabidopsis TAIR GO annotations. Simple sequence repeat molecular markers are identified within resident sequences and mapped onto the closely related Arabidopsis genome sequence. Bacterial artificial chromosome (BAC) end sequences derived from the Multinational Brassica Genome Project are also mapped onto the Arabidopsis genome sequence enabling users to identify candidate Brassica BACs corresponding to syntenic regions of Arabidopsis. This information is maintained in a MySQL database with a web interface providing the primary means of interrogation. The database is accessible at http://hornbill.cspp.latrobe.edu.au.

The need for blood alcohol concentration (BAC) legislation in Nigeria

African Journals Online (AJOL)

Dr Patrick O Erah

Tropical Journal of Pharmaceutical Research, June 2004; 3 (1): 319-327. © Pharmacotherapy Group, ... 1Department of Restorative Dentistry, Clinical therapeutic Unit, University of Benin, Benin City, .... An international survey of BAC is quite.

ABI Base Recall: Automatic Correction and Ends Trimming of DNA Sequences.

Science.gov (United States)

Elyazghi, Zakaria; Yazouli, Loubna El; Sadki, Khalid; Radouani, Fouzia

2017-12-01

Automated DNA sequencers produce chromatogram files in ABI format. When viewing chromatograms, some ambiguities are shown at various sites along the DNA sequences, because the program implemented in the sequencing machine and used to call bases cannot always precisely determine the right nucleotide, especially when it is represented by either a broad peak or a set of overlaying peaks. In such cases, a letter other than A, C, G, or T is recorded, most commonly N. Thus, DNA sequencing chromatograms need manual examination: checking for mis-calls and truncating the sequence when errors become too frequent. The purpose of this paper is to develop a program allowing the automatic correction of these ambiguities. This application is a Web-based program powered by Shiny and runs under R platform for an easy exploitation. As a part of the interface, we added the automatic ends clipping option, alignment against reference sequences, and BLAST. To develop and test our tool, we collected several bacterial DNA sequences from different laboratories within Institut Pasteur du Maroc and performed both manual and automatic correction. The comparison between the two methods was carried out. As a result, we note that our program, ABI base recall, accomplishes good correction with a high accuracy. Indeed, it increases the rate of identity and coverage and minimizes the number of mismatches and gaps, hence it provides solution to sequencing ambiguities and saves biologists' time and labor.

The Release 6 reference sequence of the Drosophila melanogaster genome.

Science.gov (United States)

Hoskins, Roger A; Carlson, Joseph W; Wan, Kenneth H; Park, Soo; Mendez, Ivonne; Galle, Samuel E; Booth, Benjamin W; Pfeiffer, Barret D; George, Reed A; Svirskas, Robert; Krzywinski, Martin; Schein, Jacqueline; Accardo, Maria Carmela; Damia, Elisabetta; Messina, Giovanni; Méndez-Lago, María; de Pablos, Beatriz; Demakova, Olga V; Andreyeva, Evgeniya N; Boldyreva, Lidiya V; Marra, Marco; Carvalho, A Bernardo; Dimitri, Patrizio; Villasante, Alfredo; Zhimulev, Igor F; Rubin, Gerald M; Karpen, Gary H; Celniker, Susan E

2015-03-01

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. © 2015 Hoskins et al.; Published by Cold Spring Harbor Laboratory Press.

A wing expressed sequence tag resource for Bicyclus anynana butterflies, an evo-devo model

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Gruber Jonathan D

2006-05-01

Full Text Available Abstract Background Butterfly wing color patterns are a key model for integrating evolutionary developmental biology and the study of adaptive morphological evolution. Yet, despite the biological, economical and educational value of butterflies they are still relatively under-represented in terms of available genomic resources. Here, we describe an Expression Sequence Tag (EST project for Bicyclus anynana that has identified the largest available collection to date of expressed genes for any butterfly. Results By targeting cDNAs from developing wings at the stages when pattern is specified, we biased gene discovery towards genes potentially involved in pattern formation. Assembly of 9,903 ESTs from a subtracted library allowed us to identify 4,251 genes of which 2,461 were annotated based on BLAST analyses against relevant gene collections. Gene prediction software identified 2,202 peptides, of which 215 longer than 100 amino acids had no homology to any known proteins and, thus, potentially represent novel or highly diverged butterfly genes. We combined gene and Single Nucleotide Polymorphism (SNP identification by constructing cDNA libraries from pools of outbred individuals, and by sequencing clones from the 3' end to maximize alignment depth. Alignments of multi-member contigs allowed us to identify over 14,000 putative SNPs, with 316 genes having at least one high confidence double-hit SNP. We furthermore identified 320 microsatellites in transcribed genes that can potentially be used as genetic markers. Conclusion Our project was designed to combine gene and sequence polymorphism discovery and has generated the largest gene collection available for any butterfly and many potential markers in expressed genes. These resources will be invaluable for exploring the potential of B. anynana in particular, and butterflies in general, as models in ecological, evolutionary, and developmental genetics.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data

Science.gov (United States)

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

Science.gov (United States)

Miller, Mark P; Knaus, Brian J; Mullins, Thomas D; Haig, Susan M

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Does the sequence of data collection influence participants' responses to closed and open-ended questions? A methodological study.

Science.gov (United States)

Covell, Christine L; Sidani, Souraya; Ritchie, Judith A

2012-06-01

The sequence used for collecting quantitative and qualitative data in concurrent mixed-methods research may influence participants' responses. Empirical evidence is needed to determine if the order of data collection in concurrent mixed methods research biases participants' responses to closed and open-ended questions. To examine the influence of the quantitative-qualitative sequence on responses to closed and open-ended questions when assessing the same variables or aspects of a phenomenon simultaneously within the same study phase. A descriptive cross-sectional, concurrent mixed-methods design was used to collect quantitative (survey) and qualitative (interview) data. The setting was a large multi-site health care centre in Canada. A convenience sample of 50 registered nurses was selected and participated in the study. Participants were randomly assigned to one of two sequences for data collection, quantitative-qualitative or qualitative-quantitative. Independent t-tests were performed to compare the two groups' responses to the survey items. Directed content analysis was used to compare the participants' responses to the interview questions. The sequence of data collection did not greatly affect the participants' responses to the closed-ended questions (survey items) or the open-ended questions (interview questions). The sequencing of data collection, when using both survey and semi-structured interviews, may not bias participants' responses to closed or open-ended questions. Additional research is required to confirm these findings. Copyright © 2011 Elsevier Ltd. All rights reserved.

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements

Science.gov (United States)

We report the stable genetic transformation of the Queensland fruit fly Bactrocera tryoni using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP.A transformation frequency of 5–10% was obtained.Inheritance of the transgenes has remained stable over eight generations despite...

PET-Tool: a software suite for comprehensive processing and managing of Paired-End diTag (PET sequence data

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Wei Chia-Lin

2006-08-01

Full Text Available Abstract Background We recently developed the Paired End diTag (PET strategy for efficient characterization of mammalian transcriptomes and genomes. The paired end nature of short PET sequences derived from long DNA fragments raised a new set of bioinformatics challenges, including how to extract PETs from raw sequence reads, and correctly yet efficiently map PETs to reference genome sequences. To accommodate and streamline data analysis of the large volume PET sequences generated from each PET experiment, an automated PET data process pipeline is desirable. Results We designed an integrated computation program package, PET-Tool, to automatically process PET sequences and map them to the genome sequences. The Tool was implemented as a web-based application composed of four modules: the Extractor module for PET extraction; the Examiner module for analytic evaluation of PET sequence quality; the Mapper module for locating PET sequences in the genome sequences; and the ProjectManager module for data organization. The performance of PET-Tool was evaluated through the analyses of 2.7 million PET sequences. It was demonstrated that PET-Tool is accurate and efficient in extracting PET sequences and removing artifacts from large volume dataset. Using optimized mapping criteria, over 70% of quality PET sequences were mapped specifically to the genome sequences. With a 2.4 GHz LINUX machine, it takes approximately six hours to process one million PETs from extraction to mapping. Conclusion The speed, accuracy, and comprehensiveness have proved that PET-Tool is an important and useful component in PET experiments, and can be extended to accommodate other related analyses of paired-end sequences. The Tool also provides user-friendly functions for data quality check and system for multi-layer data management.

Low-pass shotgun sequencing of the barley genome facilitates rapid identification of genes, conserved non-coding sequences and novel repeats

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Graner Andreas

2008-10-01

Full Text Available Abstract Background Barley has one of the largest and most complex genomes of all economically important food crops. The rise of new short read sequencing technologies such as Illumina/Solexa permits such large genomes to be effectively sampled at relatively low cost. Based on the corresponding sequence reads a Mathematically Defined Repeat (MDR index can be generated to map repetitive regions in genomic sequences. Results We have generated 574 Mbp of Illumina/Solexa sequences from barley total genomic DNA, representing about 10% of a genome equivalent. From these sequences we generated an MDR index which was then used to identify and mark repetitive regions in the barley genome. Comparison of the MDR plots with expert repeat annotation drawing on the information already available for known repetitive elements revealed a significant correspondence between the two methods. MDR-based annotation allowed for the identification of dozens of novel repeat sequences, though, which were not recognised by hand-annotation. The MDR data was also used to identify gene-containing regions by masking of repetitive sequences in eight de-novo sequenced bacterial artificial chromosome (BAC clones. For half of the identified candidate gene islands indeed gene sequences could be identified. MDR data were only of limited use, when mapped on genomic sequences from the closely related species Triticum monococcum as only a fraction of the repetitive sequences was recognised. Conclusion An MDR index for barley, which was obtained by whole-genome Illumina/Solexa sequencing, proved as efficient in repeat identification as manual expert annotation. Circumventing the labour-intensive step of producing a specific repeat library for expert annotation, an MDR index provides an elegant and efficient resource for the identification of repetitive and low-copy (i.e. potentially gene-containing sequences regions in uncharacterised genomic sequences. The restriction that a particular

Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder

NARCIS (Netherlands)

Yuen, Ryan K C; Merico, Daniele; Bookman, Matt; Howe, Jennifer L.; Thiruvahindrapuram, Bhooma; Patel, Rohan V.; Whitney, Joe; Deflaux, Nicole; Bingham, Jonathan; Wang, Zhuozhi; Pellecchia, Giovanna; Buchanan, Janet A.; Walker, Susan; Marshall, Christian R.; Uddin, Mohammed; Zarrei, Mehdi; Deneault, Eric; D'Abate, Lia; Chan, Ada J S; Koyanagi, Stephanie; Paton, Tara; Pereira, Sergio L.; Hoang, Ny; Engchuan, Worrawat; Higginbotham, Edward J.; Ho, Karen; Lamoureux, Sylvia; Li, Weili; MacDonald, Jeffrey R.; Nalpathamkalam, Thomas; Sung, Wilson W L; Tsoi, Fiona J.; Wei, John; Xu, Lizhen; Tasse, Anne Marie; Kirby, Emily; Van Etten, William; Twigger, Simon; Roberts, Wendy; Drmic, Irene; Jilderda, Sanne; Modi, Bonnie Mackinnon; Kellam, Barbara; Szego, Michael; Cytrynbaum, Cheryl; Weksberg, Rosanna; Zwaigenbaum, Lonnie; Woodbury-Smith, Marc; Brian, Jessica; Senman, Lili; Iaboni, Alana; Doyle-Thomas, Krissy; Thompson, Ann; Chrysler, Christina; Leef, Jonathan; Savion-Lemieux, Tal; Smith, Isabel M.; Liu, Xudong; Nicolson, Rob; Seifer, Vicki; Fedele, Angie; Cook, Edwin H.; Dager, Stephen; Estes, Annette; Gallagher, Louise; Malow, Beth A.; Parr, Jeremy R.; Spence, Sarah J.; Vorstman, Jacob; Frey, Brendan J.; Robinson, James T.; Strug, Lisa J.; Fernandez, Bridget A.; Elsabbagh, Mayada; Carter, Melissa T.; Hallmayer, Joachim; Knoppers, Bartha M.; Anagnostou, Evdokia; Szatmari, Peter; Ring, Robert H.; Glazer, David; Pletcher, Mathew T.; Scherer, Stephen W.

2017-01-01

We are performing whole-genome sequencing of families with autism spectrum disorder (ASD) to build a resource (MSSNG) for subcategorizing the phenotypes and underlying genetic factors involved. Here we report sequencing of 5,205 samples from families with ASD, accompanied by clinical information,

Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

Science.gov (United States)

Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2014-01-01

Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

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

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O'Brien Kimberly

2008-06-01

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

Comparison of 'time to detection' values between BacT/ALERT VIRTUO and BacT/ALERT 3D instruments for clinical blood culture samples.

Science.gov (United States)

Congestrì, Francesco; Pedna, Maria Federica; Fantini, Michela; Samuelli, Michela; Schiavone, Pasqua; Torri, Arianna; Bertini, Stefania; Sambri, Vittorio

2017-09-01

The early detection of bacteraemia and fungemia is of paramount importance to guide antimicrobial therapy in septic patients. In this study the 'time to detection' (TTD) value for the new blood culture system BacT/ALERT VIRTUO (VIRTUO) was evaluated in 1462 positive clinical bottles and compared with the TTD for 1601 positive clinical bottles incubated in the BacT/ALERT 3D system (BTA-3D). The most representative microorganisms isolated from bottles incubated in both blood culture systems were divided into eight categories (in order of frequency): coagulase-negative staphylococci (CoNS), Escherichia coli, Enterobacteriaceae (other than E. coli), Staphylococcus aureus, Enterococcus spp, viridans group streptococci, Pseudomonas aeruginosa, and Candida spp. The comparison of TTD values for the two blood culture systems strongly indicated that growth of the first five groups listed above was detected earlier with VIRTUO than with BTA-3D (p culture system can reduce the TTD for more than 75% of isolated microorganisms. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo: genome assembly and analysis.

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Rami A Dalloul

2010-09-01

Full Text Available A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo. Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers

DEFF Research Database (Denmark)

Varshney, Rajeev K.; Chen, Wenbin; Li, Yupeng

2012-01-01

Pigeonpea is an important legume food crop grown primarily by smallholder farmers in many semi-arid tropical regions of the world. We used the Illumina next-generation sequencing platform to generate 237.2 Gb of sequence, which along with Sanger-based bacterial artificial chromosome end sequences...

The need for blood alcohol concentration (BAC) legislation in Nigeria

African Journals Online (AJOL)

The pharmacology, clinical and sports implications of indulgence in alcohol and the debate on its legal status are highlighted in this article. The information presented could offer both clinical and safety benefits to psychomotor tasks executors and road safety professionals. Keywords: Blood alcohol concentration (BAC), ...

A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

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Tian Huai Shen

Full Text Available Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

[Interest of a new instrument to assess cognition in schizophrenia: The Brief Assessment of Cognition in Schizophrenia (BACS)].

Science.gov (United States)

Bralet, M C; Navarre, M; Eskenazi, A M; Lucas-Ross, M; Falissard, B

2008-12-01

administered the standard battery of cognitive tests including: the Rey Auditory-Verbal learning test, the Wechsler Adult Intelligence Scale, third edition, subtests (Digit inverse sequencing, Digit Symbol-Coding), the Trail-Making A, Verbal Fluency (Controlled Oral Word Association Test, Category Instances), and the Wisconsin Card Sort Test (128 card version). The factor structure of the French BACS A Version was determined by performing a principal components analysis with oblique rotation. The relationship between the French BACS sub-scores and the standard battery sub-scores was determined by calculating Pearson's correlations among the sub-scores, with a level of significance of alphacognitive performance, which accounted for the greatest amount of variance. The BACS thus permits an assessment of overall cognitive function as a global score, more than some individual specific cognitive domains. The sub-scores from the French BACS A Version were strongly correlated with the standard battery corresponding sub-scores. We observed significant correlations for all the subtests evaluating: verbal memory (Pearson=0.83; pinformation processing (Pearson=0.69; pcognitive impairment in patients with schizophrenia as a standard battery of tests that required over 2h to complete. Moreover, these results demonstrate that the BACS, the global score of which may be the most powerful indicator of functional outcome, can also be a good neuropsychological instrument for assessing global cognition in patients with schizophrenia.

Identification of an extensive gene cluster among a family of PPOs in Trifolium pratense L. (red clover using a large insert BAC library

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Thomas Ann

2009-07-01

Full Text Available Abstract Background Polyphenol oxidase (PPO activity in plants is a trait with potential economic, agricultural and environmental impact. In relation to the food industry, PPO-induced browning causes unacceptable discolouration in fruit and vegetables: from an agriculture perspective, PPO can protect plants against pathogens and environmental stress, improve ruminant growth by increasing nitrogen absorption and decreasing nitrogen loss to the environment through the animal's urine. The high PPO legume, red clover, has a significant economic and environmental role in sustaining low-input organic and conventional farms. Molecular markers for a range of important agricultural traits are being developed for red clover and improved knowledge of PPO genes and their structure will facilitate molecular breeding. Results A bacterial artificial chromosome (BAC library comprising 26,016 BAC clones with an average 135 Kb insert size, was constructed from Trifolium pratense L. (red clover, a diploid legume with a haploid genome size of 440–637 Mb. Library coverage of 6–8 genome equivalents ensured good representation of genes: the library was screened for polyphenol oxidase (PPO genes. Two single copy PPO genes, PPO4 and PPO5, were identified to add to a family of three, previously reported, paralogous genes (PPO1–PPO3. Multiple PPO1 copies were identified and characterised revealing a subfamily comprising three variants PPO1/2, PPO1/4 and PPO1/5. Six PPO genes clustered within the genome: four separate BAC clones could be assembled onto a predicted 190–510 Kb single BAC contig. Conclusion A PPO gene family in red clover resides as a cluster of at least 6 genes. Three of these genes have high homology, suggesting a more recent evolutionary event. This PPO cluster covers a longer region of the genome than clusters detected in rice or previously reported in tomato. Full-length coding sequences from PPO4, PPO5, PPO1/5 and PPO1/4 will facilitate

Characterization of repetitive DNA landscape in wheat homeologous group 4 chromosomes

Czech Academy of Sciences Publication Activity Database

Garbus, I.; Romero, J.R.; Valárik, Miroslav; Vanžurová, Hana; Karafiátová, Miroslava; Doležel, Jaroslav; Tranquilli, G.; Helguera, M.; Echenique, V.

2015-01-01

Roč. 16, MAY 12 (2015) ISSN 1471-2164 Institutional support: RVO:61389030 Keywords : VIRTUAL GENE ORDER * BAC END SEQUENCES * NEXT-GENERATION Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.867, year: 2015

Generation of BAC transgenic epithelial organoids.

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Gerald Schwank

Full Text Available Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome technology.

Characterization of Extracellular Penicilin G Acylase Produced by A New Local Strain of Bacillus subtilis BAC4

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SUPARTONO

2008-06-01

Full Text Available Penicillin G acylase (PGA which catalyses penicillin G hydrolysis reaction is a key enzyme for the industrial production of penicilin G derivatives used in therapeutics. A new local strain of Bacillus subtilis BAC4 was found capable of producing extracellular PGA. However, characteristics of this extracellular PGA are not known. The goal of this research was to characterize the extracellular PGA produced by B. subtilis BAC4. Enzyme production was carried out by batch fermentation, followed by enzyme purification and characterization of the PGA. The PGA activity was determined by the Kornfeld method, with optimal activity for hydrolysing penicillin G observed at 43 °C and pH 8.5. The activation energy of penicillin G hydrolysis by the PGA of B. subtilis BAC4 was determined as 4.9 kcal.mol−1 and Vmax and Km values were found to be 0.7 μmole.min−1.mg−1 and 3.5 mM respectively. PGA catalytic activity was competitively inhibited by phenylacetic acid with an inhibition constant, Ki(PAA, of 347.2 mM. It was concluded that the extracellular PGA of B. subtilis BAC4 can hydrolyse penicillin G efficiently.

Characterization of Extracellular Penicilin G Acylase Produced by A New Local Strain of Bacillus subtilis BAC4

Directory of Open Access Journals (Sweden)

SUPARTONO

2008-06-01

Full Text Available Penicillin G acylase (PGA which catalyses penicillin G hydrolysis reaction is a key enzyme for the industrial production of penicilin G derivatives used in therapeutics. A new local strain of Bacillus subtilis BAC4 was found capable of producing extracellular PGA. However, characteristics of this extracellular PGA are not known. The goal of this research was to characterize the extracellular PGA produced by B. subtilis BAC4. Enzyme production was carried out by batch fermentation, followed by enzyme purification and characterization of the PGA. The PGA activity was determined by the Kornfeld method, with optimal activity for hydrolysing penicillin G observed at 43 oC and pH 8.5. The activation energy of penicillin G hydrolysis by the PGA of B. subtilis BAC4 was determined as 4.9 kcal.mol-1 and Vmax and Km values were found to be 0.7 µmole.min-1.mg-1 and 3.5 mM respectively. PGA catalytic activity was competitively inhibited by phenylacetic acid with an inhibition constant, Ki(PAA, of 347.2 mM. It was concluded that the extracellular PGA of B. subtilis BAC4 can hydrolyse penicillin G efficiently.

Genomic analysis of a 1 Mb region near the telomere of Hessian fly chromosome X2 and avirulence gene vH13

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Chen Ming-Shun

2006-01-01

Full Text Available Abstract Background To have an insight into the Mayetiola destructor (Hessian fly genome, we performed an in silico comparative genomic analysis utilizing genetic mapping, genomic sequence and EST sequence data along with data available from public databases. Results Chromosome walking and FISH were utilized to identify a contig of 50 BAC clones near the telomere of the short arm of Hessian fly chromosome X2 and near the avirulence gene vH13. These clones enabled us to correlate physical and genetic distance in this region of the Hessian fly genome. Sequence data from these BAC ends encompassing a 760 kb region, and a fully sequenced and assembled 42.6 kb BAC clone, was utilized to perform a comparative genomic study. In silico gene prediction combined with BLAST analyses was used to determine putative orthology to the sequenced dipteran genomes of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, and to infer evolutionary relationships. Conclusion This initial effort enables us to advance our understanding of the structure, composition and evolution of the genome of this important agricultural pest and is an invaluable tool for a whole genome sequencing effort.

TE-Locate: A Tool to Locate and Group Transposable Element Occurrences Using Paired-End Next-Generation Sequencing Data

OpenAIRE

Platzer, Alexander; Nizhynska, Viktoria; Long, Quan

2012-01-01

Transposable elements (TEs) are common mobile DNA elements present in nearly all genomes. Since the movement of TEs within a genome can sometimes have phenotypic consequences, an accurate report of TE actions is desirable. To this end, we developed TE-Locate, a computational tool that uses paired-end reads to identify the novel locations of known TEs. TE-Locate can utilize either a database of TE sequences, or annotated TEs within the reference sequence of interest. This makes TE-Locate usefu...

Caring Decisions: The Development of a Written Resource for Parents Facing End-of-Life Decisions

OpenAIRE

Xafis, Vicki; Gillam, Lynn; Hynson, Jenny; Sullivan, Jane; Cossich, Mary; Wilkinson, Dominic

2015-01-01

Background: Written resources in adult intensive care have been shown to benefit families facing end of life (EoL) decisions. There are few resources for parents making EoL decisions for their child and no existing resources addressing ethical issues. TheCaring Decisionshandbook and website were developed to fill these gaps. Aim: We discuss the development of the resources, modification after reviewer feedback and findings from initial pilot implementation. Design: A targeted...

TE-Locate: A Tool to Locate and Group Transposable Element Occurrences Using Paired-End Next-Generation Sequencing Data.

