A genome-wide BAC-end sequence survey provides first insights into sweetpotato (Ipomoea batatas (L.) Lam.) genome composition.

PubMed

Si, Zengzhi; Du, Bing; Huo, Jinxi; He, Shaozhen; Liu, Qingchang; Zhai, Hong

2016-11-21

Sweetpotato, Ipomoea batatas (L.) Lam., is an important food crop widely grown in the world. However, little is known about the genome of this species because it is a highly heterozygous hexaploid. Gaining a more in-depth knowledge of sweetpotato genome is therefore necessary and imperative. In this study, the first bacterial artificial chromosome (BAC) library of sweetpotato was constructed. Clones from the BAC library were end-sequenced and analyzed to provide genome-wide information about this species. The BAC library contained 240,384 clones with an average insert size of 101 kb and had a 7.93-10.82 × coverage of the genome, and the probability of isolating any single-copy DNA sequence from the library was more than 99%. Both ends of 8310 BAC clones randomly selected from the library were sequenced to generate 11,542 high-quality BAC-end sequences (BESs), with an accumulative length of 7,595,261 bp and an average length of 658 bp. Analysis of the BESs revealed that 12.17% of the sweetpotato genome were known repetitive DNA, including 7.37% long terminal repeat (LTR) retrotransposons, 1.15% Non-LTR retrotransposons and 1.42% Class II DNA transposons etc., 18.31% of the genome were identified as sweetpotato-unique repetitive DNA and 10.00% of the genome were predicted to be coding regions. In total, 3,846 simple sequences repeats (SSRs) were identified, with a density of one SSR per 1.93 kb, from which 288 SSRs primers were designed and tested for length polymorphism using 20 sweetpotato accessions, 173 (60.07%) of them produced polymorphic bands. Sweetpotato BESs had significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum than those of Vitis vinifera, Theobroma cacao and Arabidopsis thaliana. The first BAC library for sweetpotato has been successfully constructed. The high quality BESs provide first insights into sweetpotato genome composition, and have significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum. These resources as a robust platform will be used in high-resolution mapping, gene cloning, assembly of genome sequences, comparative genomics and evolution for sweetpotato.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

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

PubMed

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.

Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

PubMed Central

Osoegawa, Kazutoyo; Tateno, Minako; Woon, Peng Yeong; Frengen, Eirik; Mammoser, Aaron G.; Catanese, Joseph J.; Hayashizaki, Yoshihide; de Jong, Pieter J.

2000-01-01

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9–14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, ∼1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research. [The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AQ797173–AQ797398.] PMID:10645956

Use of RecA protein to enrich for homologous genes in a genomic library

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taidi-Laskowski, B.; Grumet, F.C.; Tyan, D.

1988-08-25

RecA protein-coated probe has been utilized to enrich genomic digests for desired genes in order to facilitate cloning from genomic libraries. Using a previously cloned HLA-B27 gene as the recA-coated enrichment probe, the authors obtained a mean 108x increase in the ratio of specific to nonspecific plaques in lambda libraries screened for B27 variant alleles of estimated 99% homology to the probe. Class I genes of lesser homology were less enriched. Loss of genomic DNA during the enrichment procedure can, however, restrict application of this technique whenever starting genomic DNA is very limited. Nevertheless, the impressive reduction in cloning effortmore » and material makes recA enrichment a useful new tool for cloning homologous genes from genomic DNA.« less

Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

PubMed Central

Xu, Min; Wang, Yemin; Zhao, Zhilong; Gao, Guixi; Huang, Sheng-Xiong; Kang, Qianjin; He, Xinyi; Lin, Shuangjun; Pang, Xiuhua; Deng, Zixin

2016-01-01

ABSTRACT Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic from Streptomyces rochei Sal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery. PMID:27451447

SMRT sequencing of the Vitis vinifera cv. ‘Flame seedless’ genome using a SMRTbell-free library preparation from Swift Biosciences

USDA-ARS?s Scientific Manuscript database

Single Molecule Real-Time (SMRT) sequencing provides advantages to the sequencing of complex genomes. The long reads generated are superior for resolving complex genomic regions and provide highly contiguous de novo assemblies. Current SMRTbell libraries generate average read lengths of 10-15kb. How...

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

PubMed

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.

Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

PubMed Central

Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce. PMID:23409088

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.

PubMed

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.

Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and function.

PubMed

Baxter, Laura L; Hsu, Benjamin J; Umayam, Lowell; Wolfsberg, Tyra G; Larson, Denise M; Frith, Martin C; Kawai, Jun; Hayashizaki, Yoshihide; Carninci, Piero; Pavan, William J

2007-06-01

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

Improving draft genome contiguity with reference-derived in silico mate-pair libraries.