Science.gov (United States)

Platzer, Alexander; Nizhynska, Viktoria; Long, Quan

2012-09-12

Transposable elements (TEs) are common mobile DNA elements present in nearly all genomes. Since the movement of TEs within a genome can sometimes have phenotypic consequences, an accurate report of TE actions is desirable. To this end, we developed TE-Locate, a computational tool that uses paired-end reads to identify the novel locations of known TEs. TE-Locate can utilize either a database of TE sequences, or annotated TEs within the reference sequence of interest. This makes TE-Locate useful in the search for any mobile sequence, including retrotransposed gene copies. One major concern is to act on the correct hierarchy level, thereby avoiding an incorrect calling of a single insertion as multiple events of TEs with high sequence similarity. We used the (super)family level, but TE-Locate can also use any other level, right down to the individual transposable element. As an example of analysis with TE-Locate, we used the Swedish population in the 1,001 Arabidopsis genomes project, and presented the biological insights gained from the novel TEs, inducing the association between different TE superfamilies. The program is freely available, and the URL is provided in the end of the paper.

TE-Locate: A Tool to Locate and Group Transposable Element Occurrences Using Paired-End Next-Generation Sequencing Data

Directory of Open Access Journals (Sweden)

Quan Long

2012-09-01

Full Text Available Transposable elements (TEs are common mobile DNA elements present in nearly all genomes. Since the movement of TEs within a genome can sometimes have phenotypic consequences, an accurate report of TE actions is desirable. To this end, we developed TE-Locate, a computational tool that uses paired-end reads to identify the novel locations of known TEs. TE-Locate can utilize either a database of TE sequences, or annotated TEs within the reference sequence of interest. This makes TE-Locate useful in the search for any mobile sequence, including retrotransposed gene copies. One major concern is to act on the correct hierarchy level, thereby avoiding an incorrect calling of a single insertion as multiple events of TEs with high sequence similarity. We used the (superfamily level, but TE-Locate can also use any other level, right down to the individual transposable element. As an example of analysis with TE-Locate, we used the Swedish population in the 1,001 Arabidopsis genomes project, and presented the biological insights gained from the novel TEs, inducing the association between different TE superfamilies. The program is freely available, and the URL is provided in the end of the paper.

A novel resource for genomics of Triticeae: BAC library specific for the short arm of rye (Secale cereale L.) chromosome 1R (1RS)

Czech Academy of Sciences Publication Activity Database

Šimková, Hana; Šafář, Jan; Suchánková, Pavla; Kovářová, Pavlína; Bartoš, Jan; Kubaláková, Marie; Janda, Jaroslav; Čihalíková, Jarmila; Mago, R.; Lelley, T.; Doležel, Jaroslav

2008-01-01

Roč. 9, č. 237 (2008), s. 101-109 ISSN 1471-2164 R&D Projects: GA ČR GA521/04/0607; GA ČR GP521/05/P257; GA ČR GD521/05/H013; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : flow cytometry * flow-sorted chromosomes * BAC library Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.926, year: 2008

SIFTS: Structure Integration with Function, Taxonomy and Sequences resource

Science.gov (United States)

Velankar, Sameer; Dana, José M.; Jacobsen, Julius; van Ginkel, Glen; Gane, Paul J.; Luo, Jie; Oldfield, Thomas J.; O’Donovan, Claire; Martin, Maria-Jesus; Kleywegt, Gerard J.

2013-01-01

The Structure Integration with Function, Taxonomy and Sequences resource (SIFTS; http://pdbe.org/sifts) is a close collaboration between the Protein Data Bank in Europe (PDBe) and UniProt. The two teams have developed a semi-automated process for maintaining up-to-date cross-reference information to UniProt entries, for all protein chains in the PDB entries present in the UniProt database. This process is carried out for every weekly PDB release and the information is stored in the SIFTS database. The SIFTS process includes cross-references to other biological resources such as Pfam, SCOP, CATH, GO, InterPro and the NCBI taxonomy database. The information is exported in XML format, one file for each PDB entry, and is made available by FTP. Many bioinformatics resources use SIFTS data to obtain cross-references between the PDB and other biological databases so as to provide their users with up-to-date information. PMID:23203869

Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

Directory of Open Access Journals (Sweden)

Changqing Liu

2014-03-01

Full Text Available Bacterial artificial chromosome (BAC libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12, consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

KAIKObase: An integrated silkworm genome database and data mining tool

Directory of Open Access Journals (Sweden)

Nagaraju Javaregowda

2009-10-01

Full Text Available Abstract Background The silkworm, Bombyx mori, is one of the most economically important insects in many developing countries owing to its large-scale cultivation for silk production. With the development of genomic and biotechnological tools, B. mori has also become an important bioreactor for production of various recombinant proteins of biomedical interest. In 2004, two genome sequencing projects for B. mori were reported independently by Chinese and Japanese teams; however, the datasets were insufficient for building long genomic scaffolds which are essential for unambiguous annotation of the genome. Now, both the datasets have been merged and assembled through a joint collaboration between the two groups. Description Integration of the two data sets of silkworm whole-genome-shotgun sequencing by the Japanese and Chinese groups together with newly obtained fosmid- and BAC-end sequences produced the best continuity (~3.7 Mb in N50 scaffold size among the sequenced insect genomes and provided a high degree of nucleotide coverage (88% of all 28 chromosomes. In addition, a physical map of BAC contigs constructed by fingerprinting BAC clones and a SNP linkage map constructed using BAC-end sequences were available. In parallel, proteomic data from two-dimensional polyacrylamide gel electrophoresis in various tissues and developmental stages were compiled into a silkworm proteome database. Finally, a Bombyx trap database was constructed for documenting insertion positions and expression data of transposon insertion lines. Conclusion For efficient usage of genome information for functional studies, genomic sequences, physical and genetic map information and EST data were compiled into KAIKObase, an integrated silkworm genome database which consists of 4 map viewers, a gene viewer, and sequence, keyword and position search systems to display results and data at the level of nucleotide sequence, gene, scaffold and chromosome. Integration of the

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria Bac. stearothermophilus exposed to. gamma. -, UV-radiation or methylnitrosourea

Energy Technology Data Exchange (ETDEWEB)

Fomenko, L A; Kuznetsovea, E A; Gaziev, A I

1984-07-01

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to ..gamma..-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S/sub 1/-nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria.

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria Bac. stearothermophilus exposed to γ-, UV-radiation or methylnitrosourea

International Nuclear Information System (INIS)

Fomenko, L.A.; Kuznetsovea, E.A.; Gaziev, A.I.

1984-01-01

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to γ-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S 1 -nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria. (orig.)

3' end labelling of RNA with /sup 32/P suitable for rapid gel sequencing

Energy Technology Data Exchange (ETDEWEB)

Winter, G; Brownlee, G G [Medical Research Council, Cambridge (UK)

1978-09-01

A new general method of labelling the 2', 3'-diol end of RNA with /sup 32/P has been devised suitable for gel sequencing. Poly(A) polymerase (E.coli) is incubated with the RNA and limiting amounts of ..cap alpha..-/sup 32/P-ATP. The mono-addition product is then cleaved with periodate and ..beta..-eliminated with aniline, leaving the RNA terminally labelled with 3'/sup 32/P-phosphate. When applied to a model compound, tRNAsup(Phe) from E. coli, over 28 residues could be read from the 3' end.

DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

International Nuclear Information System (INIS)

Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

2003-01-01

Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

Aerobic biodegradation of a sulfonated phenylazonaphthol dye by a bacterial community immobilized in a multistage packed-bed BAC reactor.

Science.gov (United States)

Ruiz-Arias, Alfredo; Juárez-Ramírez, Cleotilde; de los Cobos-Vasconcelos, Daniel; Ruiz-Ordaz, Nora; Salmerón-Alcocer, Angélica; Ahuatzi-Chacón, Deifilia; Galíndez-Mayer, Juvencio

2010-11-01

A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (eta ( COD )) and 4-ABS (eta ( ABS )) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a-c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.

State Blood Alcohol Concentration (BAC) Testing and Reporting for Drivers Involved in Fatal Crashes : Current Practices, Results, and Strategies, 1997-2009

Science.gov (United States)

2012-08-01

This report documents current State blood alcohol concentration (BAC) testing and reporting practices and results for drivers involved in fatal crashes. It summarizes known BAC results by State for the years 1997 to 2009 for both fatally injured and ...

Genomic resources for water yam (Dioscorea alata L.): analyses of EST-Sequences, De Novo sequencing and GBS libraries

Science.gov (United States)

The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources such as SSRs, SNPs and InDels in several model and non-model plant species. Yam (Dioscorea spp.) i...

Advances in genetics and molecular breeding of three legume crops ...

Indian Academy of Sciences (India)

2012-10-15

Oct 15, 2012 ... 3. Genomic resources for SAT legumes. In the past, for genetic diversity analysis, a range of ... DNA libraries, (b) sequencing and mining the BAC (bacterial ..... spiration efficiency, biomass, specific leaf area, pod weight,.

Electricity End Uses, Energy Efficiency, and Distributed Energy Resources Baseline

Energy Technology Data Exchange (ETDEWEB)

Schwartz, Lisa [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Wei, Max [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Morrow, William [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Deason, Jeff [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Schiller, Steven R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Leventis, Greg [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Smith, Sarah [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Leow, Woei Ling [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Levin, Todd [Argonne National Lab. (ANL), Argonne, IL (United States); Plotkin, Steven [Argonne National Lab. (ANL), Argonne, IL (United States); Zhou, Yan [Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States)

2017-01-01

This report was developed by a team of analysts at Lawrence Berkeley National Laboratory, with Argonne National Laboratory contributing the transportation section, and is a DOE EPSA product and part of a series of “baseline” reports intended to inform the second installment of the Quadrennial Energy Review (QER 1.2). QER 1.2 provides a comprehensive review of the nation’s electricity system and cover the current state and key trends related to the electricity system, including generation, transmission, distribution, grid operations and planning, and end use. The baseline reports provide an overview of elements of the electricity system. This report focuses on end uses, electricity consumption, electric energy efficiency, distributed energy resources (DERs) (such as demand response, distributed generation, and distributed storage), and evaluation, measurement, and verification (EM&V) methods for energy efficiency and DERs.

Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

Science.gov (United States)

Evans, Teri; Johnson, Andrew D; Loose, Matthew

2018-01-12

Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

Resource acquisition, distribution and end-use efficiencies and the growth of industrial society

Science.gov (United States)

Jarvis, A. J.; Jarvis, S. J.; Hewitt, C. N.

2015-10-01

A key feature of the growth of industrial society is the acquisition of increasing quantities of resources from the environment and their distribution for end-use. With respect to energy, the growth of industrial society appears to have been near-exponential for the last 160 years. We provide evidence that indicates that the global distribution of resources that underpins this growth may be facilitated by the continual development and expansion of near-optimal directed networks (roads, railways, flight paths, pipelines, cables etc.). However, despite this continual striving for optimisation, the distribution efficiencies of these networks must decline over time as they expand due to path lengths becoming longer and more tortuous. Therefore, to maintain long-term exponential growth the physical limits placed on the distribution networks appear to be counteracted by innovations deployed elsewhere in the system, namely at the points of acquisition and end-use of resources. We postulate that the maintenance of the growth of industrial society, as measured by global energy use, at the observed rate of ~ 2.4 % yr-1 stems from an implicit desire to optimise patterns of energy use over human working lifetimes.

Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting.

Science.gov (United States)

Achenbach, Ute C; Tang, Xiaomin; Ballvora, Agim; de Jong, Hans; Gebhardt, Christiane

2010-02-01

Potato chromosome 5 harbours numerous genes for important qualitative and quantitative traits, such as resistance to the root cyst nematode Globodera pallida and the late blight fungus, Phytophthora infestans. The genes make up part of a "hot spot" for resistances to various pathogens covering a genetic map length of 3 cM between markers GP21 and GP179. We established the physical size and position of this region on chromosome 5 in potato and tomato using fluorescence in situ hybridization (FISH) on pachytene chromosomes. Five potato bacterial artificial chromosome (BAC) clones with the genetically anchored markers GP21, R1-contig (proximal end), CosA, GP179, and StPto were selected, labeled with different fluorophores, and hybridized in a five-colour FISH experiment. Our results showed the location of the BAC clones in the middle of the long arm of chromosome 5 in both potato and tomato. Based on chromosome measurements, we estimate the physical size of the GP21-GP179 interval at 0.85 Mb and 1.2 Mb in potato and tomato, respectively. The GP21-GP179 interval is part of a genome segment known to have inverted map positions between potato and tomato.

Cyberinfrastructure for End-to-End Environmental Explorations

Science.gov (United States)

Merwade, V.; Kumar, S.; Song, C.; Zhao, L.; Govindaraju, R.; Niyogi, D.

2007-12-01

The design and implementation of a cyberinfrastructure for End-to-End Environmental Exploration (C4E4) is presented. The C4E4 framework addresses the need for an integrated data/computation platform for studying broad environmental impacts by combining heterogeneous data resources with state-of-the-art modeling and visualization tools. With Purdue being a TeraGrid Resource Provider, C4E4 builds on top of the Purdue TeraGrid data management system and Grid resources, and integrates them through a service-oriented workflow system. It allows researchers to construct environmental workflows for data discovery, access, transformation, modeling, and visualization. Using the C4E4 framework, we have implemented an end-to-end SWAT simulation and analysis workflow that connects our TeraGrid data and computation resources. It enables researchers to conduct comprehensive studies on the impact of land management practices in the St. Joseph watershed using data from various sources in hydrologic, atmospheric, agricultural, and other related disciplines.

Defining “Binge” Drinking as Five Drinks per Occasion or Drinking to a 0.08% BAC: Which is More Sensitive to Risk?

Science.gov (United States)

Fillmore, Mark T.; Jude, Rebecca

2011-01-01

Heavy episodic or “binge” drinking is commonly defined as drinking 4–5 drinks per occasion (5/4 definition) or drinking that results in a blood alcohol concentration (BAC) of 0.08%. The present study compared the validity of each binge definition as an indicator of at-risk, problem drinking. 251 college students were classified as non-binge drinkers or as binge drinkers based on the 5/4 definition or the 0.08% BAC definition. The two definitions of binge drinking were examined in terms of their sensitivity and specificity as indicators of alcohol-related problems as determined by scores on the Alcohol Use Disorders Identification Test (AUDIT). Over half the sample (56%) were at-risk drinkers according to the AUDIT. The 0.08% definition detected only one-half of these individuals. Gender differences were also evident. Female binge drinkers actually achieved significantly higher estimated BACs per episode than their male binge drinking counterparts. The findings suggest that drinking to a sub-threshold BAC (i.e., risk independent of the BAC achieved during drinking episodes. The findings also highlight the importance of considering frequency of consumption in determining risky drinking versus relying solely on quantity measures. PMID:21838847

Functional features of a single chromosome arm in wheat (1AL) determined from its structure

Czech Academy of Sciences Publication Activity Database

Lucas, S. J.; Šimková, Hana; Šafář, Jan; Jurman, I.; Cattonaro, F.; Vautrin, S.; Bellec, A.; Berges, H.; Doležel, Jaroslav; Budak, H.

2012-01-01

Roč. 12, č. 1 (2012), s. 173-182 ISSN 1438-793X Institutional research plan: CEZ:AV0Z50380511 Keywords : Wheat * A genome * BAC end sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.292, year: 2012

'End of life' conversations, appreciation sequences, and the interaction order in cancer clinics.

Science.gov (United States)

Maynard, Douglas W; Cortez, Dagoberto; Campbell, Toby C

2016-01-01

To address the organization of conversations in oncology visits by taking an "interaction order" perspective and asking how these visits are intrinsically organized. Conversation analysis. Using audio recordings of talk in oncology visits involving patients with non-small cell lung cancer, we identify and analyze an "appreciation sequence" that is designed to elicit patients' understanding and positive assessment of treatments in terms of their prolongation of life. An "appreciation sequence," regularly initiated after the delivery of scan results and/or treatment recommendations, simultaneously reminds patients of their mortality while suggesting that the treatment received has prolonged their lives, and in some cases significantly beyond the median time of survival. We explore the functions of the appreciation sequence for cancer care and set the stage for considering where and when physicians have choices about the order and direction the talk can take and how to allocate time for end of life and quality of life conversations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Enhanced biodegradation of petrochemical wastewater using ozonation and BAC advanced treatment system

Energy Technology Data Exchange (ETDEWEB)

Lin, Chi-Kang; Tsai, Tsung-Yueh; Liu, Jiunn-Ching; Chen, Mei-Chen [Energy and Resources Labs., ITRI, Hsinchu (Taiwan)

2001-07-01

The characteristics of degradation/conversion of bio-refractory and the growth of a biofilm are investigated in laboratory-scale pre-ozonation and lifted moving-bed biological activated carbon (BAC) advanced treatment processes treating phenol, benzoic acid, aminobenzoic acid and petrochemical industry wastewater which contains acrylonitrile butadiene styrene (ABS). The optimal reaction time and ozone dosage of pre-ozonation for bio-refractory conversion were determined to be 30 min and 100-200mg O{sub 3}/hr, respectively. After pre-ozonation of 30 min treatment, BOD{sub 5}/COD ratio of influent and effluent increased apparently from 20 to 35%, approximately. However, the change of pH in pre-ozonation was inconspicuous. The optimal flow rate of influent and air were controlled at 1.6 1/h and 120-l50nl/min in lifted moving-bed BAC advanced treatment reactor. A COD removal efficiency of 85-95% and 70-90% may be maintained by using an organic loading of 3.2-6.3kg COD/m{sup 3} day and 0.6-1.6 kg-COD/m{sup 3} day with an HRT of 6.0 h as secondary and advanced treatment system, respectively. The time required for the BAC bed to be regenerated by a thermal regeneration is prolonged 4-5 times more than that of GAC system. It can be estimated that the enhanced COD removal capability of the biofilm was not only due to the increase in the COD removal capability of acclimated bacteria, but also due to species succession of bacteria in bio-film ecosystem. (Author)

Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles

Directory of Open Access Journals (Sweden)

Boussaha Mekki

2006-05-01

Full Text Available Abstract Background The Fragile Histidine Triad gene (FHIT is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. Results The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. Conclusion A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis

Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT) tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles.

Science.gov (United States)

Uboldi, Cristina; Guidi, Elena; Roperto, Sante; Russo, Valeria; Roperto, Franco; Di Meo, Giulia Pia; Iannuzzi, Leopoldo; Floriot, Sandrine; Boussaha, Mekki; Eggen, André; Ferretti, Luca

2006-05-23

The Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

Science.gov (United States)

Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

2014-12-18

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Crystal structures and electronic properties of BaC2 isomers by theoretical study based on DFT

Institute of Scientific and Technical Information of China (English)

2008-01-01

Band structures and electronic properties of two BaC2 isomers were calculated by using density func-tional theory(DFT) properly.The ionic bond features are all typical between cation(Ba) and anion clusters(C2) in both structures of the isomers.However,a much stronger covalent bond exists in anion clusters which can be seen by inspecting the electron distribution contour that has a dull bell like shape between two carbon atoms.The shortest distance between Ba2+ and C22? and the bond length in anion clusters are different in these isomers of BaC2,which are 0.2945 nm and 0.1185 nm for the structure with the I4/mmm space group and 0.2744 and 0.1136 nm with the C2/c type,respectively.Band structures were clarified by combining the DOS to indicate the ionic bonding features more clearly.Population analysis provided further evidence on these ideas.Thermodynamical calculation results reveal that the transition temperature of these two polymorphs of BaC2 locates near 132 K,which is consistent with the recent experimental results.

Support Resources Demand Parameters - Aircraft. Revision A

Science.gov (United States)

1980-01-15

Maintenance Squadron A- ST Advance Medium STOL Transport APU Auxillary Power Unit ASSY Assembly ATC Air Training Command AVG Average BAC Boeing Aerospace...entire study that will result in an organized and prioritized body of decision criteria and parameters that may be used by logistics managers, supervisors...technicians, and other decision makers in the process of predicting resource demand rates for operational and new emerging aircraft weapon systems

A genetically anchored physical framework for Theobroma cacao cv. Matina 1-6.

Science.gov (United States)

Saski, Christopher A; Feltus, Frank A; Staton, Margaret E; Blackmon, Barbara P; Ficklin, Stephen P; Kuhn, David N; Schnell, Raymond J; Shapiro, Howard; Motamayor, Juan Carlos

2011-08-16

The fermented dried seeds of Theobroma cacao (cacao tree) are the main ingredient in chocolate. World cocoa production was estimated to be 3 million tons in 2010 with an annual estimated average growth rate of 2.2%. The cacao bean production industry is currently under threat from a rise in fungal diseases including black pod, frosty pod, and witches' broom. In order to address these issues, genome-sequencing efforts have been initiated recently to facilitate identification of genetic markers and genes that could be utilized to accelerate the release of robust T. cacao cultivars. However, problems inherent with assembly and resolution of distal regions of complex eukaryotic genomes, such as gaps, chimeric joins, and unresolvable repeat-induced compressions, have been unavoidably encountered with the sequencing strategies selected. Here, we describe the construction of a BAC-based integrated genetic-physical map of the T. cacao cultivar Matina 1-6 which is designed to augment and enhance these sequencing efforts. Three BAC libraries, each comprised of 10× coverage, were constructed and fingerprinted. 230 genetic markers from a high-resolution genetic recombination map and 96 Arabidopsis-derived conserved ortholog set (COS) II markers were anchored using pooled overgo hybridization. A dense tile path consisting of 29,383 BACs was selected and end-sequenced. The physical map consists of 154 contigs and 4,268 singletons. Forty-nine contigs are genetically anchored and ordered to chromosomes for a total span of 307.2 Mbp. The unanchored contigs (105) span 67.4 Mbp and therefore the estimated genome size of T. cacao is 374.6 Mbp. A comparative analysis with A. thaliana, V. vinifera, and P. trichocarpa suggests that comparisons of the genome assemblies of these distantly related species could provide insights into genome structure, evolutionary history, conservation of functional sites, and improvements in physical map assembly. A comparison between the two T. cacao

A genetically anchored physical framework for Theobroma cacao cv. Matina 1-6

Directory of Open Access Journals (Sweden)

Kuhn David N

2011-08-01

Full Text Available Abstract Background The fermented dried seeds of Theobroma cacao (cacao tree are the main ingredient in chocolate. World cocoa production was estimated to be 3 million tons in 2010 with an annual estimated average growth rate of 2.2%. The cacao bean production industry is currently under threat from a rise in fungal diseases including black pod, frosty pod, and witches' broom. In order to address these issues, genome-sequencing efforts have been initiated recently to facilitate identification of genetic markers and genes that could be utilized to accelerate the release of robust T. cacao cultivars. However, problems inherent with assembly and resolution of distal regions of complex eukaryotic genomes, such as gaps, chimeric joins, and unresolvable repeat-induced compressions, have been unavoidably encountered with the sequencing strategies selected. Results Here, we describe the construction of a BAC-based integrated genetic-physical map of the T. cacao cultivar Matina 1-6 which is designed to augment and enhance these sequencing efforts. Three BAC libraries, each comprised of 10× coverage, were constructed and fingerprinted. 230 genetic markers from a high-resolution genetic recombination map and 96 Arabidopsis-derived conserved ortholog set (COS II markers were anchored using pooled overgo hybridization. A dense tile path consisting of 29,383 BACs was selected and end-sequenced. The physical map consists of 154 contigs and 4,268 singletons. Forty-nine contigs are genetically anchored and ordered to chromosomes for a total span of 307.2 Mbp. The unanchored contigs (105 span 67.4 Mbp and therefore the estimated genome size of T. cacao is 374.6 Mbp. A comparative analysis with A. thaliana, V. vinifera, and P. trichocarpa suggests that comparisons of the genome assemblies of these distantly related species could provide insights into genome structure, evolutionary history, conservation of functional sites, and improvements in physical map

De novo assembly and characterization of the transcriptome of seagrass Zostera marina using Illumina paired-end sequencing.

Directory of Open Access Journals (Sweden)

Fanna Kong

Full Text Available BACKGROUND: The seagrass Zostera marina is a monocotyledonous angiosperm belonging to a polyphyletic group of plants that can live submerged in marine habitats. Zostera marina L. is one of the most common seagrasses and is considered a cornerstone of marine plant molecular ecology research and comparative studies. However, the mechanisms underlying its adaptation to the marine environment still remain poorly understood due to limited transcriptomic and genomic data. PRINCIPAL FINDINGS: Here we explored the transcriptome of Z. marina leaves under different environmental conditions using Illumina paired-end sequencing. Approximately 55 million sequencing reads were obtained, representing 58,457 transcripts that correspond to 24,216 unigenes. A total of 14,389 (59.41% unigenes were annotated by blast searches against the NCBI non-redundant protein database. 45.18% and 46.91% of the unigenes had significant similarity with proteins in the Swiss-Prot database and Pfam database, respectively. Among these, 13,897 unigenes were assigned to 57 Gene Ontology (GO terms and 4,745 unigenes were identified and mapped to 233 pathways via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG. We compared the orthologous gene family of the Z. marina transcriptome to Oryza sativa and Pyropia yezoensis and 11,667 orthologous gene families are specific to Z. marina. Furthermore, we identified the photoreceptors sensing red/far-red light and blue light. Also, we identified a large number of genes that are involved in ion transporters and channels including Na+ efflux, K+ uptake, Cl- channels, and H+ pumping. CONCLUSIONS: Our study contains an extensive sequencing and gene-annotation analysis of Z. marina. This information represents a genetic resource for the discovery of genes related to light sensing and salt tolerance in this species. Our transcriptome can be further utilized in future studies on molecular adaptation to

Sur la filabilité d'une laine teinte en bac-ouvert et d'une laine teinte à la continue

NARCIS (Netherlands)

Werker, W.; Mulder, D.; Stomph, J.; Schartman, A.F.

1966-01-01

Afin de constater, si la teinture selon le procédé à la continue ou selon le procédé en bac-ouvert influence les résultats dans Ia filature, on a teint deux lots homogènes à 18OO kg par lot en quatre parties: 450 kg, procédé à la continue, teints en rouge, 450 kg, procédé bac-ouvert, teints en

High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

Science.gov (United States)

Preston, Jessica L; Royall, Ariel E; Randel, Melissa A; Sikkink, Kristin L; Phillips, Patrick C; Johnson, Eric A

2016-06-14

Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing.

Science.gov (United States)

Li, Guoxi; Zhao, Yinli; Liu, Zhonghu; Gao, Chunsheng; Yan, Fengbin; Liu, Bianzhi; Feng, Jianxin

2015-06-01

Common carp (Cyprinus carpio) is one of the most important aquacultured species of the family Cyprinidae, and breeding this species for disease resistance is becoming more and more important. However, at the genome or transcriptome levels, study of the immunogenetics of disease resistance in the common carp is lacking. In this study, 60,316,906 and 75,200,328 paired-end clean reads were obtained from two cDNA libraries of the common carp spleen by Illumina paired-end sequencing technology. Totally, 130,293 unique transcript fragments (unigenes) were assembled, with an average length of 1400.57 bp. Approximately 105,612 (81.06%) unigenes could be annotated according to their homology with matches in the Nr, Nt, Swiss-Prot, COG, GO, or KEGG databases, and they were found to represent 46,747 non-redundant genes. Comparative analysis showed that 59.82% of the unigenes have significant similarity to zebrafish Refseq proteins. Gene expression comparison revealed that 10,432 and 6889 annotated unigenes were, respectively, up- and down-regulated with at least twofold changes between two developmental stages of the common carp spleen. Gene ontology and KEGG analysis were performed to classify all unigenes into functional categories for understanding gene functions and regulation pathways. In addition, 46,847 simple sequence repeats (SSRs) were detected from 35,618 unigenes, and a large number of single nucleotide polymorphism (SNP) and insertion/deletion (INDEL) sites were identified in the spleen transcriptome of common carp. This study has characterized the spleen transcriptome of the common carp for the first time, providing a valuable resource for a better understanding of the common carp immune system and defense mechanisms. This knowledge will also facilitate future functional studies on common carp immunogenetics that may eventually be applied in breeding programs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genomic sequence around butterfly wing development genes: annotation and comparative analysis.

Directory of Open Access Journals (Sweden)

Inês C Conceição

Full Text Available BACKGROUND: Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. METHODOLOGY/PRINCIPAL FINDINGS: We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes. CONCLUSIONS: The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1 the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2 the high

Expression Analysis of CB2-GFP BAC Transgenic Mice.

Science.gov (United States)

Schmöle, Anne-Caroline; Lundt, Ramona; Gennequin, Benjamin; Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian

2015-01-01

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca)

OpenAIRE

Liu, Wei; Zhao, Yonghui; Liu, Zhaoliang; Zhang, Ying; Lian, Zhengxing; Li, Ning

2006-01-01

Abstract Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC) library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents o...

A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

Science.gov (United States)

Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

2014-01-01

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

BacHBerry:: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

DEFF Research Database (Denmark)

Dudnik, Alexey; Almeida, A. Filipa; Andrade, Ricardo

2017-01-01

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economi...

Nucleotide sequence of soybean chloroplast DNA regions which contain the psb A and trn H genes and cover the ends of the large single copy region and one end of the inverted repeats.

Science.gov (United States)

Spielmann, A; Stutz, E

1983-10-25

The soybean chloroplast psb A gene (photosystem II thylakoid membrane protein of Mr 32 000, lysine-free) and the trn H gene (tRNAHisGUG), which both map in the large single copy region adjacent to one of the inverted repeat structures (IR1), have been sequenced including flanking regions. The psb A gene shows in its structural part 92% sequence homology with the corresponding genes of spinach and N. debneyi and contains also an open reading frame for 353 aminoacids. The aminoacid sequence of a potential primary translation product (calculated Mr, 38 904, no lysine) diverges from that of spinach and N. debneyi in only two positions in the C-terminal part. The trn H gene has the same polarity as the psb A gene and the coding region is located at the very end of the large single copy region. The deduced sequence of the soybean chloroplast tRNAHisGUG is identical with that of Zea mays chloroplasts. Both ends of the large single copy region were sequenced including a small segment of the adjacent IR1 and IR2.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

Science.gov (United States)

Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron

2011-05-04

Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first

Validation of rearrangement break points identified by paired-end sequencing in natural populations of Drosophila melanogaster.

Science.gov (United States)

Cridland, Julie M; Thornton, Kevin R

2010-01-13

Several recent studies have focused on the evolution of recently duplicated genes in Drosophila. Currently, however, little is known about the evolutionary forces acting upon duplications that are segregating in natural populations. We used a high-throughput, paired-end sequencing platform (Illumina) to identify structural variants in a population sample of African D. melanogaster. Polymerase chain reaction and sequencing confirmation of duplications detected by multiple, independent paired-ends showed that paired-end sequencing reliably uncovered the break points of structural rearrangements and allowed us to identify a number of tandem duplications segregating within a natural population. Our confirmation experiments show that rates of confirmation are very high, even at modest coverage. Our results also compare well with previous studies using microarrays (Emerson J, Cardoso-Moreira M, Borevitz JO, Long M. 2008. Natural selection shapes genome wide patterns of copy-number polymorphism in Drosophila melanogaster. Science. 320:1629-1631. and Dopman EB, Hartl DL. 2007. A portrait of copy-number polymorphism in Drosophila melanogaster. Proc Natl Acad Sci U S A. 104:19920-19925.), which both gives us confidence in the results of this study as well as confirms previous microarray results.We were also able to identify whole-gene duplications, such as a novel duplication of Or22a, an olfactory receptor, and identify copy-number differences in genes previously known to be under positive selection, like Cyp6g1, which confers resistance to dichlorodiphenyltrichloroethane. Several "hot spots" of duplications were detected in this study, which indicate that particular regions of the genome may be more prone to generating duplications. Finally, population frequency analysis of confirmed events also showed an excess of rare variants in our population, which indicates that duplications segregating in the population may be deleterious and ultimately destined to be lost from the

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

Science.gov (United States)

Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

2012-11-01

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Expression Analysis of CB2-GFP BAC Transgenic Mice.

Directory of Open Access Journals (Sweden)

Anne-Caroline Schmöle

Full Text Available The endocannabinoid system (ECS is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2. As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.; Pan, Hongling; He, Yuchun; Spokony, Rebecca; Wan, Kenneth H.; Koriabine, Maxim; de Jong, Pieter J.; White, Kevin P.; Bellen, Hugo J.; Hoskins, Roger A.

2009-04-21

We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.

The specificity of memory for a highly trained finger movement sequence: Change the ending, change all.

Science.gov (United States)

Rozanov, Simon; Keren, Ofer; Karni, Avi

2010-05-17

How are highly trained movement sequences represented in long-term memory? Here we show that the gains attained in the performance of a well-trained sequence of finger movements can be expressed only when the order of the movements is exactly as practiced. Ten young adults were trained to perform a given 5-element sequence of finger-to-thumb opposition movements with their left hand. Movements were analyzed using video based tracking. Three weeks of training resulted, along with improved accuracy, in robustly shortened movement times as well as shorter finger-to-thumb touch times. However, there was little transfer of these gains in speed to the execution of the same component movements arranged in a new order. Moreover, even when the only change was the omission of the one before final movement of the trained sequence (Omit sequence), the initial movements of the sequence were significantly slowed down, although these movements were identical to the initial movements of the trained sequence. Our results support the notion that a well-trained sequence of finger movements can be represented, in the adult motor system, as a singular, co-articulated, unit of movement, in which even the initial component movements are contingent on the subsequent, anticipated, ones. Because of co-articulation related anticipatory effects, gains in fluency and accuracy acquired in training on a specific movement sequence cannot be expressed in full in the execution of the trained component movements or of a full segment of the trained sequence, if followed by a different ending segment. Copyright 2010. Published by Elsevier B.V.

Ascertainment bias in studies of human genome-wide polymorphism

DEFF Research Database (Denmark)

Clark, Andrew G.; Hubisz, Melissa J.; Bustamente, Carlos D.

2005-01-01

of the SNPs that are found are influenced by the discovery sampling effort. The International HapMap project relied on nearly any piece of information available to identify SNPs-including BAC end sequences, shotgun reads, and differences between public and private sequences-and even made use of chimpanzee...... was a resequencing-by-hybridization effort using the 24 people of diverse origin in the Polymorphism Discovery Resource. Here we take these two data sets and contrast two basic summary statistics, heterozygosity and FST, as well as the site frequency spectra, for 500-kb windows spanning the genome. The magnitude...... of disparity between these samples in these measures of variability indicates that population genetic analysis on the raw genotype data is ill advised. Given the knowledge of the discovery samples, we perform an ascertainment correction and show how the post-correction data are more consistent across...

Driving performance on the descending limb of blood alcohol concentration (BAC) in undergraduate students: a pilot study.

Science.gov (United States)

Tremblay, Mathieu; Gallant, François; Lavallière, Martin; Chiasson, Martine; Silvey, Dustin; Behm, David; Albert, Wayne J; Johnson, Michel J

2015-01-01

Young drivers are overrepresented in collisions resulting in fatalities. It is not uncommon for young drivers to socially binge drink and decide to drive a vehicle a few hours after consumption. To better understand the risks that may be associated with this behaviour, the present study has examined the effects of a social drinking bout followed by a simulated drive in undergraduate students on the descending limb of their BAC (blood alcohol concentration) curve. Two groups of eight undergraduate students (n = 16) took part in this study. Participants in the alcohol group were assessed before drinking, then at moderate and low BAC as well as 24 hours post-acute consumption. This group consumed an average of 5.3 ± 1.4 (mean ± SD) drinks in an hour in a social context and were then submitted to a driving and a predicted crash risk assessment. The control group was assessed at the same time points without alcohol intake or social context.; at 8 a.m., noon, 3 p.m. and 8 a.m. the next morning. These multiple time points were used to measure any potential learning effects from the assessment tools (i.e. driving simulator and useful field of view test (UFOV)). Diminished driving performance at moderate BAC was observed with no increases in predicted crash risk. Moderate correlations between driving variables were observed. No association exists between driving variables and UFOV variables. The control group improved measures of selective attention after the third assessment. No learning effect was observed from multiple sessions with the driving simulator. Our results show that a moderate BAC, although legal, increases the risky behaviour. Effects of alcohol expectancy could have been displayed by the experimental group. UFOV measures and predicted crash risk categories were not sensitive enough to predict crash risk for young drivers, even when intoxicated.

Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes.

Science.gov (United States)

Tweedy, Joshua G; Escriva, Eric; Topf, Maya; Gompels, Ursula A

2017-12-31

Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis.

BAC and Beer: Operationalizing Drunk Driving Laws in a Research Methods Course Exercise.

Science.gov (United States)

Taylor, Ralph B.; McConnell, Patrick

2001-01-01

Focuses on an exercise utilized in a research methods class and based on social problems that invites student interest. Explains the exercise has students determine their blood alcohol level (BAC) by asking them to estimate the number of beers it would take to have them just reach driving under the influence (DUI) status. (CMK)

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

Czech Academy of Sciences Publication Activity Database

Kasprzak, A.; Šafář, Jan; Janda, Jaroslav; Doležel, Jaroslav; Wolko, B.; Naganowska, B.

2006-01-01

Roč. 11, - (2006), s. 396-407 ISSN 1425-8153 R&D Projects: GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : BAC * genomic DNA library * Lupinus angustifolius L. Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.238, year: 2006

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library

Directory of Open Access Journals (Sweden)

Cribiu Edmond-Paul

2000-07-01

Full Text Available Abstract In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57. This study allowed us, (i, to anchor all linkage groups on sheep chromosomes, (ii, to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii, to contradict the previous orientation of the ovine × linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map.

Resource utilization and outcomes of intoxicated drivers.

Science.gov (United States)

Cherry, Robert A; Nichols, Pamela A; Snavely, Theresa M; Camera, Lindsay J; Mauger, David T

2010-08-05

The high risk behavior of intoxicated drivers, impaired reaction time, lack of seat belt use, and increased incidence of head injury raises questions of whether pre-hospital use of alcohol leads to a higher injury severity score and worse clinical outcomes. We therefore compared intoxicated and non-intoxicated drivers of motor vehicle crashes with respect to outcome measurements and also describe the resources utilized to achieve those outcomes at our Level 1 trauma center. Retrospective descriptive study (Jan 2002-June 2007) of our trauma registry and financial database comparing intoxicated drivers with blood alcohol levels (BAC) > 80 mg/dl (ETOH > 80) with drivers who had a BAC of 0 mg/dl (ETOH = 0). Drivers without a BAC drawn or who had levels ranging from 1 mg/dL to 80 mg/dL were excluded. Data was collected on demographic information (age, gender, injury severity score or ISS), outcome variables (mortality, complications, ICU and hospital LOS, ventilator days) and resource utilization (ED LOS, insurance, charges, costs, payments). p 80; stratified chi square. Out of 1732 drivers, the combined study group (n = 987) of 623 ETOH = 0 and 364 ETOH > 80 had a mean age of 38.8 +/- 17.9, ISS of 18.0 +/- 12.1, and 69.8%% male. There was no difference in ISS (p = 0.67) or complications (p = 0.38). There was a trend towards decreased mortality (p = 0.06). The ETOH = 0 group had more patients with a prolonged ICU LOS (>/= 5 days), ventilator days (>/= 8 days), and hospital LOS (> 14 days) when compared to the ETOH > 80 group (p 80 group tended to be self pay (4.9% vs. 0.7%, p pay, less likely to have charges > $50K, and less likely to pay >/= 90% of the charges. Further research using multivariable analysis is needed to determine if these apparent outcomes differences are driven by acute intoxication, and the tendency for endotracheal intubation and ICU admission, rather than injury severity.

Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

OpenAIRE

Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

2012-01-01

The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

Development of a Set of Chromosome-Specific Cytogenetic DNA Markers in Sunflower Using BAC-FISH

Science.gov (United States)

In diploid sunflower (2n=34), conventional karyotypes and various genetic linkage maps have been established. However, the relationship between genetic linkage groups and individual chromosomes of sunflower remains unknown. Recently, a set of linkage group-specific BAC and BIBAC clones were identifi...

Uncertainty of Blood Alcohol Concentration (BAC Results as Related to Instrumental Conditions: Optimization and Robustness of BAC Analysis Headspace Parameters

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Haleigh A. Boswell

2015-12-01

Full Text Available Analysis of blood alcohol concentration is a routine analysis performed in many forensic laboratories. This analysis commonly utilizes static headspace sampling, followed by gas chromatography combined with flame ionization detection (GC-FID. Studies have shown several “optimal” methods for instrumental operating conditions, which are intended to yield accurate and precise data. Given that different instruments, sampling methods, application specific columns and parameters are often utilized, it is much less common to find information on the robustness of these reported conditions. A major problem can arise when these “optimal” conditions may not also be robust, thus producing data with higher than desired uncertainty or potentially inaccurate results. The goal of this research was to incorporate the principles of quality by design (QBD in the adjustment and determination of BAC (blood alcohol concentration instrumental headspace parameters, thereby ensuring that minor instrumental variations, which occur as a matter of normal work, do not appreciably affect the final results of this analysis. This study discusses both the QBD principles as well as the results of the experiments, which allow for determination of more favorable instrumental headspace conditions. Additionally, method detection limits will also be reported in order to determine a reporting threshold and the degree of uncertainty at the common threshold value of 0.08 g/dL. Furthermore, the comparison of two internal standards, n-propanol and t-butanol, will be investigated. The study showed that an altered parameter of 85 °C headspace oven temperature and 15 psi headspace vial pressurization produces the lowest percent relative standard deviation of 1.3% when t-butanol is implemented as an internal standard, at least for one very common platform. The study also showed that an altered parameter of 100 °C headspace oven temperature and 15-psi headspace vial pressurization

End-of-life resource recovery from emerging electronic products

DEFF Research Database (Denmark)

Parajuly, Keshav; Habib, Komal; Cimpan, Ciprian

2016-01-01

Integrating product design with appropriate end-of-life (EoL) processing is widely recognized to have huge potentials in improving resource recovery from electronic products. In this study, we investigate both the product characteristics and EoL processing of robotic vacuum cleaner (RVC), as a case...... of emerging electronic product, in order to understand the recovery fate of different materials and its linkage to product design. Ten different brands of RVC were dismantled and their material composition and design profiles were studied. Another 125 RVCs (349 kg) were used for an experimental trial...... at a conventional ‘shred-and-separate’ type preprocessing plant in Denmark. A detailed material flow analysis was performed throughout the recycling chain. The results show a mismatch between product design and EoL processing, and the lack of practical implementation of ‘Design for EoL’ thinking. In the best...

Building a model: developing genomic resources for common milkweed (Asclepias syriaca with low coverage genome sequencing

Directory of Open Access Journals (Sweden)

Weitemier Kevin

2011-05-01

Full Text Available Abstract Background Milkweeds (Asclepias L. have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L. could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp and 5S rDNA (120 bp sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp, with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae unigenes (median coverage of 0.29× and 66% of single copy orthologs (COSII in asterids (median coverage of 0.14×. From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites and phylogenetics (low-copy nuclear genes studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species

A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance

Directory of Open Access Journals (Sweden)

Scalabrin Simone

2008-06-01

Full Text Available Abstract Background Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. Results The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32% were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. Conclusion Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and

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

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Thomas Kai Wolfgruber

2016-03-01

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

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies.

Science.gov (United States)

Tulpová, Zuzana; Luo, Ming-Cheng; Toegelová, Helena; Visendi, Paul; Hayashi, Satomi; Vojta, Petr; Paux, Etienne; Kilian, Andrzej; Abrouk, Michaël; Bartoš, Jan; Hajdúch, Marián; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2018-03-08

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere. Copyright © 2018. Published by Elsevier B.V.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

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Hyun-Kyoung Kim

Full Text Available BACKGROUND: The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs, which are abundant in solid tumors, can be utilized for identification of rearranged ends. METHOD: As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP microarray method entailing CNB-region refinement by competitor DNA. RESULT: Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9% were identified, and two polymerase chain reaction (PCR-amplifiable rearrangements were obtained in six cases (66.7%. And significantly, TNGS-CNB, with its high positive identification rate (82.6% of PCR-amplifiable rearrangements at candidate sites (19/23, just from filtering of aligned sequences, requires little effort for validation. CONCLUSION: Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

Science.gov (United States)

Kim, Hyun-Kyoung; Park, Won Cheol; Lee, Kwang Man; Hwang, Hai-Li; Park, Seong-Yeol; Sorn, Sungbin; Chandra, Vishal; Kim, Kwang Gi; Yoon, Woong-Bae; Bae, Joon Seol; Shin, Hyoung Doo; Shin, Jong-Yeon; Seoh, Ju-Young; Kim, Jong-Il; Hong, Kyeong-Man

2014-01-01

The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences.

Science.gov (United States)

Gao, Song; Sung, Wing-Kin; Nagarajan, Niranjan

2011-11-01

Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/ ).

Feasibility of a novel positive feedback effect of 131I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

International Nuclear Information System (INIS)

Guo Rui; Tian Lipeng; Han Bing; Xu Haoping; Zhang Miao; Li Biao

2011-01-01

Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by 131 I and may promote human NIS (hNIS) expression, hNIS also induces 131 I uptake and activates Egr1, so the existence of a positive feedback effect of 131 I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test 131 I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without 131 I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of 131 I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without 131 I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with 131 I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of 131 I after 131 I stimulation. The amount of 131 I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the 131 I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of 131 I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher 131 I uptake. It might provide a reference for the existence of a positive feedback effect in 131 I-promoted Bac-Egr1-h

An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives.

Science.gov (United States)

Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun

2017-05-08

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.

Caring Decisions: The Development of a Written Resource for Parents Facing End-of-Life Decisions.

Science.gov (United States)

Xafis, Vicki; Gillam, Lynn; Hynson, Jenny; Sullivan, Jane; Cossich, Mary; Wilkinson, Dominic

2015-11-01

Written resources in adult intensive care have been shown to benefit families facing end of life (EoL) decisions. There are few resources for parents making EoL decisions for their child and no existing resources addressing ethical issues. The Caring Decisions handbook and website were developed to fill these gaps. We discuss the development of the resources, modification after reviewer feedback and findings from initial pilot implementation. A targeted literature review-to identify resources and factors that impact on parental EoL decision-making; development phase-guided by the literature and the researchers' expertise; consultation process-comprised a multi-disciplinary panel of experts and parents; pilot evaluation study-hard-copy handbook was distributed as part of routine care at an Australian Children's Hospital. Twelve experts and parents formed the consultation panel. Eight parents of children with life-limiting conditions and clinicians were interviewed in the pilot study. Numerous factors supporting/impeding EoL decisions were identified. Caring Decisions addressed issues identified in the literature and by the multidisciplinary research team. The consultation panel provided overwhelmingly positive feedback. Pilot study parents found the resources helpful and comforting. Most clinicians viewed the resources as very beneficial to parents and identified them as ideal for training purposes. The development of the resources addressed many of the gaps in existing resources. The consultation process and the pilot study suggest these resources could be of significant benefit to parents and clinicians.

Cross-species BAC-FISH painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements

NARCIS (Netherlands)

Tang, X.; Szinay, D.; Ramanna, M.S.; Vossen, van der E.A.G.; Datema, E.; Klein Lankhorst, R.M.; Boer, de J.M.; Peters, S.A.; Bachem, C.W.B.; Stiekema, W.J.; Visser, R.G.F.; Jong, de J.H.; Bai, Y.