PubMed

Grau, José Horacio; Hackl, Thomas; Koepfli, Klaus-Peter; Hofreiter, Michael

2018-05-01

Contiguous genome assemblies are a highly valued biological resource because of the higher number of completely annotated genes and genomic elements that are usable compared to fragmented draft genomes. Nonetheless, contiguity is difficult to obtain if only low coverage data and/or only distantly related reference genome assemblies are available. In order to improve genome contiguity, we have developed Cross-Species Scaffolding-a new pipeline that imports long-range distance information directly into the de novo assembly process by constructing mate-pair libraries in silico. We show how genome assembly metrics and gene prediction dramatically improve with our pipeline by assembling two primate genomes solely based on ∼30x coverage of shotgun sequencing data.

Analysis of an RNA-seq Strand-Specific Library from an East Timorese Cucumber Sample Reveals a Complete Cucurbit aphid-borne yellows virus Genome

PubMed Central

Maina, Solomon; Edwards, Owain R.; de Almeida, Luis; Ximenes, Abel

2017-01-01

ABSTRACT Analysis of an RNA-seq library from cucumber leaf RNA extracted from a fast technology for analysis of nucleic acids (FTA) card revealed the first complete genome of Cucurbit aphid-borne yellows virus (CABYV) from East Timor. We compare it with 35 complete CABYV genomes from other world regions. It most resembled the genome of the South Korean isolate HD118. PMID:28495776

CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries.

PubMed

Heigwer, Florian; Zhan, Tianzuo; Breinig, Marco; Winter, Jan; Brügemann, Dirk; Leible, Svenja; Boutros, Michael

2016-03-24

Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld.

DNAism: exploring genomic datasets on the web with Horizon Charts.

PubMed

Rio Deiros, David; Gibbs, Richard A; Rogers, Jeffrey

2016-01-27

Computational biologists daily face the need to explore massive amounts of genomic data. New visualization techniques can help researchers navigate and understand these big data. Horizon Charts are a relatively new visualization method that, under the right circumstances, maximizes data density without losing graphical perception. Horizon Charts have been successfully applied to understand multi-metric time series data. We have adapted an existing JavaScript library (Cubism) that implements Horizon Charts for the time series domain so that it works effectively with genomic datasets. We call this new library DNAism. Horizon Charts can be an effective visual tool to explore complex and large genomic datasets. Researchers can use our library to leverage these techniques to extract additional insights from their own datasets.

Analysis of an RNA-seq Strand-Specific Library from an East Timorese Cucumber Sample Reveals a Complete Cucurbit aphid-borne yellows virus Genome.

PubMed

Maina, Solomon; Edwards, Owain R; de Almeida, Luis; Ximenes, Abel; Jones, Roger A C

2017-05-11

Analysis of an RNA-seq library from cucumber leaf RNA extracted from a fast technology for analysis of nucleic acids (FTA) card revealed the first complete genome of Cucurbit aphid-borne yellows virus (CABYV) from East Timor. We compare it with 35 complete CABYV genomes from other world regions. It most resembled the genome of the South Korean isolate HD118. Copyright © 2017 Maina et al.

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

USDA-ARS?s Scientific Manuscript database

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 ...

Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

PubMed Central

Rondon, Michelle R.; Raffel, Sandra J.; Goodman, Robert M.; Handelsman, Jo

1999-01-01

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. PMID:10339608

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project.

PubMed

Park, Tae-Ho; Park, Beom-Seok; Kim, Jin-A; Hong, Joon Ki; Jin, Mina; Seol, Young-Joo; Mun, Jeong-Hwan

2011-01-01

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa. Copyright © 2011. Published by Elsevier Ltd.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing

PubMed Central

2011-01-01

Background Lupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species. Results A NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers. Conclusions The NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species. PMID:22014081

A Deep-Coverage Tomato BAC Library and Prospects Toward Development of an STC Framework for Genome Sequencing

PubMed Central

Budiman, Muhammad A.; Mao, Long; Wood, Todd C.; Wing, Rod A.

2000-01-01

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10−6, and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed. [The BAC end sequences described in this paper have been deposited in the GenBank data library under accession nos. AQ367111–AQ368361.] PMID:10645957

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

PubMed Central

Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

2017-01-01

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

A new single-nucleotide polymorphism database for rainbow trout generated through whole genome re-sequencing

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

PubMed Central

McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

1993-01-01

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

Dominant genetics using a yeast genomic library under the control of a strong inducible promoter.

PubMed

Ramer, S W; Elledge, S J; Davis, R W

1992-12-01

In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 x 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.