2008-01-01

Ongoing genomics projects of tomato (Solanum lycopersicum ) and potato (Solanum tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, BACs can be positioned on pachytene comple-ments by fluorescent in situ hybridization (FISH) on homoeologous

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

Science.gov (United States)

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

GDR (Genome Database for Rosaceae: integrated web resources for Rosaceae genomics and genetics research

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Ficklin Stephen

2004-09-01

Full Text Available Abstract Background Peach is being developed as a model organism for Rosaceae, an economically important family that includes fruits and ornamental plants such as apple, pear, strawberry, cherry, almond and rose. The genomics and genetics data of peach can play a significant role in the gene discovery and the genetic understanding of related species. The effective utilization of these peach resources, however, requires the development of an integrated and centralized database with associated analysis tools. Description The Genome Database for Rosaceae (GDR is a curated and integrated web-based relational database. GDR contains comprehensive data of the genetically anchored peach physical map, an annotated peach EST database, Rosaceae maps and markers and all publicly available Rosaceae sequences. Annotations of ESTs include contig assembly, putative function, simple sequence repeats, and anchored position to the peach physical map where applicable. Our integrated map viewer provides graphical interface to the genetic, transcriptome and physical mapping information. ESTs, BACs and markers can be queried by various categories and the search result sites are linked to the integrated map viewer or to the WebFPC physical map sites. In addition to browsing and querying the database, users can compare their sequences with the annotated GDR sequences via a dedicated sequence similarity server running either the BLAST or FASTA algorithm. To demonstrate the utility of the integrated and fully annotated database and analysis tools, we describe a case study where we anchored Rosaceae sequences to the peach physical and genetic map by sequence similarity. Conclusions The GDR has been initiated to meet the major deficiency in Rosaceae genomics and genetics research, namely a centralized web database and bioinformatics tools for data storage, analysis and exchange. GDR can be accessed at http://www.genome.clemson.edu/gdr/.

GDR (Genome Database for Rosaceae): integrated web resources for Rosaceae genomics and genetics research.

Science.gov (United States)

Jung, Sook; Jesudurai, Christopher; Staton, Margaret; Du, Zhidian; Ficklin, Stephen; Cho, Ilhyung; Abbott, Albert; Tomkins, Jeffrey; Main, Dorrie

2004-09-09

Peach is being developed as a model organism for Rosaceae, an economically important family that includes fruits and ornamental plants such as apple, pear, strawberry, cherry, almond and rose. The genomics and genetics data of peach can play a significant role in the gene discovery and the genetic understanding of related species. The effective utilization of these peach resources, however, requires the development of an integrated and centralized database with associated analysis tools. The Genome Database for Rosaceae (GDR) is a curated and integrated web-based relational database. GDR contains comprehensive data of the genetically anchored peach physical map, an annotated peach EST database, Rosaceae maps and markers and all publicly available Rosaceae sequences. Annotations of ESTs include contig assembly, putative function, simple sequence repeats, and anchored position to the peach physical map where applicable. Our integrated map viewer provides graphical interface to the genetic, transcriptome and physical mapping information. ESTs, BACs and markers can be queried by various categories and the search result sites are linked to the integrated map viewer or to the WebFPC physical map sites. In addition to browsing and querying the database, users can compare their sequences with the annotated GDR sequences via a dedicated sequence similarity server running either the BLAST or FASTA algorithm. To demonstrate the utility of the integrated and fully annotated database and analysis tools, we describe a case study where we anchored Rosaceae sequences to the peach physical and genetic map by sequence similarity. The GDR has been initiated to meet the major deficiency in Rosaceae genomics and genetics research, namely a centralized web database and bioinformatics tools for data storage, analysis and exchange. GDR can be accessed at http://www.genome.clemson.edu/gdr/.

The Resource Benefits Evaluation Model on Remanufacturing Processes of End-of-Life Construction Machinery under the Uncertainty in Recycling Price

Directory of Open Access Journals (Sweden)

Qian-wang Deng

2017-02-01

Full Text Available In the process of end-of-life construction machinery remanufacturing, the existence of uncertainties in all aspects of the remanufacturing process increase the difficulty and complexity of resource benefits evaluation for them. To quantify the effects of those uncertainty factors, this paper makes a mathematical analysis of the recycling and remanufacturing processes, building a resource benefits evaluation model for the end-of-life construction machinery. The recycling price and the profits of remanufacturers can thereby be obtained with a maximum remanufacturing resource benefit. The study investigates the change regularity of the resource benefits, recycling price, and profits of remanufacturers when the recycling price, quality fluctuation coefficient, demand coefficient, and the reusing ratio of products or parts are varying. In the numerical experiment, we explore the effects of uncertainties on the remanufacturing decisions and the total expected costs. The simulated analysis shows when the quality fluctuation coefficient is approaching to 1, the values of the profits of remanufacturer, the maximal resource benefits and recycling price grade into constants.

The Effect of Uncertain End-of-Life Product Quality and Consumer Incentives on Partial Disassembly Sequencing in Value Recovery Operations

OpenAIRE

Rickli, Jeremy Lewis

2013-01-01

This dissertation addresses gaps in the interaction between End-of-Life (EoL) product acquisition systems and disassembly sequencing. The research focuses on two remanufacturing research problems; 1) modeling uncertain EoL product quality, quantity, and timing in regards to EoL product acquisition and disassembly sequencing and 2) designing EoL product acquisition schemes considering EoL product uncertainty. The main research objectives within these areas are; analyzing, predicting, and contr...

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis.

Science.gov (United States)

Pan, Luyuan; Shah, Arish N; Phelps, Ian G; Doherty, Dan; Johnson, Eric A; Moens, Cecilia B

2015-02-14

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive. Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250 bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified. Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

Science.gov (United States)

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

OpenAIRE

Zuccolo, Andrea; Bowers, John E; Estill, James C; Xiong, Zhiyong; Luo, Meizhong; Sebastian, Aswathy; Goicoechea, Jos? Luis; Collura, Kristi; Yu, Yeisoo; Jiao, Yuannian; Duarte, Jill; Tang, Haibao; Ayyampalayam, Saravanaraj; Rounsley, Steve; Kudrna, Dave

2011-01-01

Background Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome....

Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs

Directory of Open Access Journals (Sweden)

Sugano Sumio

2009-07-01

Full Text Available Abstract Background Apicomplexan parasites are causative agents of various diseases including malaria and have been targets of extensive genomic sequencing. We generated 5'-EST collections for six apicomplexa parasites using our full-length oligo-capping cDNA library method. To improve upon the current genome annotations, as well as to validate the importance for physical cDNA clone resources, we generated a large-scale collection of full-length cDNAs for several apicomplexa parasites. Results In this study, we used a total of 61,056 5'-end-single-pass cDNA sequences from Plasmodium falciparum, P. vivax, P. yoelii, P. berghei, Cryptosporidium parvum, and Toxoplasma gondii. We compared these partially sequenced cDNA sequences with the currently annotated gene models and observed significant inconsistencies between the two datasets. In particular, we found that on average 14% of the exons in the current gene models were not supported by any cDNA evidence, and that 16% of the current gene models may contain at least one mis-annotation and should be re-evaluated. We also identified a large number of transcripts that had been previously unidentified. For 732 cDNAs in T. gondii, the entire sequences were determined in order to evaluate the annotated gene models at the complete full-length transcript level. We found that 41% of the T. gondii gene models contained at least one inconsistency. We also identified and confirmed by RT-PCR 140 previously unidentified transcripts found in the intergenic regions of the current gene annotations. We show that the majority of these discrepancies are due to questionable predictions of one or two extra exons in the upstream or downstream regions of the genes. Conclusion Our data indicates that the current gene models are likely to still be incomplete and have much room for improvement. Our unique full-length cDNA information is especially useful for further refinement of the annotations for the genomes of

Identification and analysis of common bean (Phaseolus vulgaris L. transcriptomes by massively parallel pyrosequencing

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Thimmapuram Jyothi

2011-10-01

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

A new normalizing algorithm for BAC CGH arrays with quality control metrics.

Science.gov (United States)

Miecznikowski, Jeffrey C; Gaile, Daniel P; Liu, Song; Shepherd, Lori; Nowak, Norma

2011-01-01

The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose "SmoothArray", a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package "aCGHplus," this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

A New Normalizing Algorithm for BAC CGH Arrays with Quality Control Metrics

Directory of Open Access Journals (Sweden)

Jeffrey C. Miecznikowski

2011-01-01

Full Text Available The main focus in pin-tip (or print-tip microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose “SmoothArray”, a new method to preprocess comparative genomic hybridization (CGH bacterial artificial chromosome (BAC arrays and we show the effects on a cancer dataset. As part of our R software package “aCGHplus,” this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

Science.gov (United States)

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic

A PiggyBac mediated approach for lactoferricin gene transfer in bovine mammary epithelial stem cells for management of bovine mastitis.

Science.gov (United States)

Sharma, Neelesh; Huynh, Do Luong; Kim, Sung Woo; Ghosh, Mrinmoy; Sodhi, Simrinder Singh; Singh, Amit Kumar; Kim, Nam Eun; Lee, Sung Jin; Hussain, Kafil; Oh, Sung Jong; Jeong, Dong Kee

2017-11-28

The antibacterial and anti-inflammatory properties of lactoferricin have been ascribed to its ability to sequester essential iron. The objective of the study was to clone bovine lactoferricin ( LFcinB ) gene into PiggyBac Transposon vector, expression study in the bovine mammary epithelial stem cells (bMESCs) and also to determine the antimicrobial property of recombinant LFcinB against bovine mastitis-causing organisms. The PiggyBac-LFcinB was transfected into bMESCs by electroporation and a three fold of LFcinB secretion was observed in the transfected bMESCs medium by ELISA assay. Furthermore, the assessment of antimicrobial activity against mastitis causing pathogens Staphylococcus aureus and Escherichia coli demonstrated convincing evidence to prove strong antibacterial activity of LFcinB with 14.0±1.0 mm and 18.0±1.5 mm zone of inhibition against both organisms, respectively. The present study provides the convincing evidence to suggest the potential of PiggyBac transposon system to transfer antibacterial peptide into bMESCs or cow mammary gland and also pave the way to use bovine mammary gland as the bioreactors. Simultaneously, it also suggest toward commercial utilization of LFcinB bioreactor system in pharmaceutical industry.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc. Using a Hybrid Assembly Approach

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Tokurou Shimizu

2017-12-01

Full Text Available Satsuma (Citrus unshiu Marc. is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase” was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

Distribution and uses of legume DNA clone resources

International Nuclear Information System (INIS)

Young, N.D.

2001-01-01

Since 1990, my lab has developed and distributed various DNA clone resources for the legumes. In the first several years, the focus was on members of the tropical genus, Vigna, including the widely cultivated species, mungbean (V. radiata) and cowpea (V. unguiculata). Both of these grain legumes play key roles in agriculture in developing countries of Asia (mungbean) and Africa (cowpea). Moreover, because there is substantial genome conservation among legumes, these genetic resources have also been utilized by a wide range of researchers in other crop species. In 1997, my lab began to focus on the development and distribution of a new generation of DNA clone resources; Bacterial Artificial Chromosomes (BAC). A library of these clones was constructed in soybean (Glycine max) the most important legume species worldwide in terms of economic value. Again, the library has become a valuable resource for the legume research community and has been widely used in studies of legume genomics. (author)

Contributions of Bacău to the economic literature and culture – The life and work of Professor Vasile Pătruţ

OpenAIRE

Mihai Deju

2012-01-01

The economic culture and accounting theory in Bacău area has its beginnings in the setting up of the first practical school of agriculture, by Ion Ionescu de la Brad, who included in the curriculum an accounting course, as well. Over the years, the economic education of Bacău area education has evolved from “The School of Accounting and Co-operative Education” (1919) to the modern economic higher education, in our days. During an important period of the evolution of education and culture in B...

Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms

Science.gov (United States)

Forbes, Sarah; Cowley, Nicola; Mistry, Hitesh; Amézquita, Alejandro

2017-01-01

ABSTRACT The current investigation aimed to generate data to inform the development of risk assessments of biocide usage. Stabilized domestic drain biofilm microcosms were exposed daily over 6 months to increasing concentrations (0.01% to 1%) of the biocide benzalkonium chloride (BAC) in a simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analyzed by culture, differentiating by bacterial functional group and by BAC or antibiotic susceptibility. Bacterial isolates were identified by 16S rRNA sequencing, and changes in biofilm composition were assessed by high-throughput sequencing. Exposure to BAC-f resulted in significantly larger reductions in levels of viable bacteria than exposure to BAC-s, while bacterial diversity greatly decreased during exposure to both BAC-s and BAC-f, as evidenced by sequencing and viable counts. Increases in the abundance of bacteria exhibiting reduced antibiotic or BAC susceptibility following exposure to BAC at 0.1% were significantly greater for BAC-s than BAC-f. Bacteria with reduced BAC and antibiotic susceptibility were generally suppressed by higher BAC concentrations, and formulation significantly enhanced this effect. Significant decreases in the antimicrobial susceptibility of bacteria isolated from the systems before and after long-term BAC exposure were not detected. In summary, dose-dependent suppression of bacterial viability by BAC was enhanced by formulation. Biocide exposure decreased bacterial diversity and transiently enriched populations of organisms with lower antimicrobial susceptibility, and the effects were subsequently suppressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products. IMPORTANCE Assessment of the risks of biocide use has been based mainly on the exposure of axenic cultures of bacteria to biocides in simple aqueous solutions. The current investigation aimed to assess the

Network resource management

NARCIS (Netherlands)

2009-01-01

The invention provides real time dynamic resource management to improve end-to-end QoS by mobile devices regularly updating a resource availability server (RAS) with resource update information. Examples of resource update information are device battery status, available memory, session bandwidth,

Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud.

Directory of Open Access Journals (Sweden)

Malachi Griffith

2015-08-01

Full Text Available Massively parallel RNA sequencing (RNA-seq has rapidly become the assay of choice for interrogating RNA transcript abundance and diversity. This article provides a detailed introduction to fundamental RNA-seq molecular biology and informatics concepts. We make available open-access RNA-seq tutorials that cover cloud computing, tool installation, relevant file formats, reference genomes, transcriptome annotations, quality-control strategies, expression, differential expression, and alternative splicing analysis methods. These tutorials and additional training resources are accompanied by complete analysis pipelines and test datasets made available without encumbrance at www.rnaseq.wiki.

Features of the organization of bread wheat chromosome 5BS based on physical mapping.

Science.gov (United States)

Salina, Elena A; Nesterov, Mikhail A; Frenkel, Zeev; Kiseleva, Antonina A; Timonova, Ekaterina M; Magni, Federica; Vrána, Jan; Šafář, Jan; Šimková, Hana; Doležel, Jaroslav; Korol, Abraham; Sergeeva, Ekaterina M

2018-02-09

The IWGSC strategy for construction of the reference sequence of the bread wheat genome is based on first obtaining physical maps of the individual chromosomes. Our aim is to develop and use the physical map for analysis of the organization of the short arm of wheat chromosome 5B (5BS) which bears a number of agronomically important genes, including genes conferring resistance to fungal diseases. A physical map of the 5BS arm (290 Mbp) was constructed using restriction fingerprinting and LTC software for contig assembly of 43,776 BAC clones. The resulting physical map covered ~ 99% of the 5BS chromosome arm (111 scaffolds, N50 = 3.078 Mb). SSR, ISBP and zipper markers were employed for anchoring the BAC clones, and from these 722 novel markers were developed based on previously obtained data from partial sequencing of 5BS. The markers were mapped using a set of Chinese Spring (CS) deletion lines, and F2 and RICL populations from a cross of CS and CS-5B dicoccoides. Three approaches have been used for anchoring BAC contigs on the 5BS chromosome, including clone-by-clone screening of BACs, GenomeZipper analysis, and comparison of BAC-fingerprints with in silico fingerprinting of 5B pseudomolecules of T. dicoccoides. These approaches allowed us to reach a high level of BAC contig anchoring: 96% of 5BS BAC contigs were located on 5BS. An interesting pattern was revealed in the distribution of contigs along the chromosome. Short contigs (200-999 kb) containing markers for the regions interrupted by tandem repeats, were mainly localized to the 5BS subtelomeric block; whereas the distribution of larger 1000-3500 kb contigs along the chromosome better correlated with the distribution of the regions syntenic to rice, Brachypodium, and sorghum, as detected by the Zipper approach. The high fingerprinting quality, LTC software and large number of BAC clones selected by the informative markers in screening of the 43,776 clones allowed us to significantly increase the

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava.

Science.gov (United States)

López, C E; Zuluaga, A P; Cooke, R; Delseny, M; Tohme, J; Verdier, V

2003-08-01

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

NNAlign: A Web-Based Prediction Method Allowing Non-Expert End-User Discovery of Sequence Motifs in Quantitative Peptide Data

DEFF Research Database (Denmark)

Andreatta, Massimo; Schafer-Nielsen, Claus; Lund, Ole

2011-01-01

Recent advances in high-throughput technologies have made it possible to generate both gene and protein sequence data at an unprecedented rate and scale thereby enabling entirely new "omics"-based approaches towards the analysis of complex biological processes. However, the amount and complexity...... to interpret large data sets. We have recently developed a method, NNAlign, which is generally applicable to any biological problem where quantitative peptide data is available. This method efficiently identifies underlying sequence patterns by simultaneously aligning peptide sequences and identifying motifs...... associated with quantitative readouts. Here, we provide a web-based implementation of NNAlign allowing non-expert end-users to submit their data (optionally adjusting method parameters), and in return receive a trained method (including a visual representation of the identified motif) that subsequently can...

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Science.gov (United States)

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Directory of Open Access Journals (Sweden)

Olivier Arnaiz

Full Text Available Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a

Standardizing an End-to-end Accounting Service

Science.gov (United States)

Greenberg, Edward; Kazz, Greg

2006-01-01

Currently there are no space system standards available for space agencies to accomplish end-to-end accounting. Such a standard does not exist for spacecraft operations nor for tracing the relationship between the mission planning activities, the command sequences designed to perform those activities, the commands formulated to initiate those activities and the mission data and specifically the mission data products created by those activities. In order for space agencies to cross-support one another for data accountability/data tracing and for inter agency spacecraft to interoperate with each other, an international CCSDS standard for end-to-end data accountability/tracing needs to be developed. We will first describe the end-to-end accounting service model and functionality that supports the service. This model will describe how science plans that are ultimately transformed into commands can be associated with the telemetry products generated as a result of their execution. Moreover, the interaction between end-to-end accounting and service management will be explored. Finally, we will show how the standard end-to-end accounting service can be applied to a real life flight project i.e., the Mars Reconnaissance Orbiter project.

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii

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Patel Hardip R

2011-08-01

Full Text Available Abstract Background The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. Results A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH, (b End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Conclusions Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii).

Science.gov (United States)

Wang, Chenwei; Webley, Lee; Wei, Ke-jun; Wakefield, Matthew J; Patel, Hardip R; Deakin, Janine E; Alsop, Amber; Marshall Graves, Jennifer A; Cooper, Desmond W; Nicholas, Frank W; Zenger, Kyall R

2011-08-19

The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat

Czech Academy of Sciences Publication Activity Database

Bartoš, Jan; Vlček, Čestmír; Choulet, F.; Džunková, Mária; Cviková, Kateřina; Šafář, Jan; Šimková, Hana; Pačes, Jan; Strnad, Hynek; Sourdille, P.; Berges, H.; Cattonaro, F.; Feuillet, C.; Doležel, Jaroslav

2012-01-01

Roč. 12, č. 155 (2012), s. 1-10 ISSN 1471-2229 R&D Projects: GA ČR GAP501/10/1778 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514 Keywords : Wheat * BAC sequencing * Homoeologous genomes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.354, year: 2012

A Label Correcting Algorithm for Partial Disassembly Sequences in the Production Planning for End-of-Life Products

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Pei-Fang (Jennifer Tsai

2012-01-01

Full Text Available Remanufacturing of used products has become a strategic issue for cost-sensitive businesses. Due to the nature of uncertain supply of end-of-life (EoL products, the reverse logistic can only be sustainable with a dynamic production planning for disassembly process. This research investigates the sequencing of disassembly operations as a single-period partial disassembly optimization (SPPDO problem to minimize total disassembly cost. AND/OR graph representation is used to include all disassembly sequences of a returned product. A label correcting algorithm is proposed to find an optimal partial disassembly plan if a specific reusable subpart is retrieved from the original return. Then, a heuristic procedure that utilizes this polynomial-time algorithm is presented to solve the SPPDO problem. Numerical examples are used to demonstrate the effectiveness of this solution procedure.

BacHbpred: Support Vector Machine Methods for the Prediction of Bacterial Hemoglobin-Like Proteins

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MuthuKrishnan Selvaraj

2016-01-01

Full Text Available The recent upsurge in microbial genome data has revealed that hemoglobin-like (HbL proteins may be widely distributed among bacteria and that some organisms may carry more than one HbL encoding gene. However, the discovery of HbL proteins has been limited to a small number of bacteria only. This study describes the prediction of HbL proteins and their domain classification using a machine learning approach. Support vector machine (SVM models were developed for predicting HbL proteins based upon amino acid composition (AC, dipeptide composition (DC, hybrid method (AC + DC, and position specific scoring matrix (PSSM. In addition, we introduce for the first time a new prediction method based on max to min amino acid residue (MM profiles. The average accuracy, standard deviation (SD, false positive rate (FPR, confusion matrix, and receiver operating characteristic (ROC were analyzed. We also compared the performance of our proposed models in homology detection databases. The performance of the different approaches was estimated using fivefold cross-validation techniques. Prediction accuracy was further investigated through confusion matrix and ROC curve analysis. All experimental results indicate that the proposed BacHbpred can be a perspective predictor for determination of HbL related proteins. BacHbpred, a web tool, has been developed for HbL prediction.

Simultaneous Structural Variation Discovery in Multiple Paired-End Sequenced Genomes

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Hormozdiari, Fereydoun; Hajirasouliha, Iman; McPherson, Andrew; Eichler, Evan E.; Sahinalp, S. Cenk

Next generation sequencing technologies have been decreasing the costs and increasing the world-wide capacity for sequence production at an unprecedented rate, making the initiation of large scale projects aiming to sequence almost 2000 genomes [1]. Structural variation detection promises to be one of the key diagnostic tools for cancer and other diseases with genomic origin. In this paper, we study the problem of detecting structural variation events in two or more sequenced genomes through high throughput sequencing . We propose to move from the current model of (1) detecting genomic variations in single next generation sequenced (NGS) donor genomes independently, and (2) checking whether two or more donor genomes indeed agree or disagree on the variations (in this paper we name this framework Independent Structural Variation Discovery and Merging - ISV&M), to a new model in which we detect structural variation events among multiple genomes simultaneously.

Progress report Waste Resources Utilization Program period ending March 31, 1976

International Nuclear Information System (INIS)

1976-06-01

This report describes the work on the Waste Resources Utilization Program for the quarter ending March 31, 1976. The purpose of this program is to develop technologies to utilize a 137 Cs γ source to modify sewage sludge for safe application as a fertilizer or an animal feed supplement. Results are reported from studies in microbiology, virology, and physical-chemical studies. Determinations were made of inactivation rates for Salmonella species, coliforms, and fecal strep in sewage sludge when radiation and thermoradiation were applied while bubbling oxygen through the sludge. Virology studies were continued investigating virucidal characteristics of anaerobically digested sludge. Another area of study was the dewatering of sewage sludge to reduce the drying time of the sewage sludge in the drying beds. A centrifuge was also installed to dewater treated sludge to approximately 30 percent solids

Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyBac Transposon-Engineered T Cells

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Daisuke Morita

2018-03-01

Full Text Available Adoptive T cell therapy using chimeric antigen receptor (CAR-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs as feeder cells and virus-specific T cell receptor (TCR stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.