The Essential Genome of Escherichia coli K-12

PubMed Central

2018-01-01

ABSTRACT Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry. PMID:29463657

Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.

PubMed

Pan, Jin; Wang, Baosheng; Pei, Zhi-Yong; Zhao, Wei; Gao, Jie; Mao, Jian-Feng; Wang, Xiao-Ru

2015-07-01

Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking. © 2014 John Wiley & Sons Ltd.

Library Resources for Bac End Sequencing. Final Technical Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pieter J. de Jong

2000-10-01

Studies directed towards the specific aims outlined for this research award are summarized. The RPCI II Human Bac Library has been expanded by the addition of 6.9-fold genomic coverage. This segment has been generated from a MBOI partial digest of the same anonymous donor DNA used for the rest of the library. A new cloning vector, pTARBAC1, has been constructed and used in the construction of RPCI-II segment 5. This new cloning vector provides a new strategy in identifying targeted genomic regions and will greatly facilitate a large-scale analysis for positional cloning. A new maleCS7BC/6J mouse BAC library has beenmore » constructed. RPCI-23 contain 576 plates (approx 210,000 clones) and represents approximately 11-fold coverage of the mouse genome.« less

SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

PubMed Central

Manlig, Erika; Wahlberg, Per

2017-01-01

Abstract Sodium bisulphite treatment of DNA combined with next generation sequencing (NGS) is a powerful combination for the interrogation of genome-wide DNA methylation profiles. Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method. PMID:27899585

A new single-nucleotide polymorphisms database for rainbow trout generated through whole genome resequencing of selected samples

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Sequencing and assembly of the 22-gb loblolly pine genome.

PubMed

Zimin, Aleksey; Stevens, Kristian A; Crepeau, Marc W; Holtz-Morris, Ann; Koriabine, Maxim; Marçais, Guillaume; Puiu, Daniela; Roberts, Michael; Wegrzyn, Jill L; de Jong, Pieter J; Neale, David B; Salzberg, Steven L; Yorke, James A; Langley, Charles H

2014-03-01

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

PubMed Central

2010-01-01

Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries. Conclusions The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes. PMID:20170511

Scaling up the 454 Titanium Library Construction and Pooling of Barcoded Libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phung, Wilson; Hack, Christopher; Shapiro, Harris

2009-03-23

We have been developing a high throughput 454 library construction process at the Joint Genome Institute to meet the needs of de novo sequencing a large number of microbial and eukaryote genomes, EST, and metagenome projects. We have been focusing efforts in three areas: (1) modifying the current process to allow the construction of 454 standard libraries on a 96-well format; (2) developing a robotic platform to perform the 454 library construction; and (3) designing molecular barcodes to allow pooling and sorting of many different samples. In the development of a high throughput process to scale up the number ofmore » libraries by adapting the process to a 96-well plate format, the key process change involves the replacement of gel electrophoresis for size selection with Solid Phase Reversible Immobilization (SPRI) beads. Although the standard deviation of the insert sizes increases, the overall quality sequence and distribution of the reads in the genome has not changed. The manual process of constructing 454 shotgun libraries on 96-well plates is a time-consuming, labor-intensive, and ergonomically hazardous process; we have been experimenting to program a BioMek robot to perform the library construction. This will not only enable library construction to be completed in a single day, but will also minimize any ergonomic risk. In addition, we have implemented a set of molecular barcodes (AKA Multiple Identifiers or MID) and a pooling process that allows us to sequence many targets simultaneously. Here we will present the testing of pooling a set of selected fosmids derived from the endomycorrhizal fungus Glomus intraradices. By combining the robotic library construction process and the use of molecular barcodes, it is now possible to sequence hundreds of fosmids that represent a minimal tiling path of this genome. Here we present the progress and the challenges of developing these scaled-up processes.« less

In Situ Hi-C Library Preparation for Plants to Study Their Three-Dimensional Chromatin Interactions on a Genome-Wide Scale.

PubMed

Liu, Chang

2017-01-01

The spatial organization of the genome in the nucleus is critical for many cellular processes. It has been broadly accepted that the packing of chromatin inside the nucleus is not random, but structured at several hierarchical levels. The Hi-C method combines Chromatin Conformation Capture and high-throughput sequencing, which allows interrogating genome-wide chromatin interactions. Depending on the sequencing depth, chromatin packing patterns derived from Hi-C experiments can be viewed on a chromosomal scale or at a local genic level. Here, I describe a protocol of plant in situ Hi-C library preparation, which covers procedures starting from tissue fixation to library amplification.

Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs.