Limited resources of genome sequencing in developing countries: Challenges and solutions

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Mohamed Helmy

2016-06-01

Full Text Available The differences between countries in national income, growth, human development and many other factors are used to classify countries into developed and developing countries. There are several classification systems that use different sets of measures and criteria. The most common classifications are the United Nations (UN and the World Bank (WB systems. The UN classification system uses the UN Human Development Index (HDI, an indicator that uses statistic of life expectancy, education, and income per capita for countries' classification. While the WB system uses gross national income (GNI per capita that is calculated using the World Bank Atlas method. According to the UN and WB classification systems, there are 151 and 134 developing countries, respectively, with 89% overlap between the two systems. Developing countries have limited human development, and limited expenditure in education and research, among several other limitations. The biggest challenge facing genomic researchers and clinicians is limited resources. As a result, genomic tools, specifically genome sequencing technologies, which are rapidly becoming indispensable, are not widely available. In this report, we explore the current status of sequencing technologies in developing countries, describe the associated challenges and emphasize potential solutions.

Legume Genome Initiative at the University of Oklahoma

Energy Technology Data Exchange (ETDEWEB)

Bruce A. Roe

2004-02-27

Consolidated Appropriations Resolution, 2003 Conference Report for the Department of Energy's Biological and Environmental Research (BER) program provided $481,000 for the Legume Genome Initiative at the University of Oklahoma. These funds were used to support our research that is aimed at determining the entire sequence of the gene rich regions of the genome of the legume, Medicago truncatula, by allowing us to obtain a greater degree of finished BAC sequences from the draft sequences we have already obtained through research funded by the Noble Foundation. During the funding period we increased the number of Medicago truncatula BACs with finished (Bermuda standard) sequences from 109 to 359, and the total number of BACs for which we collected sequence data from 584 to 842, 359 of which reached phase 2 (ordered and oriented contigs). We also sequenced a series of pooled BAC clones that cover additional euchromatic (gene rich) genomic regions. This work resulted in 6 refereed publications, see below. Genes whose sequence was determined during this study included multiple members of the plant disease resistance (R-gene) family as well as several genes involved in flavinoid biosynthesis, nitrogen fixation and plant-microbial symbosis. This work also served as a prelude to obtaining NSF funding for the international collaborative effort to complete the entire sequence of the Medicago truncatula genomic euchromatic regions using a BAC based approach.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

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Misako Yajima

2018-04-01

Full Text Available Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.

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Yajima, Misako; Ikuta, Kazufumi; Kanda, Teru

2018-04-03

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Voltage-Gated Sodium Channels: Evolutionary History and Distinctive Sequence Features.

Science.gov (United States)

Kasimova, M A; Granata, D; Carnevale, V

2016-01-01

Voltage-gated sodium channels (Nav) are responsible for the rising phase of the action potential. Their role in electrical signal transmission is so relevant that their emergence is believed to be one of the crucial factors enabling development of nervous system. The presence of voltage-gated sodium-selective channels in bacteria (BacNav) has raised questions concerning the evolutionary history of the ones in animals. Here we review some of the milestones in the field of Nav phylogenetic analysis and discuss some of the most important sequence features that distinguish these channels from voltage-gated potassium channels and transient receptor potential channels. Copyright © 2016 Elsevier Inc. All rights reserved.

Time-to-detection of bacteria and yeast with the BACTEC FX versus BacT/Alert Virtuo blood culture systems.

Science.gov (United States)

Somily, Ali Mohammed; Habib, Hanan Ahmed; Torchyan, Armen Albert; Sayyed, Samina B; Absar, Muhammed; Al-Aqeel, Rima; Binkhamis, A Khalifa

2018-01-01

Bloodstream infections are associated with high rates of morbidity and mortality. Rapid detection of bloodstream infections is important in achieving better patient outcomes. Compare the time-to-detection (TTD) of the new BacT/Alert Virtuo and the BACTEC FX automated blood culture systems. Prospective simulated comparison of two instruments using seeded samples. Medical microbiology laboratory. Blood culture bottles were seeded in triplicate with each of the standard ATCC strains of aerobes, anaerobes and yeast. TTD was calculated as the length of time from the beginning of culture incubation to the detection of bacterial growth. TTD for the various tested organisms on the two microbial detection systems. The 99 bottles of seeded blood cultures incubated in each of the blood culture systems included 21 anaerobic, 39 aerobic and 39 pediatric bottles. The BacT/Alert Virtuo system exhibited significantly shorter TTD for 72.7 % of the tested organisms compared to BACTEC FX system with a median difference in mean TTD of 2.1 hours (interquartile range: 1.5-3.5 hours). The BACTEC FX system was faster in 15.2% (5/33) of microorganisms, with a median difference in mean TTD of 25.9 hours (IQR: 9.1-29.2 hours). TTD was significantly shorter for most of the microorganisms tested on the new BacT/Alert Virtuo system compared to the BACTEC FX system. Use of simulated cultures to assess TTD may not precisely represent clinical blood cultures. None.

AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

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Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

2017-11-14

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

A Study of End Users' Perceived Usefulness of and Satisfaction with Enterprise Resource Planning Systems in Higher Education

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Su, Shau E. Eve

2012-01-01

Enterprise resource planning (ERP) systems have become necessities in today's educational arena and are used extensively to improve office productivity and institutional efficacy. As the implementation of ERP systems usually is costly and time-consuming, it is imperative for administrators to understand how end users adopt the introduced…

Enriching Genomic Resources and Transcriptional Profile Analysis of Miscanthus sinensis under Drought Stress Based on RNA Sequencing

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Gang Nie

2017-01-01

Full Text Available Miscanthus × giganteus is wildly cultivated as a potential biofuel feedstock around the world; however, the narrow genetic basis and sterile characteristics have become a limitation for its utilization. As a progenitor of M. × giganteus, M. sinensis is widely distributed around East Asia providing well abiotic stress tolerance. To enrich the M. sinensis genomic databases and resources, we sequenced and annotated the transcriptome of M. sinensis by using an Illumina HiSeq 2000 platform. Approximately 316 million high-quality trimmed reads were generated from 349 million raw reads, and a total of 114,747 unigenes were obtained after de novo assembly. Furthermore, 95,897 (83.57% unigenes were annotated to at least one database including NR, Swiss-Prot, KEGG, COG, GO, and NT, supporting that the sequences obtained were annotated properly. Differentially expressed gene analysis indicates that drought stress 15 days could be a critical period for M. sinensis response to drought stress. The high-throughput transcriptome sequencing of M. sinensis under drought stress has greatly enriched the current genomic available resources. The comparison of DEGs under different periods of drought stress identified a wealth of candidate genes involved in drought tolerance regulatory networks, which will facilitate further genetic improvement and molecular studies of the M. sinensis.

Unusual Gene Order and Organization of the Sea Urchin HoxCluster

Energy Technology Data Exchange (ETDEWEB)

Richardson, Paul M.; Lucas, Susan; Cameron, R. Andrew; Rowen,Lee; Nesbitt, Ryan; Bloom, Scott; Rast, Jonathan P.; Berney, Kevin; Arenas-Mena, Cesar; Martinez, Pedro; Davidson, Eric H.; Peterson, KevinJ.; Hood, Leroy

2005-05-10

The highly consistent gene order and axial colinear expression patterns found in vertebrate hox gene clusters are less well conserved across the rest of bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is : 5'-Hox1,2, 3, 11/13c, 11/13b, '11/13a, 9/10, 8, 7, 6, 5 - 3)'. The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.

A bacterial artificial chromosome-based physical map of Manihot esculenta ssp.flabellifolia

Institute of Scientific and Technical Information of China (English)

Yuhua FU; Zhiqiang XIA; Shujuan WANG; Xin CHEN; Cheng LU; Mingcheng LUO; Hongbin ZHANG; Wenquan WANG

2016-01-01

Cassava (Manihot esculenta) is known as the third most important food crop in the tropics and also used for industrial feedstock for biofuels.Two new bacterial artificial chromosome (BAC) libraries were constructed for W14 (M.Esculenta ssp.flabellifolia),a wild ancestor of domesticated cassava.The libraries were constructed with EcoRI and HindⅢ insertion vectors,respectively.The EcoRI library has 29952 clones with an average insert size of 115 kb,while the HindⅢ library consists of 29952 clones with an average insert of 129 kb.The combined libraries contain a total of 59904 clones with an average insert size of 125 kb,representing approximately 10×haploid genome equivalents.A total of 29952 clones were fingerprinted and resulted in a cassava physical map composed of 2485 contigs with an average physical length of 336 kb and 2909 singletons,representing approximately 762 Mb of the cassava genome.5000 clones located at the ends of BAC contigs were selected and sequenced.A total of 6077 SNPs and 231 indels were identified,that covered 459 gene sequences,of which 6 genes were associated with starch and sucrose metabolism.This BAC-based physical map provides valuable tools to understand the genetics and evolution of cassava.

Multicenter Clinical Evaluation of BacT/Alert Virtuo Blood Culture System.

Science.gov (United States)

Jacobs, Michael R; Mazzulli, Tony; Hazen, Kevin C; Good, Caryn E; Abdelhamed, Ayman M; Lo, Pauline; Shum, Bianche; Roman, Katharine P; Robinson, Danielle C

2017-08-01

BacT/Alert Virtuo is an advanced, automated blood culture system incorporating improved automation and an enhanced detection algorithm to shorten time to detection. A multicenter study of the investigational Virtuo system (bioMérieux, Inc., Durham, NC) compared to BacT/Alert 3D (BTA3D) for detection of bacteremia/fungemia in four bottle types, SA and FA Plus (aerobic) and SN and FN Plus (anaerobic), was performed in a clinical setting with patient samples in a matched system design clinical trial. Blood was added to paired aerobic or anaerobic bottles, with the volume in each bottle in each pair required to be ≤10 ml and with the volumes required to be within 30% of each other. Of 5,709 bottle sets (52.5% aerobic pairs and 47.5% anaerobic pairs), 430 (7.5%) were positive for bacterial or fungal growth, with 342 (6.0%) clinically significant and 83 (1.5%) contaminated. A total of 3,539 sets (62.0%) were volume compliant, with 203 sets (5.7%) clinically significant. The positivity rates for volume-compliant bottle pairs determined by the two systems were comparable, with 68.7% of clinically significant isolates detected by both instruments, 15.7% by Virtuo only, and 15.7% by BTA3D only. Virtuo detected microbial growth nearly 2 h sooner overall than BTA3D (mean, 15.9 h versus 17.7 h). Shorter time to detection by Virtuo was related to organism group, with the time to detection being significantly shorter for enteric Gram-negative bacilli and enterococci (means, 3.6 h and 2.3 h shorter, respectively). This large clinical study demonstrated that the Virtuo blood culture system produced results comparable to those seen with the long-established BTA3D system, with significantly shorter time to detection. Copyright © 2017 Jacobs et al.

A sensitive short read homology search tool for paired-end read sequencing data.

Science.gov (United States)

Techa-Angkoon, Prapaporn; Sun, Yanni; Lei, Jikai

2017-10-16

Homology search is still a significant step in functional analysis for genomic data. Profile Hidden Markov Model-based homology search has been widely used in protein domain analysis in many different species. In particular, with the fast accumulation of transcriptomic data of non-model species and metagenomic data, profile homology search is widely adopted in integrated pipelines for functional analysis. While the state-of-the-art tool HMMER has achieved high sensitivity and accuracy in domain annotation, the sensitivity of HMMER on short reads declines rapidly. The low sensitivity on short read homology search can lead to inaccurate domain composition and abundance computation. Our experimental results showed that half of the reads were missed by HMMER for a RNA-Seq dataset. Thus, there is a need for better methods to improve the homology search performance for short reads. We introduce a profile homology search tool named Short-Pair that is designed for short paired-end reads. By using an approximate Bayesian approach employing distribution of fragment lengths and alignment scores, Short-Pair can retrieve the missing end and determine true domains. In particular, Short-Pair increases the accuracy in aligning short reads that are part of remote homologs. We applied Short-Pair to a RNA-Seq dataset and a metagenomic dataset and quantified its sensitivity and accuracy on homology search. The experimental results show that Short-Pair can achieve better overall performance than the state-of-the-art methodology of profile homology search. Short-Pair is best used for next-generation sequencing (NGS) data that lack reference genomes. It provides a complementary paired-end read homology search tool to HMMER. The source code is freely available at https://sourceforge.net/projects/short-pair/ .

Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

Science.gov (United States)

Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T

2017-01-01

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat

Czech Academy of Sciences Publication Activity Database

Sehgal, S. K.; Li, W.; Rabinowicz, P. D.; Chan, A.; Šimková, Hana; Doležel, Jaroslav; Gill, B. S.

2012-01-01

Roč. 12, č. 64 (2012) ISSN 1471-2229 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : BREAD WHEAT * BRACHYPODIUM-DISTACHYON * REPETITIVE ELEMENTS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.354, year: 2012

Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

Directory of Open Access Journals (Sweden)

de Koning-Ward Tania F

2011-03-01

Full Text Available Abstract The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism. See research article: http://www.biomedcentral.com/1471-2164/12/155

Characterization of pig farms in Hung Yen, Hai Duong and Bac Ninh provinces

OpenAIRE

Nguyen Van Duy; Vu Dinh; Dang Vu, Binh; Vo Trong, Thanh; Nguyen Cong, oanh; Phan Van, Chung

2007-01-01

In order to characterization of pig farms in the Red River Delta, a study was conducted on 90 pig farms in Hung Yen, Hai Duong and Bac Ninh provinces from June to December 2006. Results show that most of the pig farms had been built for five years with a small size (0.5 hectare per farm). The invested capital was about 300-400 millions VND per farm. Four main sow groups used in the farms included crossbred exotic sows (51.1%), crossbred sow between local and exotic breeds (14.4%), purebred La...

A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.

Science.gov (United States)

Dolman, Nick J; Kilgore, Jason A; Davidson, Michael W

2013-07-01

Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. © 2013 by John Wiley & Sons, Inc.

The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.

Directory of Open Access Journals (Sweden)

Mohammed Almuhayawi

Full Text Available Detection and identification of anaerobic bacteria in blood cultures (BC is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux, BACTEC-Plus and -Lytic (Becton Dickinson BioSciences BC bottles in detection and time to detection (TTD of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94% than BacT/ALERT FN Plus (80/100, 80% (p<0.01 in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001. The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h, BACTEC Plus (27 h and finally BacT/ALERT FN Plus (38 h bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76% BacT/ALERT FN, 51/67 (76% BacT/ALERT FN Plus, 53/67 (79% BACTEC Plus and 50/67 (75% BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.

MUTATION ON Bacillus subtilis BAC4 USING ACRIDINE ORANGE AS AN EFFORT FOR INCREASING ANTIBIOTIC PRODUCTION

Directory of Open Access Journals (Sweden)

Supartono Supartono

2010-06-01

Full Text Available The efforts to get a new antibiotic require to be done continuously, because infection diseases still become the main health problems in Indonesia. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibites Serratia marcescens ATCC 27117 growth. Nevertheless, the optimum conditions have not been studied seriously. The objective of this research was to conduct mutation on B. subtilis BAC4 in order to obtain a mutant cell that overproduct in producing antibiotic. The mutation process was performed by using acridine orange of 1 g.L-1 randomly at various volumes. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with agar absorption method using S.  marcescens ATCC 27117 as bacteria assay. Research result provided a B. subtilis M10 mutant with overproduction of antibiotic. Characterization of B. subtilis M10 mutant showed that the mutant cell has size of (0.5-1.0 µm x (1.85-2.5 µm; spore has the form of ellipse with thick wavy wall, positive reaction for catalase, and forming acid from glucose and xylose.   Keywords: mutant, Bacillus, acridin, and antibiotics

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

Science.gov (United States)

2011-01-01

Background Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

Directory of Open Access Journals (Sweden)

Wynhoven Brian

2011-03-01

Full Text Available Abstract Background Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt, we have generated Expressed Sequence Tags (ESTs by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores and asexual (germinated urediniospores stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum, 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs. Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt and stripe rust, P. striiformis f. sp

Aspects of coverage in medical DNA sequencing

Directory of Open Access Journals (Sweden)

Wilson Richard K

2008-05-01

Full Text Available Abstract Background DNA sequencing is now emerging as an important component in biomedical studies of diseases like cancer. Short-read, highly parallel sequencing instruments are expected to be used heavily for such projects, but many design specifications have yet to be conclusively established. Perhaps the most fundamental of these is the redundancy required to detect sequence variations, which bears directly upon genomic coverage and the consequent resolving power for discerning somatic mutations. Results We address the medical sequencing coverage problem via an extension of the standard mathematical theory of haploid coverage. The expected diploid multi-fold coverage, as well as its generalization for aneuploidy are derived and these expressions can be readily evaluated for any project. The resulting theory is used as a scaling law to calibrate performance to that of standard BAC sequencing at 8× to 10× redundancy, i.e. for expected coverages that exceed 99% of the unique sequence. A differential strategy is formalized for tumor/normal studies wherein tumor samples are sequenced more deeply than normal ones. In particular, both tumor alleles should be detected at least twice, while both normal alleles are detected at least once. Our theory predicts these requirements can be met for tumor and normal redundancies of approximately 26× and 21×, respectively. We explain why these values do not differ by a factor of 2, as might intuitively be expected. Future technology developments should prompt even deeper sequencing of tumors, but the 21× value for normal samples is essentially a constant. Conclusion Given the assumptions of standard coverage theory, our model gives pragmatic estimates for required redundancy. The differential strategy should be an efficient means of identifying potential somatic mutations for further study.

Shedding genomic light on Aristotle's lantern.

Science.gov (United States)

Sodergren, Erica; Shen, Yufeng; Song, Xingzhi; Zhang, Lan; Gibbs, Richard A; Weinstock, George M

2006-12-01

Sea urchins have proved fascinating to biologists since the time of Aristotle who compared the appearance of their bony mouth structure to a lantern in The History of Animals. Throughout modern times it has been a model system for research in developmental biology. Now, the genome of the sea urchin Strongylocentrotus purpuratus is the first echinoderm genome to be sequenced. A high quality draft sequence assembly was produced using the Atlas assembler to combine whole genome shotgun sequences with sequences from a collection of BACs selected to form a minimal tiling path along the genome. A formidable challenge was presented by the high degree of heterozygosity between the two haplotypes of the selected male representative of this marine organism. This was overcome by use of the BAC tiling path backbone, in which each BAC represents a single haplotype, as well as by improvements in the Atlas software. Another innovation introduced in this project was the sequencing of pools of tiling path BACs rather than individual BAC sequencing. The Clone-Array Pooled Shotgun Strategy greatly reduced the cost and time devoted to preparing shotgun libraries from BAC clones. The genome sequence was analyzed with several gene prediction methods to produce a comprehensive gene list that was then manually refined and annotated by a volunteer team of sea urchin experts. This latter annotation community edited over 9000 gene models and uncovered many unexpected aspects of the sea urchin genetic content impacting transcriptional regulation, immunology, sensory perception, and an organism's development. Analysis of the basic deuterostome genetic complement supports the sea urchin's role as a model system for deuterostome and, by extension, chordate development.

INTERACTION BETWEEN DIFFERENT MOLECULAR FORMS OF IMMUNOGLOBULIN A AND RECOMBINANT DERIVATIVES POLYPEPTIDES OF BAC RECEPTOR PROTEINS FROM GROUP B STREPTOCOCCI

Directory of Open Access Journals (Sweden)

A. S. Korzhueva

2008-01-01

Full Text Available Abstract. The article concerns interactions between immunoglobulin A and recombinant P6, P7, P8 polypeptides, designed on the basis of externally localized Bac protein of the Group B streptococci, possessing IgA-binding activity.There is a current demand for immunochemical reagents that are strictly specific for IgA, in order to develop antigenic standards for detection of IgA levels in biological fluids, as well as for affinity purification of IgA and its fragments.To analyze an opportunity of the abovementioned application ways for these proteins, a special study was performed to assay an interaction capability of recombinant P6, P7, P8 polypeptides binding to Fc regions of different IgA forms (serum IgA, secretory IgA, subclasses of serum IgA – IgA1, IgA2. Selectivity of ligand binding was specially confirmed.It was found out that, among three presented polypeptides, the structure of recombinant P6 derivative proved to be optimal for IgA-binding ability of Bac protein.Structural features of IgA-binding fragments of Bac protein, i.e., binding site position on the IgA molecule (proximity to epitopes for three monoclonal antibodies, variability of the site structure, as well as resistance of binding site for P6, P7, P8 in IgA molecule against partial disulfide bonds reduction. (Med. Immunol., vol. 10, N 4-5, pp 327-336.

THE DISCOVERY OF SOLAR-LIKE ACTIVITY CYCLES BEYOND THE END OF THE MAIN SEQUENCE?

Energy Technology Data Exchange (ETDEWEB)

Route, Matthew, E-mail: mroute@purdue.edu [Research Computing, Information Technology at Purdue, Purdue University, 155 S. Grant Street, West Lafayette, IN 47907 (United States)

2016-10-20

The long-term magnetic behavior of objects near the cooler end of the stellar main sequence is poorly understood. Most theoretical work on the generation of magnetism in these ultracool dwarfs (spectral type ≥M7 stars and brown dwarfs) suggests that their magnetic fields should not change in strength and direction. Using polarized radio emission measurements of their magnetic field orientations, I demonstrate that these cool, low-mass, fully convective objects appear to undergo magnetic polarity reversals analogous to those that occur on the Sun. This powerful new technique potentially indicates that the patterns of magnetic activity displayed by the Sun continue to exist, despite the fully convective interiors of these objects, in contravention of several leading theories of the generation of magnetic fields by internal dynamos.

Overlapping genomic sequences: a treasure trove of single-nucleotide polymorphisms.

Science.gov (United States)

Taillon-Miller, P; Gu, Z; Li, Q; Hillier, L; Kwok, P Y

1998-07-01

An efficient strategy to develop a dense set of single-nucleotide polymorphism (SNP) markers is to take advantage of the human genome sequencing effort currently under way. Our approach is based on the fact that bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) used in long-range sequencing projects come from diploid libraries. If the overlapping clones sequenced are from different lineages, one is comparing the sequences from 2 homologous chromosomes in the overlapping region. We have analyzed in detail every SNP identified while sequencing three sets of overlapping clones found on chromosome 5p15.2, 7q21-7q22, and 13q12-13q13. In the 200.6 kb of DNA sequence analyzed in these overlaps, 153 SNPs were identified. Computer analysis for repetitive elements and suitability for STS development yielded 44 STSs containing 68 SNPs for further study. All 68 SNPs were confirmed to be present in at least one of the three (Caucasian, African-American, Hispanic) populations studied. Furthermore, 42 of the SNPs tested (62%) were informative in at least one population, 32 (47%) were informative in two or more populations, and 23 (34%) were informative in all three populations. These results clearly indicate that developing SNP markers from overlapping genomic sequence is highly efficient and cost effective, requiring only the two simple steps of developing STSs around the known SNPs and characterizing them in the appropriate populations.

A resource of genome-wide single-nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

DEFF Research Database (Denmark)

Pujolar, J.M.; Jacobsen, M.W.; Frydenberg, J.

2013-01-01

Reduced representation genome sequencing such as restriction-site-associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single-nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the Eu...... 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome...

Supporting end-to-end resource virtualization for Web 2.0 applications using Service Oriented Architecture

NARCIS (Netherlands)

Papagianni, C.; Karagiannis, Georgios; Tselikas, N. D.; Sfakianakis, E.; Chochliouros, I. P.; Kabilafkas, D.; Cinkler, T.; Westberg, L.; Sjödin, P.; Hidell, M.; Heemstra de Groot, S.M.; Kontos, T.; Katsigiannis, C.; Pappas, C.; Antonakopoulou, A.; Venieris, I.S.

2008-01-01

In recent years, technologies have been introduced offering a large amount of computing and networking resources. New applications such as Google AdSense and BitTorrent can profit from the use of these resources. An efficient way of discovering and reserving these resources is by using the Service

Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C receptor with a short amino-terminal domain

DEFF Research Database (Denmark)

Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

2000-01-01

Query of GenBank with the amino acid sequence of human metabotropic glutamate receptor subtype 2 (mGluR2) identified a predicted gene product of unknown function on BAC clone CIT987SK-A-69G12 (located on chromosome band 16p12) as a homologous protein. The transcript, entitled GPRC5B, was cloned f...... from an expressed sequence tag clone that contained the entire open reading frame of the transcript encoding a protein of 395 amino acids. Analysis of the protein sequence reveal that GPRC5B contains a signal peptide and seven transmembrane alpha-helices, which is a hallmark of G...

System for Informatics in the Molecular Pathology Laboratory: An Open-Source End-to-End Solution for Next-Generation Sequencing Clinical Data Management.

Science.gov (United States)

Kang, Wenjun; Kadri, Sabah; Puranik, Rutika; Wurst, Michelle N; Patil, Sushant A; Mujacic, Ibro; Benhamed, Sonia; Niu, Nifang; Zhen, Chao Jie; Ameti, Bekim; Long, Bradley C; Galbo, Filipo; Montes, David; Iracheta, Crystal; Gamboa, Venessa L; Lopez, Daisy; Yourshaw, Michael; Lawrence, Carolyn A; Aisner, Dara L; Fitzpatrick, Carrie; McNerney, Megan E; Wang, Y Lynn; Andrade, Jorge; Volchenboum, Samuel L; Furtado, Larissa V; Ritterhouse, Lauren L; Segal, Jeremy P

2018-04-24

Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive. Herein, we present the System for Informatics in the Molecular Pathology Laboratory, a free and open-source Laboratory Information System/Laboratory Information Management System for academic and nonprofit molecular pathology NGS laboratories, developed at the Genomic and Molecular Pathology Division at the University of Chicago Medicine. The System for Informatics in the Molecular Pathology Laboratory was designed as a modular end-to-end information system to handle all stages of the NGS laboratory workload from test order to reporting. We describe the features of the system, its clinical validation at the Genomic and Molecular Pathology Division at the University of Chicago Medicine, and its installation and testing within a different academic center laboratory (University of Colorado), and we propose a platform for future community co-development and interlaboratory data sharing. Copyright © 2018. Published by Elsevier Inc.

Sifting through genomes with iterative-sequence clustering produces a large, phylogenetically diverse protein-family resource.

Science.gov (United States)

Sharpton, Thomas J; Jospin, Guillaume; Wu, Dongying; Langille, Morgan G I; Pollard, Katherine S; Eisen, Jonathan A

2012-10-13

New computational resources are needed to manage the increasing volume of biological data from genome sequencing projects. One fundamental challenge is the ability to maintain a complete and current catalog of protein diversity. We developed a new approach for the identification of protein families that focuses on the rapid discovery of homologous protein sequences. We implemented fully automated and high-throughput procedures to de novo cluster proteins into families based upon global alignment similarity. Our approach employs an iterative clustering strategy in which homologs of known families are sifted out of the search for new families. The resulting reduction in computational complexity enables us to rapidly identify novel protein families found in new genomes and to perform efficient, automated updates that keep pace with genome sequencing. We refer to protein families identified through this approach as "Sifting Families," or SFams. Our analysis of ~10.5 million protein sequences from 2,928 genomes identified 436,360 SFams, many of which are not represented in other protein family databases. We validated the quality of SFam clustering through statistical as well as network topology-based analyses. We describe the rapid identification of SFams and demonstrate how they can be used to annotate genomes and metagenomes. The SFam database catalogs protein-family quality metrics, multiple sequence alignments, hidden Markov models, and phylogenetic trees. Our source code and database are publicly available and will be subject to frequent updates (http://edhar.genomecenter.ucdavis.edu/sifting_families/).

Radiation hybrid maps of the D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes.

Science.gov (United States)

Kumar, Ajay; Seetan, Raed; Mergoum, Mohamed; Tiwari, Vijay K; Iqbal, Muhammad J; Wang, Yi; Al-Azzam, Omar; Šimková, Hana; Luo, Ming-Cheng; Dvorak, Jan; Gu, Yong Q; Denton, Anne; Kilian, Andrzej; Lazo, Gerard R; Kianian, Shahryar F

2015-10-16

The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay. In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii. A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average

Sequence assembly

DEFF Research Database (Denmark)

Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

2009-01-01

Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

Mobilization and integration of bacterial phenotypic data-Enabling next generation biodiversity analysis through the BacDive metadatabase.

Science.gov (United States)

Reimer, Lorenz C; Söhngen, Carola; Vetcininova, Anna; Overmann, Jörg

2017-11-10

Microbial data and metadata are scattered throughout the scientific literature, databases and unpublished lab notes and thereby often are difficult to access. Hot spots of (meta)data are internal descriptions of culture collections and initial descriptions of novel taxa in primary literature. Here we describe three exemplary mobilization projects which yielded metadata published through the prokaryotic metadatabase BacDive. The Reichenbach collection of myxobacteria includes information on 12,535 typewritten index cards which were digitized. A total of 37,156 data points were extracted by text mining. In the second mobilization project, Analytical Profile Index (API) tests on paper forms were targeted. Overall 6820 API tests were digitized, which provide physiological data of 4524 microbial strains. Thirdly, the extraction of metadata from 523 new species descriptions of the International Journal of Systematic and Evolutionary Microbiology, yielding 35,651 data points, is described. All data sets were integrated and published in BacDive. Thereby these metadata not only became accessible and searchable but were also linked to strain taxonomy, isolation source, cultivation condition, and molecular biology data. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of the Azotobacter vinelandii Poly-β-Hydroxybutyrate Biosynthetic phbBAC Operon Is Driven by Two Overlapping Promoters and Is Dependent on the Transcriptional Activator PhbR

OpenAIRE

Peralta-Gil, Martín; Segura, Daniel; Guzmán, Josefina; Servín-González, Luis; Espín, Guadalupe

2002-01-01

The Azotobacter vinelandii phbBAC genes encode the enzymes for poly-β-hydroxybutyrate (PHB) synthesis. The phbR gene, which is located upstream of and in the opposite direction of phbBAC, encodes PhbR, a transcriptional activator which is a member of the AraC family of activators. Here we report that a mutation in phbR reduced PHB accumulation and transcription of a phbB-lacZ fusion. We also report that phbB is transcribed from two overlapping promoters, pB1 and pB2. The region corresponding ...

Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

Directory of Open Access Journals (Sweden)

Martin Beránek

2012-01-01

Full Text Available Dideoxynucleotide DNA sequencing is one of the principal procedures in molecular biology. Loss of an initial part of nucleotides behind the 3' end of the sequencing primer limits the readability of sequenced amplicons. We present a method which extends the readability by using sequencing primers modified by polyadenylated tails attached to their 5' ends. Performing a polymerase chain reaction, we amplified eight amplicons of six human genes (AMELX, APOE, HFE, MBL2, SERPINA1 and TGFB1 ranging from 106 bp to 680 bp. Polyadenylation of the sequencing primers minimized the loss of bases in all amplicons. Complete sequences of shorter products (AMELX 106 bp, SERPINA1 121 bp, HFE 208 bp, APOE 244 bp, MBL2 317 bp were obtained. In addition, in the case of TGFB1 products (366 bp, 432 bp, and 680 bp, respectively, the lengths of sequencing readings were significantly longer if adenylated primers were used. Thus, single strand dideoxynucleotide sequencing with adenylated primers enables complete or near complete readability of short PCR amplicons.

Gene : CBRC-HSAP-06-0003 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-HSAP-06-0003 Novel 6 D UNKNOWN CN165_HUMAN 2e-21 63% dbj|BAC85887.1| unnamed protein product [Homo sapi...ens] 7e-35 68% gnl|UG|Hs#S28297950 DA109160 BRACE3 Homo sapiens cDNA clone BRACE302...5623 5', mRNA sequence /clone=BRACE3025623 /clone_end=5' /gb=DA109160 /gi=80475782 /ug=Hs.690917 /len=598 1e-39 69% MAPI...YRHMPPYPTNFCIFSRDRLSLCWPGWSPFLISGDPAALVSQSAGITGVSHGTWPFDCFSSFELGLFLPPHSPIAILEILGYQILPGQVSAPIDAYSNAVPFSHGF ...

The genome of the pear (Pyrus bretschneideri Rehd.)

DEFF Research Database (Denmark)

Wu, Jun; Wang, Zhiwen; Shi, Zebin

2013-01-01

The draft genome of the pear (Pyrus bretschneideri) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194× coverage. High-density genetic maps ...

30 CFR 57.19028 - End attachment replacement.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false End attachment replacement. 57.19028 Section 57.19028 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Hoisting Wire Ropes § 57.19028 End attachment replacement. Wire rope attachments shall be replaced when...

30 CFR 56.19028 - End attachment replacement.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false End attachment replacement. 56.19028 Section 56.19028 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Wire Ropes § 56.19028 End attachment replacement. Wire rope attachments shall be replaced when cracked...

30 CFR 77.1438 - End attachment replacement.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false End attachment replacement. 77.1438 Section 77.1438 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND... Personnel Hoisting Wire Ropes § 77.1438 End attachment replacement. Wire rope attachments shall be replaced...

Evaluation of BacT/Alert 3D Liquid Culture System for Recovery of Mycobacteria from Clinical Specimens Using Sodium Dodecyl (Lauryl) Sulfate-NaOH Decontamination

Science.gov (United States)

Carricajo, A.; Fonsale, N.; Vautrin, A. C.; Aubert, G.

2001-01-01

A total of 52 mycobacterial isolates were recovered from 1,197 clinical specimens decontaminated by a sodium dodecyl (lauryl) sulfate (SDS)-NaOH protocol. Of these, 94% were recovered with the BacT/Alert 3D system (Organon Teknika, Durham, N.C.) and 79% were recovered on Löwenstein-Jensen (LJ) medium. Mean times to detection of organisms of the Mycobacterium tuberculosis complex (n = 47) were 22.8 days with LJ medium and 16.2 days with the system. The BacT/Alert 3D system is a rapid and efficient detection system which can be used with an SDS-NaOH decontamination procedure. PMID:11574623

Enriching Genomic Resources and Marker Development from Transcript Sequences of Jatropha curcas for Microgravity Studies

Science.gov (United States)

Tian, Wenlan; Paudel, Dev

2017-01-01

Jatropha (Jatropha curcas L.) is an economically important species with a great potential for biodiesel production. To enrich the jatropha genomic databases and resources for microgravity studies, we sequenced and annotated the transcriptome of jatropha and developed SSR and SNP markers from the transcriptome sequences. In total 1,714,433 raw reads with an average length of 441.2 nucleotides were generated. De novo assembling and clustering resulted in 115,611 uniquely assembled sequences (UASs) including 21,418 full-length cDNAs and 23,264 new jatropha transcript sequences. The whole set of UASs were fully annotated, out of which 59,903 (51.81%) were assigned with gene ontology (GO) term, 12,584 (10.88%) had orthologs in Eukaryotic Orthologous Groups (KOG), and 8,822 (7.63%) were mapped to 317 pathways in six different categories in Kyoto Encyclopedia of Genes and Genome (KEGG) database, and it contained 3,588 putative transcription factors. From the UASs, 9,798 SSRs were discovered with AG/CT as the most frequent (45.8%) SSR motif type. Further 38,693 SNPs were detected and 7,584 remained after filtering. This UAS set has enriched the current jatropha genomic databases and provided a large number of genetic markers, which can facilitate jatropha genetic improvement and many other genetic and biological studies. PMID:28154822

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

Science.gov (United States)

Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.

2017-09-01

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., 60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. [Figure not available: see fulltext.

Sifting through genomes with iterative-sequence clustering produces a large, phylogenetically diverse protein-family resource

Directory of Open Access Journals (Sweden)

Sharpton Thomas J

2012-10-01

Full Text Available Abstract Background New computational resources are needed to manage the increasing volume of biological data from genome sequencing projects. One fundamental challenge is the ability to maintain a complete and current catalog of protein diversity. We developed a new approach for the identification of protein families that focuses on the rapid discovery of homologous protein sequences. Results We implemented fully automated and high-throughput procedures to de novo cluster proteins into families based upon global alignment similarity. Our approach employs an iterative clustering strategy in which homologs of known families are sifted out of the search for new families. The resulting reduction in computational complexity enables us to rapidly identify novel protein families found in new genomes and to perform efficient, automated updates that keep pace with genome sequencing. We refer to protein families identified through this approach as “Sifting Families,” or SFams. Our analysis of ~10.5 million protein sequences from 2,928 genomes identified 436,360 SFams, many of which are not represented in other protein family databases. We validated the quality of SFam clustering through statistical as well as network topology–based analyses. Conclusions We describe the rapid identification of SFams and demonstrate how they can be used to annotate genomes and metagenomes. The SFam database catalogs protein-family quality metrics, multiple sequence alignments, hidden Markov models, and phylogenetic trees. Our source code and database are publicly available and will be subject to frequent updates (http://edhar.genomecenter.ucdavis.edu/sifting_families/.

BACS: The Brussels Artificial Character Sets for studies in cognitive psychology and neuroscience.

Science.gov (United States)

Vidal, Camille; Content, Alain; Chetail, Fabienne

2017-12-01

Written symbols such as letters have been used extensively in cognitive psychology, whether to understand their contributions to written word recognition or to examine the processes involved in other mental functions. Sometimes, however, researchers want to manipulate letters while removing their associated characteristics. A powerful solution to do so is to use new characters, devised to be highly similar to letters, but without the associated sound or name. Given the growing use of artificial characters in experimental paradigms, the aim of the present study was to make available the Brussels Artificial Character Sets (BACS): two full, strictly controlled, and portable sets of artificial characters for a broad range of experimental situations.

BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors

International Nuclear Information System (INIS)

Samuelson, Emma; Karlsson, Sara; Partheen, Karolina; Nilsson, Staffan; Szpirer, Claude; Behboudi, Afrouz

2012-01-01

Development of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis. Mammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform. Tumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors. Some of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic

Toxicity and genotoxicity of the quaternary ammonium compound benzalkonium chloride (BAC) using Daphnia magna and Ceriodaphnia dubia as model systems

International Nuclear Information System (INIS)

Lavorgna, Margherita; Russo, Chiara; D'Abrosca, Brigida; Parrella, Alfredo; Isidori, Marina

2016-01-01

The toxicity and genotoxicity of the cationic surfactant benzalkonium chloride (BAC) were studied using Daphnia magna and Ceriodaphnia dubia as model systems. Acute and chronic toxicity testing were performed according to the international standard guidelines and the genotoxicity was detected through the comet assay on cells from whole organisms in vivo exposed. Acute effects occurred at concentrations in the order of tens of μg/L in D. magna and hundreds of μg/L in C. dubia. Chronic effects were found at one order of magnitude less than short-term effects maintaining the same difference in sensitivity between D. magna and C. dubia. BAC induced relevant DNA damage, in both cladocerans; the lowest adverse effect levels were 0.4 and 4 ng/L for D. magna and C. dubia, respectively. As these effective concentrations are far lower than BAC occurrence in surface waters (units of μg/L) a concerning environmental risk cannot be excluded. The findings of this study showed that D. magna and C. dubia, could be used as model organisms to detect acute and chronic toxicity as well as genotoxicity at the whole organism level. - Highlights: • Benzalkonium chloride chronic effect in C. dubia was found at dozens of μg/L. • The LOAEC detected by comet assay in D. magna is in the order of hundreds of pg/L. • D. magna and C. dubia are useful model organisms to detect toxicity and genotoxicity. - Benzalkonium chloride showed chronic toxicity and genotoxicity in Daphnia magna and Ceriodaphnia dubia at concentrations of environmental concern. Daphnids are useful model organisms.

Retrospective studies of end-of-life resource utilization and costs in cancer care using health administrative data: a systematic review.

Science.gov (United States)

Langton, Julia M; Blanch, Bianca; Drew, Anna K; Haas, Marion; Ingham, Jane M; Pearson, Sallie-Anne

2014-12-01

There has been an increase in observational studies using health administrative data to examine the nature, quality, and costs of care at life's end, particularly in cancer care. To synthesize retrospective observational studies on resource utilization and/or costs at the end of life in cancer patients. We also examine the methods and outcomes of studies assessing the quality of end-of-life care. A systematic review according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and AMSTAR (A Measurement Tool to Assess Systematic Reviews) methodology. We searched MEDLINE, Embase, CINAHL, and York Centre for Research and Dissemination (1990-2011). Independent reviewers screened abstracts of 14,424 articles, and 835 full-text manuscripts were further reviewed. Inclusion criteria were English-language; at least one resource utilization or cost outcome in adult cancer decedents with solid tumors; outcomes derived from health administrative data; and an exclusive end-of-life focus. We reviewed 78 studies examining end-of-life care in over 3.7 million cancer decedents; 33 were published since 2008. We observed exponential increases in service use and costs as death approached; hospital services being the main cost driver. Palliative services were relatively underutilized and associated with lower expenditures than hospital-based care. The 15 studies using quality indicators demonstrated that up to 38% of patients receive chemotherapy or life-sustaining treatments in the last month of life and up to 66% do not receive hospice/palliative services. Observational studies using health administrative data have the potential to drive evidence-based palliative care practice and policy. Further development of quality care markers will enhance benchmarking activities across health care jurisdictions, providers, and patient populations. © The Author(s) 2014.

The soybean-Phytophthora resistance locus Rps1-k encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive sequences

Directory of Open Access Journals (Sweden)

Bhattacharyya Madan K

2008-03-01

Full Text Available Abstract Background A series of Rps (resistance to Pytophthora sojae genes have been protecting soybean from the root and stem rot disease caused by the Oomycete pathogen, Phytophthora sojae. Five Rps genes were mapped to the Rps1 locus located near the 28 cM map position on molecular linkage group N of the composite genetic soybean map. Among these five genes, Rps1-k was introgressed from the cultivar, Kingwa. Rps1-k has been providing stable and broad-spectrum Phytophthora resistance in the major soybean-producing regions of the United States. Rps1-k has been mapped and isolated. More than one functional Rps1-k gene was identified from the Rps1-k locus. The clustering feature at the Rps1-k locus might have facilitated the expansion of Rps1-k gene numbers and the generation of new recognition specificities. The Rps1-k region was sequenced to understand the possible evolutionary steps that shaped the generation of Phytophthora resistance genes in soybean. Results Here the analyses of sequences of three overlapping BAC clones containing the 184,111 bp Rps1-k region are reported. A shotgun sequencing strategy was applied in sequencing the BAC contig. Sequence analysis predicted a few full-length genes including two Rps1-k genes, Rps1-k-1 and Rps1-k-2. Previously reported Rps1-k-3 from this genomic region 1 was evolved through intramolecular recombination between Rps1-k-1 and Rps1-k-2 in Escherichia coli. The majority of the predicted genes are truncated and therefore most likely they are nonfunctional. A member of a highly abundant retroelement, SIRE1, was identified from the Rps1-k region. The Rps1-k region is primarily composed of repetitive sequences. Sixteen simple repeat and 63 tandem repeat sequences were identified from the locus. Conclusion These data indicate that the Rps1 locus is located in a gene-poor region. The abundance of repetitive sequences in the Rps1-k region suggested that the location of this locus is in or near a

Analysis of Multiple Genomic Sequence Alignments: A Web Resource, Online Tools, and Lessons Learned From Analysis of Mammalian SCL Loci

Science.gov (United States)

Chapman, Michael A.; Donaldson, Ian J.; Gilbert, James; Grafham, Darren; Rogers, Jane; Green, Anthony R.; Göttgens, Berthold

2004-01-01

Comparative analysis of genomic sequences is becoming a standard technique for studying gene regulation. However, only a limited number of tools are currently available for the analysis of multiple genomic sequences. An extensive data set for the testing and training of such tools is provided by the SCL gene locus. Here we have expanded the data set to eight vertebrate species by sequencing the dog SCL locus and by annotating the dog and rat SCL loci. To provide a resource for the bioinformatics community, all SCL sequences and functional annotations, comprising a collation of the extensive experimental evidence pertaining to SCL regulation, have been made available via a Web server. A Web interface to new tools specifically designed for the display and analysis of multiple sequence alignments was also implemented. The unique SCL data set and new sequence comparison tools allowed us to perform a rigorous examination of the true benefits of multiple sequence comparisons. We demonstrate that multiple sequence alignments are, overall, superior to pairwise alignments for identification of mammalian regulatory regions. In the search for individual transcription factor binding sites, multiple alignments markedly increase the signal-to-noise ratio compared to pairwise alignments. PMID:14718377

A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity.

Science.gov (United States)

Hobbs, Matthew; Pavasovic, Ana; King, Andrew G; Prentis, Peter J; Eldridge, Mark D B; Chen, Zhiliang; Colgan, Donald J; Polkinghorne, Adam; Wilkins, Marc R; Flanagan, Cheyne; Gillett, Amber; Hanger, Jon; Johnson, Rebecca N; Timms, Peter

2014-09-11

dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org.

Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector

Directory of Open Access Journals (Sweden)

Riccardo De Santis

2018-05-01

Full Text Available Human pluripotent stem cells (PSCs are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP. The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions. Keywords: Spinal motor neuron, Cranial motor neuron, Induced pluripotent stem cells, Amyotrophic lateral sclerosis, Phox2a, piggyBac

BACs-on-Beads Technology: A Reliable Test for Rapid Detection of Aneuploidies and Microdeletions in Prenatal Diagnosis

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Sandra García-Herrero

2014-01-01

Full Text Available The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF or chorionic villus (CV samples based on BACs-on-Beads (BoBs technology and to compare the results with classical karyotyping by Giemsa banding (G-banding of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.

Desarrollo de un modelo de evaluación financiera de proyectos para BAC Credomatic Network

OpenAIRE

Sandí Piedra, Allan Adolfo

2016-01-01

Universidad de Costa Rica. Posgrado en Administración y Dirección de Empresas. Maestría Profesional en Administración y Dirección de Empresas con énfasis en Finanzas, 2016 El presente trabajo final de investigación contiene los aspectos que fundamentan un modelo de Evaluación Financiera de Proyectos que permite solventar la problemática identificada en BAC Credomatic Network respecto al limitado valor agregado del proceso existente de valoración financiera de los proyectos de la empresa. P...

Brain activation during anticipation of sound sequences.

Science.gov (United States)

Leaver, Amber M; Van Lare, Jennifer; Zielinski, Brandon; Halpern, Andrea R; Rauschecker, Josef P

2009-02-25

Music consists of sound sequences that require integration over time. As we become familiar with music, associations between notes, melodies, and entire symphonic movements become stronger and more complex. These associations can become so tight that, for example, hearing the end of one album track can elicit a robust image of the upcoming track while anticipating it in total silence. Here, we study this predictive "anticipatory imagery" at various stages throughout learning and investigate activity changes in corresponding neural structures using functional magnetic resonance imaging. Anticipatory imagery (in silence) for highly familiar naturalistic music was accompanied by pronounced activity in rostral prefrontal cortex (PFC) and premotor areas. Examining changes in the neural bases of anticipatory imagery during two stages of learning conditional associations between simple melodies, however, demonstrates the importance of fronto-striatal connections, consistent with a role of the basal ganglia in "training" frontal cortex (Pasupathy and Miller, 2005). Another striking change in neural resources during learning was a shift between caudal PFC earlier to rostral PFC later in learning. Our findings regarding musical anticipation and sound sequence learning are highly compatible with studies of motor sequence learning, suggesting common predictive mechanisms in both domains.