PubMed

Sanders, Ashley D; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Lansdorp, Peter M

2017-06-01

The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5'-3' orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU + strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.

Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools.

PubMed

Guizard, Sébastien; Piégu, Benoît; Arensburger, Peter; Guillou, Florian; Bigot, Yves

2016-08-19

The program RepeatMasker and the database Repbase-ISB are part of the most widely used strategy for annotating repeats in animal genomes. They have been used to show that avian genomes have a lower repeat content (8-12 %) than the sequenced genomes of many vertebrate species (30-55 %). However, the efficiency of such a library-based strategies is dependent on the quality and completeness of the sequences in the database that is used. An alternative to these library based methods are methods that identify repeats de novo. These alternative methods have existed for a least a decade and may be more powerful than the library based methods. We have used an annotation strategy involving several complementary de novo tools to determine the repeat content of the model genome galGal4 (1.04 Gbp), including identifying simple sequence repeats (SSRs), tandem repeats and transposable elements (TEs). We annotated over one Gbp. of the galGal4 genome and showed that it is composed of approximately 19 % SSRs and TEs repeats. Furthermore, we estimate that the actual genome of the red jungle fowl contains about 31-35 % repeats. We find that library-based methods tend to overestimate TE diversity. These results have a major impact on the current understanding of repeats distributions throughout chromosomes in the red jungle fowl. Our results are a proof of concept of the reliability of using de novo tools to annotate repeats in large animal genomes. They have also revealed issues that will need to be resolved in order to develop gold-standard methodologies for annotating repeats in eukaryote genomes.

Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization

PubMed Central

Hasterok, Robert; Marasek, Agnieszka; Donnison, Iain S.; Armstead, Ian; Thomas, Ann; King, Ian P.; Wolny, Elzbieta; Idziak, Dominika; Draper, John; Jenkins, Glyn

2006-01-01

As part of an initiative to develop Brachypodium distachyon as a genomic “bridge” species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice. PMID:16489232

Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization.

PubMed

Hasterok, Robert; Marasek, Agnieszka; Donnison, Iain S; Armstead, Ian; Thomas, Ann; King, Ian P; Wolny, Elzbieta; Idziak, Dominika; Draper, John; Jenkins, Glyn

2006-05-01

As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

Evaluation of anonymous and expressed sequence tag derived polymorphic microsatellite markers in the tobacco budworm Heliothis virescens (Lepidoptera: noctuidae)

USDA-ARS?s Scientific Manuscript database

Polymorphic genetic markers were identified and characterized using a partial genomic library of Heliothis virescens enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 u...

Genomic sequencing of Pleistocene cave bears

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noonan, James P.; Hofreiter, Michael; Smith, Doug

2005-04-01

Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome,more » the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.« less

Library construction for next-generation sequencing: Overviews and challenges

PubMed Central

Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

2014-01-01

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

PubMed Central

2013-01-01

Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome. PMID:24010766

Exploring Pandora's Box: Potential and Pitfalls of Low Coverage Genome Surveys for Evolutionary Biology

PubMed Central

Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.

2012-01-01

High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309

Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

PubMed

Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

2008-06-01

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

The development and characterisation of a bacterial artificial chromosome library for Fragaria vesca

PubMed Central

Bonet, Julio; Girona, Elena Lopez; Sargent, Daniel J; Muñoz-Torres, Monica C; Monfort, Amparo; Abbott, Albert G; Arús, Pere; Simpson, David W; Davik, Jahn

2009-01-01

Background The cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library. Methods A BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN). Findings Clones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size. Conclusion This BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR. PMID:19772672

Characterization of Three Maize Bacterial Artificial Chromosome Libraries toward Anchoring of the Physical Map to the Genetic Map Using High-Density Bacterial Artificial Chromosome Filter Hybridization1

PubMed Central

Yim, Young-Sun; Davis, Georgia L.; Duru, Ngozi A.; Musket, Theresa A.; Linton, Eric W.; Messing, Joachim W.; McMullen, Michael D.; Soderlund, Carol A.; Polacco, Mary L.; Gardiner, Jack M.; Coe, Edward H.

2002-01-01

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction. PMID:12481051

CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.