Automatic provisioning of end-to-end QoS into the home

DEFF Research Database (Denmark)

Brewka, Lukasz Jerzy; Skoldström, Pontus; Nelis, Jelle

2011-01-01

Due to a growing number of high bandwidth applications today (such as HDTV), and an increasing amount of network and cloud based applications, service providers need to pay attention to QoS in their networks. We believe there is a need for an end-to-end approach reaching into the home as well....... The Home Gateway (HG) as a key component of the home network is crucial for enabling the end-to-end solutions. UPnP-QoS has been proposed as an inhome solution for resource reservations. In this paper we assess a solution for automatic QoS reservations, on behalf of non-UPnP-QoS aware applications....... Additionally we focus on an integrated end-to-end solution, combining GMPLS-based reservations in e.g., access/metro and UPnP-QoS based reservation in the home network....

Analyses of Hypomethylated Oil Palm Gene Space

Science.gov (United States)

Jayanthi, Nagappan; Mohd-Amin, Ab Halim; Azizi, Norazah; Chan, Kuang-Lim; Maqbool, Nauman J.; Maclean, Paul; Brauning, Rudi; McCulloch, Alan; Moraga, Roger; Ong-Abdullah, Meilina; Singh, Rajinder

2014-01-01

Demand for palm oil has been increasing by an average of ∼8% the past decade and currently accounts for about 59% of the world's vegetable oil market. This drives the need to increase palm oil production. Nevertheless, due to the increasing need for sustainable production, it is imperative to increase productivity rather than the area cultivated. Studies on the oil palm genome are essential to help identify genes or markers that are associated with important processes or traits, such as flowering, yield and disease resistance. To achieve this, 294,115 and 150,744 sequences from the hypomethylated or gene-rich regions of Elaeis guineensis and E. oleifera genome were sequenced and assembled into contigs. An additional 16,427 shot-gun sequences and 176 bacterial artificial chromosomes (BAC) were also generated to check the quality of libraries constructed. Comparison of these sequences revealed that although the methylation-filtered libraries were sequenced at low coverage, they still tagged at least 66% of the RefSeq supported genes in the BAC and had a filtration power of at least 2.0. A total 33,752 microsatellites and 40,820 high-quality single nucleotide polymorphism (SNP) markers were identified. These represent the most comprehensive collection of microsatellites and SNPs to date and would be an important resource for genetic mapping and association studies. The gene models predicted from the assembled contigs were mined for genes of interest, and 242, 65 and 14 oil palm transcription factors, resistance genes and miRNAs were identified respectively. Examples of the transcriptional factors tagged include those associated with floral development and tissue culture, such as homeodomain proteins, MADS, Squamosa and Apetala2. The E. guineensis and E. oleifera hypomethylated sequences provide an important resource to understand the molecular mechanisms associated with important agronomic traits in oil palm. PMID:24497974

A genome sequence resource for the aye-aye (Daubentonia madagascariensis), a nocturnal lemur from Madagascar.

Science.gov (United States)

Perry, George H; Reeves, Darryl; Melsted, Páll; Ratan, Aakrosh; Miller, Webb; Michelini, Katelyn; Louis, Edward E; Pritchard, Jonathan K; Mason, Christopher E; Gilad, Yoav

2012-01-01

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ~3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ~2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ~10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage-a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests.

Proceso para transferir el conocimiento en la adquisición de software en la Gerencia de Tecnologías de Información del BAC San José

OpenAIRE

Castillo Rivera, Roberto

2016-01-01

Proyecto final de graduación, Maestría profesional El presente proyecto es el resultado de la aplicación de las mejores prácticas tomando como base la metodología de ITIL en su versión 3 para la liberación final de productos de software, específicamente en cuanto a la transferencia del conocimiento obtenido durante el proceso de adquisición de software aplicativo en la Gerencia de Tecnologías de Información del Banco BAC San José. El Banco BAC San José es una entidad financiera privada,...

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

Science.gov (United States)

Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

2015-10-29

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

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Gong Shiaochin

2009-03-01

Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

Science.gov (United States)

Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

2009-03-13

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

Analyzing AbrB-Knockout Effects through Genome and Transcriptome Sequencing of Bacillus licheniformis DW2

Science.gov (United States)

Shu, Cheng-Cheng; Wang, Dong; Guo, Jing; Song, Jia-Ming; Chen, Shou-Wen; Chen, Ling-Ling; Gao, Jun-Xiang

2018-01-01

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (ΔAbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102∼4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ΔAbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ΔAbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ΔAbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression. PMID:29599755

Weighted-DESYNC and Its Application to End-to-End Throughput Fairness in Wireless Multihop Network

Directory of Open Access Journals (Sweden)

Ui-Seong Yu

2017-01-01

Full Text Available The end-to-end throughput of a routing path in wireless multihop network is restricted by a bottleneck node that has the smallest bandwidth among the nodes on the routing path. In this study, we propose a method for resolving the bottleneck-node problem in multihop networks, which is based on multihop DESYNC (MH-DESYNC algorithm that is a bioinspired resource allocation method developed for use in multihop environments and enables fair resource allocation among nearby (up to two hops neighbors. Based on MH-DESYNC, we newly propose weighted-DESYNC (W-DESYNC as a tool artificially to control the amount of resource allocated to the specific user and thus to achieve throughput fairness over a routing path. Proposed W-DESYNC employs the weight factor of a link to determine the amount of bandwidth allocated to a node. By letting the weight factor be the link quality of a routing path and making it the same across a routing path via Cucker-Smale flocking model, we can obtain throughput fairness over a routing path. The simulation results show that the proposed algorithm achieves throughput fairness over a routing path and can increase total end-to-end throughput in wireless multihop networks.

Novel porcine repetitive elements

Directory of Open Access Journals (Sweden)

Nonneman Dan J

2006-12-01

Full Text Available Abstract Background Repetitive elements comprise ~45% of mammalian genomes and are increasingly known to impact genomic function by contributing to the genomic architecture, by direct regulation of gene expression and by affecting genomic size, diversity and evolution. The ubiquity and increasingly understood importance of repetitive elements contribute to the need to identify and annotate them. We set out to identify previously uncharacterized repetitive DNA in the porcine genome. Once found, we characterized the prevalence of these repeats in other mammals. Results We discovered 27 repetitive elements in 220 BACs covering 1% of the porcine genome (Comparative Vertebrate Sequencing Initiative; CVSI. These repeats varied in length from 55 to 1059 nucleotides. To estimate copy numbers, we went to an independent source of data, the BAC-end sequences (Wellcome Trust Sanger Institute, covering approximately 15% of the porcine genome. Copy numbers in BAC-ends were less than one hundred for 6 repeat elements, between 100 and 1000 for 16 and between 1,000 and 10,000 for 5. Several of the repeat elements were found in the bovine genome and we have identified two with orthologous sites, indicating that these elements were present in their common ancestor. None of the repeat elements were found in primate, rodent or dog genomes. We were unable to identify any of the replication machinery common to active transposable elements in these newly identified repeats. Conclusion The presence of both orthologous and non-orthologous sites indicates that some sites existed prior to speciation and some were generated later. The identification of low to moderate copy number repetitive DNA that is specific to artiodactyls will be critical in the assembly of livestock genomes and studies of comparative genomics.

De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

Science.gov (United States)

2013-01-01

Background Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. Results The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. Conclusion The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration. PMID:23815514

Detection of an inversion in the Ty-2 region between S. lycopersicum and S. habrochaites by a combination of de novo genome assembly and BAC cloning.

Science.gov (United States)

Wolters, Anne-Marie A; Caro, Myluska; Dong, Shufang; Finkers, Richard; Gao, Jianchang; Visser, Richard G F; Wang, Xiaoxuan; Du, Yongchen; Bai, Yuling

2015-10-01

A chromosomal inversion associated with the tomato Ty - 2 gene for TYLCV resistance is the cause of severe suppression of recombination in a tomato Ty - 2 introgression line. Among tomato and its wild relatives inversions are often observed, which result in suppression of recombination. Such inversions hamper the transfer of important traits from a related species to the crop by introgression breeding. Suppression of recombination was reported for the TYLCV resistance gene, Ty-2, which has been introgressed in cultivated tomato (Solanum lycopersicum) from the wild relative S. habrochaites accession B6013. Ty-2 was mapped to a 300-kb region on the long arm of chromosome 11. The suppression of recombination in the Ty-2 region could be caused by chromosomal rearrangements in S. habrochaites compared with S. lycopersicum. With the aim of visualizing the genome structure of the Ty-2 region, we compared the draft de novo assembly of S. habrochaites accession LYC4 with the sequence of cultivated tomato ('Heinz'). Furthermore, using populations derived from intraspecific crosses of S. habrochaites accessions, the order of markers in the Ty-2 region was studied. Results showed the presence of an inversion of approximately 200 kb in the Ty-2 region when comparing S. lycopersicum and S. habrochaites. By sequencing a BAC clone from the Ty-2 introgression line, one inversion breakpoint was identified. Finally, the obtained results are discussed with respect to introgression breeding and the importance of a priori de novo sequencing of the species involved.

De novo assembly and phasing of a Korean human genome.

Science.gov (United States)

Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

2016-10-13

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of

Deletion of the budBAC operon in Klebsiella pneumoniae to understand the physiological role of 2,3-butanediol biosynthesis.

Science.gov (United States)

Jeong, Daun; Yang, Jeongmo; Lee, Soojin; Kim, Borim; Um, Youngsoon; Kim, Youngrok; Ha, Kyoung-Su; Lee, Jinwon

2016-05-18

Klebsiella pneumoniae is known to produce 2,3-butanediol (2,3-BDO), a valuable chemical. In K. pneumoniae, the 2,3-BDO operon (budBAC) is involved in the production of 2,3-BDO. To observe the physiological role of the 2,3-BDO operon in a mixed acid fermentation, we constructed a budBAC-deleted strain (SGSB109). The production of extracellular metabolites, CO2 emission, carbon distribution, and NADH/NAD(+) balance of SGSB109 were compared with the parent strain (SGSB100). When comparing the carbon distribution at 15 hr, four significant differences were observed: in 2,3-BDO biosynthesis, lactate and acetate production (lactate and acetate production increased 2.3-fold and 4.1-fold in SGSB109 compared to SGSB100), CO2 emission (higher in SGSB100), and carbon substrate uptake (higher in SGSB100). Previous studies on the inactivation of the 2,3-BDO operon were focused on the increase of 1,3-propanediol production. Few studies have been done observing the role of 2,3-BDO biosynthesis. This study provides a prime insight into the role of 2,3-BDO biosynthesis of K. pneumoniae.

Overlapping Genomic Sequences: A Treasure Trove of Single-Nucleotide Polymorphisms

Science.gov (United States)

Taillon-Miller, Patricia; Gu, Zhijie; Li, Qun; Hillier, LaDeana; Kwok, Pui-Yan

1998-01-01

An efficient strategy to develop a dense set of single-nucleotide polymorphism (SNP) markers is to take advantage of the human genome sequencing effort currently under way. Our approach is based on the fact that bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) used in long-range sequencing projects come from diploid libraries. If the overlapping clones sequenced are from different lineages, one is comparing the sequences from 2 homologous chromosomes in the overlapping region. We have analyzed in detail every SNP identified while sequencing three sets of overlapping clones found on chromosome 5p15.2, 7q21–7q22, and 13q12–13q13. In the 200.6 kb of DNA sequence analyzed in these overlaps, 153 SNPs were identified. Computer analysis for repetitive elements and suitability for STS development yielded 44 STSs containing 68 SNPs for further study. All 68 SNPs were confirmed to be present in at least one of the three (Caucasian, African-American, Hispanic) populations studied. Furthermore, 42 of the SNPs tested (62%) were informative in at least one population, 32 (47%) were informative in two or more populations, and 23 (34%) were informative in all three populations. These results clearly indicate that developing SNP markers from overlapping genomic sequence is highly efficient and cost effective, requiring only the two simple steps of developing STSs around the known SNPs and characterizing them in the appropriate populations. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AC003015 (for GS113423), AC002380 (GS330J10), AC000066 (RG293F11), AC003086 (RG104F04), AC002525 (257C22A), and U73331 (96A18A).] PMID:9685323

Optimizing End-to-End Big Data Transfers over Terabits Network Infrastructure

International Nuclear Information System (INIS)

Kim, Youngjae; Vallee, Geoffroy R.; Lee, Sangkeun; Shipman, Galen M.

2016-01-01

While future terabit networks hold the promise of significantly improving big-data motion among geographically distributed data centers, significant challenges must be overcome even on today's 100 gigabit networks to realize end-to-end performance. Multiple bottlenecks exist along the end-to-end path from source to sink, for instance, the data storage infrastructure at both the source and sink and its interplay with the wide-area network are increasingly the bottleneck to achieving high performance. In this study, we identify the issues that lead to congestion on the path of an end-to-end data transfer in the terabit network environment, and we present a new bulk data movement framework for terabit networks, called LADS. LADS exploits the underlying storage layout at each endpoint to maximize throughput without negatively impacting the performance of shared storage resources for other users. LADS also uses the Common Communication Interface (CCI) in lieu of the sockets interface to benefit from hardware-level zero-copy, and operating system bypass capabilities when available. It can further improve data transfer performance under congestion on the end systems using buffering at the source using flash storage. With our evaluations, we show that LADS can avoid congested storage elements within the shared storage resource, improving input/output bandwidth, and data transfer rates across the high speed networks. We also investigate the performance degradation problems of LADS due to I/O contention on the parallel file system (PFS), when multiple LADS tools share the PFS. We design and evaluate a meta-scheduler to coordinate multiple I/O streams while sharing the PFS, to minimize the I/O contention on the PFS. Finally, with our evaluations, we observe that LADS with meta-scheduling can further improve the performance by up to 14 percent relative to LADS without meta-scheduling.

Efficient identification of Y chromosome sequences in the human and Drosophila genomes

Science.gov (United States)

Carvalho, Antonio Bernardo; Clark, Andrew G.

2013-01-01

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes. PMID:23921660

Efficient identification of Y chromosome sequences in the human and Drosophila genomes.

Science.gov (United States)

Carvalho, Antonio Bernardo; Clark, Andrew G

2013-11-01

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.

Automated Design of Propellant-Optimal, End-to-End, Low-Thrust Trajectories for Trojan Asteroid Tours

Science.gov (United States)

Stuart, Jeffrey; Howell, Kathleen; Wilson, Roby

2013-01-01

The Sun-Jupiter Trojan asteroids are celestial bodies of great scientific interest as well as potential resources offering water and other mineral resources for longterm human exploration of the solar system. Previous investigations under this project have addressed the automated design of tours within the asteroid swarm. This investigation expands the current automation scheme by incorporating options for a complete trajectory design approach to the Trojan asteroids. Computational aspects of the design procedure are automated such that end-to-end trajectories are generated with a minimum of human interaction after key elements and constraints associated with a proposed mission concept are specified.

Leveraging long read sequencing from a single individual to provide a comprehensive resource for benchmarking variant calling methods.

Science.gov (United States)

Mu, John C; Tootoonchi Afshar, Pegah; Mohiyuddin, Marghoob; Chen, Xi; Li, Jian; Bani Asadi, Narges; Gerstein, Mark B; Wong, Wing H; Lam, Hugo Y K

2015-09-28

A high-confidence, comprehensive human variant set is critical in assessing accuracy of sequencing algorithms, which are crucial in precision medicine based on high-throughput sequencing. Although recent works have attempted to provide such a resource, they still do not encompass all major types of variants including structural variants (SVs). Thus, we leveraged the massive high-quality Sanger sequences from the HuRef genome to construct by far the most comprehensive gold set of a single individual, which was cross validated with deep Illumina sequencing, population datasets, and well-established algorithms. It was a necessary effort to completely reanalyze the HuRef genome as its previously published variants were mostly reported five years ago, suffering from compatibility, organization, and accuracy issues that prevent their direct use in benchmarking. Our extensive analysis and validation resulted in a gold set with high specificity and sensitivity. In contrast to the current gold sets of the NA12878 or HS1011 genomes, our gold set is the first that includes small variants, deletion SVs and insertion SVs up to a hundred thousand base-pairs. We demonstrate the utility of our HuRef gold set to benchmark several published SV detection tools.

The Technology Acceptance Model for Resource-Limited Settings (TAM-RLS): A Novel Framework for Mobile Health Interventions Targeted to Low-Literacy End-Users in Resource-Limited Settings.

Science.gov (United States)

Campbell, Jeffrey I; Aturinda, Isaac; Mwesigwa, Evans; Burns, Bridget; Santorino, Data; Haberer, Jessica E; Bangsberg, David R; Holden, Richard J; Ware, Norma C; Siedner, Mark J

2017-11-01

Although mobile health (mHealth) technologies have shown promise in improving clinical care in resource-limited settings (RLS), they are infrequently brought to scale. One limitation to the success of many mHealth interventions is inattention to end-user acceptability, which is an important predictor of technology adoption. We conducted in-depth interviews with 43 people living with HIV in rural Uganda who had participated in a clinical trial of a short messaging system (SMS)-based intervention designed to prompt return to clinic after an abnormal laboratory test. Interviews focused on established features of technology acceptance models, including perceived ease of use and perceived usefulness, and included open-ended questions to gain insight into unexplored issues related to the intervention's acceptability. We used conventional (inductive) and direct content analysis to derive categories describing use behaviors and acceptability. Interviews guided development of a proposed conceptual framework, the technology acceptance model for resource-limited settings (TAM-RLS). This framework incorporates both classic technology acceptance model categories as well as novel factors affecting use in this setting. Participants described how SMS message language, phone characteristics, and experience with similar technologies contributed to the system's ease of use. Perceived usefulness was shaped by the perception that the system led to augmented HIV care services and improved access to social support from family and colleagues. Emergent themes specifically related to mHealth acceptance among PLWH in Uganda included (1) the importance of confidentiality, disclosure, and stigma, and (2) the barriers and facilitators downstream from the intervention that impacted achievement of the system's target outcome. The TAM-RLS is a proposed model of mHealth technology acceptance based upon end-user experiences in rural Uganda. Although the proposed model requires validation, the TAM

Simultaneous identification of long similar substrings in large sets of sequences

Directory of Open Access Journals (Sweden)

Wittig Burghardt

2007-05-01

Full Text Available Abstract Background Sequence comparison faces new challenges today, with many complete genomes and large libraries of transcripts known. Gene annotation pipelines match these sequences in order to identify genes and their alternative splice forms. However, the software currently available cannot simultaneously compare sets of sequences as large as necessary especially if errors must be considered. Results We therefore present a new algorithm for the identification of almost perfectly matching substrings in very large sets of sequences. Its implementation, called ClustDB, is considerably faster and can handle 16 times more data than VMATCH, the most memory efficient exact program known today. ClustDB simultaneously generates large sets of exactly matching substrings of a given minimum length as seeds for a novel method of match extension with errors. It generates alignments of maximum length with a considered maximum number of errors within each overlapping window of a given size. Such alignments are not optimal in the usual sense but faster to calculate and often more appropriate than traditional alignments for genomic sequence comparisons, EST and full-length cDNA matching, and genomic sequence assembly. The method is used to check the overlaps and to reveal possible assembly errors for 1377 Medicago truncatula BAC-size sequences published at http://www.medicago.org/genome/assembly_table.php?chr=1. Conclusion The program ClustDB proves that window alignment is an efficient way to find long sequence sections of homogenous alignment quality, as expected in case of random errors, and to detect systematic errors resulting from sequence contaminations. Such inserts are systematically overlooked in long alignments controlled by only tuning penalties for mismatches and gaps. ClustDB is freely available for academic use.

EndNote 7.0

Directory of Open Access Journals (Sweden)

Eapen Bell

2006-01-01

Full Text Available EndNote is a useful software for online literature search and efficient bibliography management. It helps to format the bibliography according to the citation style of each journal. EndNote stores references in a library file, which can be shared with others. It can connect to online resources like PubMed and retrieve search results as per the search criteria. It can also effortlessly integrate with popular word processors like MS Word. The Indian Journal of Dermatology, Venereology and Leprology website has a provision to import references to EndNote.

Comparison of BacT/Alert FAN and FAN Plus Bottles with Conventional Medium for Culturing Cerebrospinal Fluid.

Science.gov (United States)

Yoo, In Young; Chun, Sejong; Song, Dong Joon; Huh, Hee Jae; Lee, Nam Yong

2016-11-01

We compared the BacT/Alert system FAN and FAN Plus media to conventional media for culturing cerebrospinal fluid (CSF) with 2,545 samples. FAN/FAN Plus bottles showed better performance for isolating microorganisms in CSF than conventional media (positive rate, 7.2% [182/2,545] versus 3.1% [80/2,545]). The incremental recovery rate of Cryptococcus neoformans from FAN Plus bottles was higher than that from FAN bottles. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Semantic distributed resource discovery for multiple resource providers

NARCIS (Netherlands)

Pittaras, C.; Ghijsen, M.; Wibisono, A.; Grosso, P.; van der Ham, J.; de Laat, C.

2012-01-01

An emerging modus operandi among providers of cloud infrastructures is the one where they share and combine their heterogenous resources to offer end user services tailored to specific scientific and business needs. A challenge to overcome is the discovery of suitable resources among these multiple

User-oriented end-to-end transport protocols for the real-time distribution of telemetry data from NASA spacecraft

Science.gov (United States)

Hooke, A. J.

1979-01-01

A set of standard telemetry protocols for downlink data flow facilitating the end-to-end transport of instrument data from the spacecraft to the user in real time is proposed. The direct switching of data by autonomous message 'packets' that are assembled by the source instrument on the spacecraft is discussed. The data system consists thus of a format on a message rather than word basis, and such packet telemetry would include standardized protocol headers. Standards are being developed within the NASA End-to-End Data System (NEEDS) program for the source packet and transport frame protocols. The source packet protocol contains identification of both the sequence number of the packet as it is generated by the source and the total length of the packet, while the transport frame protocol includes a sequence count defining the serial number of the frame as it is generated by the spacecraft data system, and a field specifying any 'options' selected in the format of the frame itself.

MiSeq: A Next Generation Sequencing Platform for Genomic Analysis.

Science.gov (United States)

Ravi, Rupesh Kanchi; Walton, Kendra; Khosroheidari, Mahdieh

2018-01-01

MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs. A single run can produce output data of up to 15 Gb in as little as 4 h of runtime and can output up to 25 M single reads and 50 M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.

Now and next-generation sequencing techniques: future of sequence analysis using cloud computing.

Science.gov (United States)

Thakur, Radhe Shyam; Bandopadhyay, Rajib; Chaudhary, Bratati; Chatterjee, Sourav

2012-01-01

Advances in the field of sequencing techniques have resulted in the greatly accelerated production of huge sequence datasets. This presents immediate challenges in database maintenance at datacenters. It provides additional computational challenges in data mining and sequence analysis. Together these represent a significant overburden on traditional stand-alone computer resources, and to reach effective conclusions quickly and efficiently, the virtualization of the resources and computation on a pay-as-you-go concept (together termed "cloud computing") has recently appeared. The collective resources of the datacenter, including both hardware and software, can be available publicly, being then termed a public cloud, the resources being provided in a virtual mode to the clients who pay according to the resources they employ. Examples of public companies providing these resources include Amazon, Google, and Joyent. The computational workload is shifted to the provider, which also implements required hardware and software upgrades over time. A virtual environment is created in the cloud corresponding to the computational and data storage needs of the user via the internet. The task is then performed, the results transmitted to the user, and the environment finally deleted after all tasks are completed. In this discussion, we focus on the basics of cloud computing, and go on to analyze the prerequisites and overall working of clouds. Finally, the applications of cloud computing in biological systems, particularly in comparative genomics, genome informatics, and SNP detection are discussed with reference to traditional workflows.

Enhancing faba bean (Vicia faba L.) genome resources.

Science.gov (United States)

Cooper, James W; Wilson, Michael H; Derks, Martijn F L; Smit, Sandra; Kunert, Karl J; Cullis, Christopher; Foyer, Christine H

2017-04-01

Grain legume improvement is currently impeded by a lack of genomic resources. The paucity of genome information for faba bean can be attributed to the intrinsic difficulties of assembling/annotating its giant (~13 Gb) genome. In order to address this challenge, RNA-sequencing analysis was performed on faba bean (cv. Wizard) leaves. Read alignment to the faba bean reference transcriptome identified 16 300 high quality unigenes. In addition, Illumina paired-end sequencing was used to establish a baseline for genomic information assembly. Genomic reads were assembled de novo into contigs with a size range of 50-5000 bp. Over 85% of sequences did not align to known genes, of which ~10% could be aligned to known repetitive genetic elements. Over 26 000 of the reference transcriptome unigenes could be aligned to DNA-sequencing (DNA-seq) reads with high confidence. Moreover, this comparison identified 56 668 potential splice points in all identified unigenes. Sequence length data were extended at 461 putative loci through alignment of DNA-seq contigs to full-length, publicly available linkage marker sequences. Reads also yielded coverages of 3466× and 650× for the chloroplast and mitochondrial genomes, respectively. Inter- and intraspecies organelle genome comparisons established core legume organelle gene sets, and revealed polymorphic regions of faba bean organelle genomes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Performance of two resin-containing blood culture media in detection of bloodstream infections and in direct matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) broth assays for isolate identification: clinical comparison of the BacT/Alert Plus and Bactec Plus systems.

Science.gov (United States)

Fiori, Barbara; D'Inzeo, Tiziana; Di Florio, Viviana; De Maio, Flavio; De Angelis, Giulia; Giaquinto, Alessia; Campana, Lara; Tanzarella, Eloisa; Tumbarello, Mario; Antonelli, Massimo; Sanguinetti, Maurizio; Spanu, Teresa

2014-10-01

We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥ 16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤ 5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P direct matrix-assisted laser desorption ionization-time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings. Copyright �

Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

A bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0-613-2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384-well microtiter plate. Random samples of BACs digested with the Notl enzyme indicated that the average insert size is approximately 130 kb, with a range of 80-275 kb,and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 x haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hindlll enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.

Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

Science.gov (United States)

Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

1993-01-01

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.

Science.gov (United States)

Li, Guotian; Jain, Rashmi; Chern, Mawsheng; Pham, Nikki T; Martin, Joel A; Wei, Tong; Schackwitz, Wendy S; Lipzen, Anna M; Duong, Phat Q; Jones, Kyle C; Jiang, Liangrong; Ruan, Deling; Bauer, Diane; Peng, Yi; Barry, Kerrie W; Schmutz, Jeremy; Ronald, Pamela C

2017-06-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake ( Oryza sativa ssp japonica ), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. © 2017 American Society of Plant Biologists. All rights reserved.

A High Resolution Genetic Map Anchoring Scaffolds of the Sequenced Watermelon Genome

Science.gov (United States)

Kou, Qinghe; Jiang, Jiao; Guo, Shaogui; Zhang, Haiying; Hou, Wenju; Zou, Xiaohua; Sun, Honghe; Gong, Guoyi; Levi, Amnon; Xu, Yong

2012-01-01

As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F8 population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ∼800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits. PMID:22247776

HIV Sequence Compendium 2015

Energy Technology Data Exchange (ETDEWEB)

Foley, Brian Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Leitner, Thomas Kenneth [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Apetrei, Cristian [Univ. of Pittsburgh, PA (United States); Hahn, Beatrice [Univ. of Pennsylvania, Philadelphia, PA (United States); Mizrachi, Ilene [National Center for Biotechnology Information, Bethesda, MD (United States); Mullins, James [Univ. of Washington, Seattle, WA (United States); Rambaut, Andrew [Univ. of Edinburgh, Scotland (United Kingdom); Wolinsky, Steven [Northwestern Univ., Evanston, IL (United States); Korber, Bette Tina Marie [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-10-05

This compendium is an annual printed summary of the data contained in the HIV sequence database. We try to present a judicious selection of the data in such a way that it is of maximum utility to HIV researchers. Each of the alignments attempts to display the genetic variability within the different species, groups and subtypes of the virus. This compendium contains sequences published before January 1, 2015. Hence, though it is published in 2015 and called the 2015 Compendium, its contents correspond to the 2014 curated alignments on our website. The number of sequences in the HIV database is still increasing. In total, at the end of 2014, there were 624,121 sequences in the HIV Sequence Database, an increase of 7% since the previous year. This is the first year that the number of new sequences added to the database has decreased compared to the previous year. The number of near complete genomes (>7000 nucleotides) increased to 5834 by end of 2014. However, as in previous years, the compendium alignments contain only a fraction of these. A more complete version of all alignments is available on our website, http://www.hiv.lanl.gov/ content/sequence/NEWALIGN/align.html As always, we are open to complaints and suggestions for improvement. Inquiries and comments regarding the compendium should be addressed to seq-info@lanl.gov.

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: Isolation, RFLP marker development, and physical mapping

Directory of Open Access Journals (Sweden)

Souza Manoel T

2008-01-01

deposited in GenBank and assigned numbers ER935972 – ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

Tracking the Flow of Resources in Electronic Waste - The Case of End-of-Life Computer Hard Disk Drives.

Science.gov (United States)

Habib, Komal; Parajuly, Keshav; Wenzel, Henrik

2015-10-20

Recovery of resources, in particular, metals, from waste flows is widely seen as a prioritized option to reduce their potential supply constraints in the future. The current waste electrical and electronic equipment (WEEE) treatment system is more focused on bulk metals, where the recycling rate of specialty metals, such as rare earths, is negligible compared to their increasing use in modern products, such as electronics. This study investigates the challenges in recovering these resources in the existing WEEE treatment system. It is illustrated by following the material flows of resources in a conventional WEEE treatment plant in Denmark. Computer hard disk drives (HDDs) containing neodymium-iron-boron (NdFeB) magnets were selected as the case product for this experiment. The resulting output fractions were tracked until their final treatment in order to estimate the recovery potential of rare earth elements (REEs) and other resources contained in HDDs. The results further show that out of the 244 kg of HDDs treated, 212 kg comprising mainly of aluminum and steel can be finally recovered from the metallurgic process. The results further demonstrate the complete loss of REEs in the existing shredding-based WEEE treatment processes. Dismantling and separate processing of NdFeB magnets from their end-use products can be a more preferred option over shredding. However, it remains a technological and logistic challenge for the existing system.

Genome sequence of the olive tree, Olea europaea.

Science.gov (United States)

Cruz, Fernando; Julca, Irene; Gómez-Garrido, Jèssica; Loska, Damian; Marcet-Houben, Marina; Cano, Emilio; Galán, Beatriz; Frias, Leonor; Ribeca, Paolo; Derdak, Sophia; Gut, Marta; Sánchez-Fernández, Manuel; García, Jose Luis; Gut, Ivo G; Vargas, Pablo; Alioto, Tyler S; Gabaldón, Toni

2016-06-27

The Mediterranean olive tree (Olea europaea subsp. europaea) was one of the first trees to be domesticated and is currently of major agricultural importance in the Mediterranean region as the source of olive oil. The molecular bases underlying the phenotypic differences among domesticated cultivars, or between domesticated olive trees and their wild relatives, remain poorly understood. Both wild and cultivated olive trees have 46 chromosomes (2n). A total of 543 Gb of raw DNA sequence from whole genome shotgun sequencing, and a fosmid library containing 155,000 clones from a 1,000+ year-old olive tree (cv. Farga) were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 443 kb, and a total length of 1.31 Gb, which represents 95 % of the estimated genome length (1.38 Gb). In addition, the associated fungus Aureobasidium pullulans was partially sequenced. Genome annotation, assisted by RNA sequencing from leaf, root, and fruit tissues at various stages, resulted in 56,349 unique protein coding genes, suggesting recent genomic expansion. Genome completeness, as estimated using the CEGMA pipeline, reached 98.79 %. The assembled draft genome of O. europaea will provide a valuable resource for the study of the evolution and domestication processes of this important tree, and allow determination of the genetic bases of key phenotypic traits. Moreover, it will enhance breeding programs and the formation of new varieties.

Brucella detection in blood: comparison of the BacT/Alert standard aerobic bottle, BacT/Alert FAN aerobic bottle and BacT/Alert enhanced FAN aerobic bottle in simulated blood culture.

Science.gov (United States)

Sümerkan, B; Gökahmetoglu, S; Esel, D

2001-07-01

The objective of this study was to compare the performances of the standard aerobic bottle (StAe), FAN aerobic (FANAe) and enhanced FAN aerobic (E-FANAe) (the charcoal component of the FANAe was revised recently to improve the feasibility of Gram smear interpretation) blood culture bottles for BacT/Alert system for the detection of Brucella melitensis in simulated blood culture. Triplicate strains of eight clinical isolates of B. melitensis were studied. Each bottle was inoculated with 5 mL of freshly collected human blood at three different targeted bacterial inocula (10(1), 10(2) and 10(3) CFU/bottle). All bottles were monitored for up to 21 days or until they became positive. The results of time to detection (TTD) on the eight B. melitensis samples were as follows: at 10(1) CFU/bottle, the E-FANAe had a mean TTD significantly shorter than the StAe (48 h vs. 56.2 h, P StAe (41.2 h and 40 h vs. 45.6 h, P StAe, FANAe and E-FANAe were 96, 83 and 58%, respectively. At 10(3) CFU/bottle, the reproducibilities of StAe, FANAe and E-FANAe were 95, 95 and 91%, respectively. Positive results for the presence of bacteria in Gram smears were confirmed in 68% of StAe, 54% of FANAe and 90% of E-FANAe. In case of suspected brucellosis, the combination of one StAe bottle and one E-FANAe bottle seems to provide the highest and fastest recovery of the organism.

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

Science.gov (United States)

Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria Mar; Receveur, Aline; Smaïli, Sadek; Roest Crollius, Hugues; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

2004-07-29

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

DEEPre: sequence-based enzyme EC number prediction by deep learning

KAUST Repository

Li, Yu

2017-10-20

Annotation of enzyme function has a broad range of applications, such as metagenomics, industrial biotechnology, and diagnosis of enzyme deficiency-caused diseases. However, the time and resource required make it prohibitively expensive to experimentally determine the function of every enzyme. Therefore, computational enzyme function prediction has become increasingly important. In this paper, we develop such an approach, determining the enzyme function by predicting the Enzyme Commission number.We propose an end-to-end feature selection and classification model training approach, as well as an automatic and robust feature dimensionality uniformization method, DEEPre, in the field of enzyme function prediction. Instead of extracting manuallycrafted features from enzyme sequences, our model takes the raw sequence encoding as inputs, extracting convolutional and sequential features from the raw encoding based on the classification result to directly improve the prediction performance. The thorough cross-fold validation experiments conducted on two large-scale datasets show that DEEPre improves the prediction performance over the previous state-of-the-art methods. In addition, our server outperforms five other servers in determining the main class of enzymes on a separate low-homology dataset. Two case studies demonstrate DEEPre\\'s ability to capture the functional difference of enzyme isoforms.The server could be accessed freely at http://www.cbrc.kaust.edu.sa/DEEPre.

DEEPre: sequence-based enzyme EC number prediction by deep learning

KAUST Repository

Li, Yu; Wang, Sheng; Umarov, Ramzan; Xie, Bingqing; Fan, Ming; Li, Lihua; Gao, Xin

2017-01-01

Annotation of enzyme function has a broad range of applications, such as metagenomics, industrial biotechnology, and diagnosis of enzyme deficiency-caused diseases. However, the time and resource required make it prohibitively expensive to experimentally determine the function of every enzyme. Therefore, computational enzyme function prediction has become increasingly important. In this paper, we develop such an approach, determining the enzyme function by predicting the Enzyme Commission number.We propose an end-to-end feature selection and classification model training approach, as well as an automatic and robust feature dimensionality uniformization method, DEEPre, in the field of enzyme function prediction. Instead of extracting manuallycrafted features from enzyme sequences, our model takes the raw sequence encoding as inputs, extracting convolutional and sequential features from the raw encoding based on the classification result to directly improve the prediction performance. The thorough cross-fold validation experiments conducted on two large-scale datasets show that DEEPre improves the prediction performance over the previous state-of-the-art methods. In addition, our server outperforms five other servers in determining the main class of enzymes on a separate low-homology dataset. Two case studies demonstrate DEEPre's ability to capture the functional difference of enzyme isoforms.The server could be accessed freely at http://www.cbrc.kaust.edu.sa/DEEPre.

Proficiency Testing for Bacterial Whole Genome Sequencing: An End-User Survey of Current Capabilities, Requirements and Priorities

DEFF Research Database (Denmark)

Moran-Gilad, Jacob; Sintchenko, Vitali; Karlsmose Pedersen, Susanne

2015-01-01

The advent of next-generation sequencing (NGS) has revolutionised public health microbiology. Given the potential impact of NGS, it is paramount to ensure standardisation of ‘wet’ laboratory and bioinformatic protocols and promote comparability of methods employed by different laboratories...... and their outputs. Therefore, one of the ambitious goals of the Global Microbial Identifier (GMI) initiative (http://​www.​globalmicrobiali​dentifier.​org/​) has been to establish a mechanism for inter-laboratory NGS proficiency testing (PT). This report presents findings from the survey recently conducted...... by Working Group 4 among GMI members in order to ascertain NGS end-use requirements and attitudes towards NGS PT. The survey identified the high professional diversity of laboratories engaged in NGS-based public health projects and the wide range of capabilities within institutions, at a notable range...

Proficiency testing for bacterial whole genome sequencing: an end-user survey of current capabilities, requirements and priorities

DEFF Research Database (Denmark)

Moran-Gilad, Jacob; Sintchenko, Vitali; Karlsmose Pedersen, Susanne

2015-01-01

The advent of next-generation sequencing (NGS) has revolutionised public health microbiology. Given the potential impact of NGS, it is paramount to ensure standardisation of 'wet' laboratory and bioinformatic protocols and promote comparability of methods employed by different laboratories...... and their outputs. Therefore, one of the ambitious goals of the Global Microbial Identifier (GMI) initiative (http://www.globalmicrobialidentifier.org/) has been to establish a mechanism for inter-laboratory NGS proficiency testing (PT). This report presents findings from the survey recently conducted by Working...... Group 4 among GMI members in order to ascertain NGS end-use requirements and attitudes towards NGS PT. The survey identified the high professional diversity of laboratories engaged in NGS-based public health projects and the wide range of capabilities within institutions, at a notable range of costs...

Transcriptome sequencing and De Novo analysis of Youngia japonica using the illumina platform.

Directory of Open Access Journals (Sweden)

Yulan Peng

Full Text Available Youngia japonica, a weed species distributed worldwide, has been widely used in traditional Chinese medicine. It is an ideal plant for studying the evolution of Asteraceae plants because of its short life history and abundant source. However, little is known about its evolution and genetic diversity. In this study, de novo transcriptome sequencing was conducted for the first time for the comprehensive analysis of the genetic diversity of Y. japonica. The Y. japonica transcriptome was sequenced using Illumina paired-end sequencing technology. We produced 21,847,909 high-quality reads for Y. japonica and assembled them into contigs. A total of 51,850 unigenes were identified, among which 46,087 were annotated in the NCBI non-redundant protein database and 41,752 were annotated in the Swiss-Prot database. We mapped 9,125 unigenes onto 163 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. In addition, 3,648 simple sequence repeats (SSRs were detected. Our data provide the most comprehensive transcriptome resource currently available for Y. japonica. C4 photosynthesis unigenes were found in the biological process of Y. japonica. There were 5596 unigenes related to defense response and 1344 ungienes related to signal transduction mechanisms (10.95%. These data provide insights into the genetic diversity of Y. japonica. Numerous SSRs contributed to the development of novel markers. These data may serve as a new valuable resource for genomic studies on Youngia and, more generally, Cichoraceae.

GenBank blastn search result: AK288534 [KOME

Lifescience Database Archive (English)

Full Text Available AK288534 J090045E15 AC095876.6 AC095876 Rattus norvegicus BAC CH230-10G12 (Children's Hospital Oakland Resea...rch Institute Rat (BN/SsNHsd/MCW) BAC library) complete sequence. ROD 3e-81 1 -1 ...

GenBank blastn search result: AK060330 [KOME

Lifescience Database Archive (English)

Full Text Available AK060330 001-008-B01 AC125292.4 Drosophila melanogaster X BAC CH223-5O10 (Children's Hospital Oakland Resear...ch Institute Drosophila melanogaster sheared DNA BAC Library) complete sequence.|INV INV 3e-16 Plus Minus ...

De novo transcriptome sequencing and sequence analysis of the malaria vector Anopheles sinensis (Diptera: Culicidae)

Science.gov (United States)

2014-01-01

Background Anopheles sinensis is the major malaria vector in China and Southeast Asia. Vector control is one of the most effective measures to prevent malaria transmission. However, there is little transcriptome information available for the malaria vector. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to build a transcriptome dataset for functional genomics analysis by large-scale RNA sequencing (RNA-seq). Methods To provide a more comprehensive and complete transcriptome of An. sinensis, eggs, larvae, pupae, male adults and female adults RNA were pooled together for cDNA preparation, sequenced using the Illumina paired-end sequencing technology and assembled into unigenes. These unigenes were then analyzed in their genome mapping, functional annotation, homology, codon usage bias and simple sequence repeats (SSRs). Results Approximately 51.6 million clean reads were obtained, trimmed, and assembled into 38,504 unigenes with an average length of 571 bp, an N50 of 711 bp, and an average GC content 51.26%. Among them, 98.4% of unigenes could be mapped onto the reference genome, and 69% of unigenes could be annotated with known biological functions. Homology analysis identified certain numbers of An. sinensis unigenes that showed homology or being putative 1:1 orthologues with genomes of other Dipteran species. Codon usage bias was analyzed and 1,904 SSRs were detected, which will provide effective molecular markers for the population genetics of this species. Conclusions Our data and analysis provide the most comprehensive transcriptomic resource and characteristics currently available for An. sinensis, and will facilitate genetic, genomic studies, and further vector control of An. sinensis. PMID:25000941

An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS.

Science.gov (United States)

Loonen, A J M; Jansz, A R; Stalpers, J; Wolffs, P F G; van den Brule, A J C

2012-07-01

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.

Restoring San Xavier del Bac, "Our Church": Tohono O'odham Work to Restore the 200-Year-Old Church Built by Their Ancestors.

Science.gov (United States)

Fontana, Bernard L.

1995-01-01

The Tohono O'odham built Mission San Xavier del Bac for Franciscan missionaries in the late 1700s and have protected and cared for it through changing circumstances ever since. As part of a massive restoration project, outstanding experts have been restoring the church's painted and sculpted interior and training local Tohono O'odham to be…

A new genomic resource dedicated to wood formation in Eucalyptus

Directory of Open Access Journals (Sweden)

Couloux Arnaud

2009-03-01

Full Text Available Abstract Background Renowned for their fast growth, valuable wood properties and wide adaptability, Eucalyptus species are amongst the most planted hardwoods in the world, yet they are still at the early stages of domestication because conventional breeding is slow and costly. Thus, there is huge potential for marker-assisted breeding programs to improve traits such as wood properties. To this end, the sequencing, analysis and annotation of a large collection of expressed sequences tags (ESTs from genes involved in wood formation in Eucalyptus would provide a valuable resource. Results We report here the normalization and sequencing of a cDNA library from developing Eucalyptus secondary xylem, as well as the construction and sequencing of two subtractive libraries (juvenile versus mature wood and vice versa. A total of 9,222 high quality sequences were collected from about 10,000 cDNA clones. The EST assembly generated a set of 3,857 wood-related unigenes including 2,461 contigs (Cg and 1,396 singletons (Sg that we named 'EUCAWOOD'. About 65% of the EUCAWOOD sequences produced matches with poplar, grapevine, Arabidopsis and rice protein sequence databases. BlastX searches of the Uniref100 protein database allowed us to allocate gene ontology (GO and protein family terms to the EUCAWOOD unigenes. This annotation of the EUCAWOOD set revealed key functional categories involved in xylogenesis. For instance, 422 sequences matched various gene families involved in biosynthesis and assembly of primary and secondary cell walls. Interestingly, 141 sequences were annotated as transcription factors, some of them being orthologs of regulators known to be involved in xylogenesis. The EUCAWOOD dataset was also mined for genomic simple sequence repeat markers, yielding a total of 639 putative microsatellites. Finally, a publicly accessible database was created, supporting multiple queries on the EUCAWOOD dataset. Conclusion In this work, we have identified a

Including 10-Gigabit-capable Passive Optical Network under End-to-End Generalized Multi-Protocol Label Switching Provisioned Quality of Service

DEFF Research Database (Denmark)

Brewka, Lukasz Jerzy; Gavler, Anders; Wessing, Henrik

2012-01-01

of the network where quality of service signaling is bridged. This article proposes strategies for generalized multi-protocol label switching control over next emerging passive optical network standard, i.e., the 10-gigabit-capable passive optical network. Node management and resource allocation approaches...... are discussed, and possible issues are raised. The analysis shows that consideration of a 10-gigabit-capable passive optical network as a generalized multi-protocol label switching controlled domain is valid and may advance end-to-end quality of service provisioning for passive optical network based customers.......End-to-end quality of service provisioning is still a challenging task despite many years of research and development in this area. Considering a generalized multi-protocol label switching based core/metro network and resource reservation protocol capable home gateways, it is the access part...

Spanning k-ended trees of 3-regular connected graphs

Directory of Open Access Journals (Sweden)

Hamed Ghasemian Zoeram

2017-10-01

Full Text Available A vertex of degree one is called an end-vertex and the set of end-vertices of G is denoted by End(G. For a positive integer k, a tree T be called k-ended tree if $|End(T| \\leq k$. In this paper, we obtain sufficient conditions for spanning k-trees of 3-regular connected graphs. We give a construction sequence of graphs satisfying the condition. At the end, we present a conjecture about spanning k-ended trees of 3-regular connected graphs.

Short Linear Sequence Motif LxxPTPh Targets Diverse Proteins to Growing Microtubule Ends

NARCIS (Netherlands)

Kumar, Anil; Manatschal, Cristina; Rai, Ankit; Grigoriev, Ilya; Degen, Miriam Steiner; Jaussi, Rolf; Kretzschmar, Ines; Prota, Andrea E; Volkmer, Rudolf; Kammerer, Richard A.; Akhmanova, Anna; Steinmetz, Michel O.

2017-01-01

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End-binding (EB) proteins are considered master regulators of +TIP interaction networks, since they autonomously track growing microtubule ends and recruit a plethora of proteins to this

Next Generation Sequencing of Classical Swine Fever Virus and Border Disease virus cloned in Bacterial Artificial Chromosomes

DEFF Research Database (Denmark)

Fahnøe, Ulrik; Höper, Dirk; Beer, martin

2012-01-01

artificial chromosomes (BACs). From these BACs, RNA copies of the viral genomes can be transcribed in vitro and upon transfection of these RNAs into mammalian cells, autonomous replication of the viral genome occurs and infectious progeny can be rescued. However, we have observed that virus progeny can...

Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.

Science.gov (United States)

Srichaisupakit, Akkaraphol; Ohashi, Takao; Fujiyama, Kazuhito

2014-09-01

Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

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Suvi Jain

2016-04-01

Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.