PubMed

Köferle, Anna; Worf, Karolina; Breunig, Christopher; Baumann, Valentin; Herrero, Javier; Wiesbeck, Maximilian; Hutter, Lukas H; Götz, Magdalena; Fuchs, Christiane; Beck, Stephan; Stricker, Stefan H

2016-11-14

The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

PubMed

Kim, Heon Seok; Lee, Kyungjin; Bae, Sangsu; Park, Jeongbin; Lee, Chong-Kyo; Kim, Meehyein; Kim, Eunji; Kim, Minju; Kim, Seokjoong; Kim, Chonsaeng; Kim, Jin-Soo

2017-06-23

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes ( i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Developing new microsatellite markers in walnut (Juglans regia L.) from Juglans nigra genomic GA enriched library

Treesearch

Hayat Topcu; Nergiz Coban; Keith Woeste; Mehmet Sutyemez; Salih Kafkas

2015-01-01

We attempted to develop new polymorphic SSR primer pairs in walnut using sequences derived from Juglans nigra L. genomic enriched library with GA repeat. The designed 94 SSR primer pairs were subjected to gradient PCR in 12 walnut cultivars to determine their optimum annealing temperatures and to determine whether they produce bands. Then, the...

Democratizing Human Genome Project Information: A Model Program for Education, Information and Debate in Public Libraries.

ERIC Educational Resources Information Center

Pollack, Miriam

The "Mapping the Human Genome" project demonstrated that librarians can help whomever they serve in accessing information resources in the areas of biological and health information, whether it is the scientists who are developing the information or a member of the public who is using the information. Public libraries can guide library…

Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christian, A T; Coleman, M A; Tucker, J D

2001-02-08

Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

Next-generation sequencing library construction on a surface.

PubMed

Feng, Kuan; Costa, Justin; Edwards, Jeremy S

2018-05-30

Next-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface. The surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates. We described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

zipHMMlib: a highly optimised HMM library exploiting repetitions in the input to speed up the forward algorithm.

PubMed

Sand, Andreas; Kristiansen, Martin; Pedersen, Christian N S; Mailund, Thomas

2013-11-22

Hidden Markov models are widely used for genome analysis as they combine ease of modelling with efficient analysis algorithms. Calculating the likelihood of a model using the forward algorithm has worst case time complexity linear in the length of the sequence and quadratic in the number of states in the model. For genome analysis, however, the length runs to millions or billions of observations, and when maximising the likelihood hundreds of evaluations are often needed. A time efficient forward algorithm is therefore a key ingredient in an efficient hidden Markov model library. We have built a software library for efficiently computing the likelihood of a hidden Markov model. The library exploits commonly occurring substrings in the input to reuse computations in the forward algorithm. In a pre-processing step our library identifies common substrings and builds a structure over the computations in the forward algorithm which can be reused. This analysis can be saved between uses of the library and is independent of concrete hidden Markov models so one preprocessing can be used to run a number of different models.Using this library, we achieve up to 78 times shorter wall-clock time for realistic whole-genome analyses with a real and reasonably complex hidden Markov model. In one particular case the analysis was performed in less than 8 minutes compared to 9.6 hours for the previously fastest library. We have implemented the preprocessing procedure and forward algorithm as a C++ library, zipHMM, with Python bindings for use in scripts. The library is available at http://birc.au.dk/software/ziphmm/.

Library preparation and data analysis packages for rapid genome sequencing.

PubMed

Pomraning, Kyle R; Smith, Kristina M; Bredeweg, Erin L; Connolly, Lanelle R; Phatale, Pallavi A; Freitag, Michael

2012-01-01

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.

Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway

PubMed Central

2012-01-01

Background Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. Results Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. Conclusions Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens. PMID:23006893

YAC cloning Mus musculus telomeric DNA: physical, genetic, in situ and STS markers for the distal telomere of chromosome 10.

PubMed

Kipling, D; Wilson, H E; Thomson, E J; Cooke, H J

1995-06-01

Three Mus musculus DBA/2 YAC libraries were constructed using a half-YAC telomere cloning vector. This functional complementation approach yields libraries which include terminal restriction fragments of the mouse genome. Screening all three libraries led to the isolation of 32 independent clones which carry linear YACs containing the mouse terminal repeat sequence, (TTAGGG)n. These YACs provide a resource to isolate regions of the mouse genome close to chromosome termini and excluded from existing conventional YAC libraries. To demonstrate their utility, a hybridization probe was isolated from Mtel-1, the first (TTAGGG)n-containing YAC isolated. This probe detects a approximately 70 kb Kpnl fragment in the mouse genome which is sensitive to pretreatment with BAL31 exonuclease. A PCR-based genetic marker generated from the sequence of this probe maps 4.4 cM from the most distal anchor locus on chromosome 10 in the EUCIB interspecific backcross. STS primers for this locus, D10Hgu1, were used to isolate YAC 110F4 from a commercially available mouse YAC library. Fluorescence in situ hybridization demonstrates that YAC 110F4 hybridizes to the distal telomere of chromosome 10. Clones in this collection of telomere YACs therefore partially overlap clones in conventional YAC libraries, and thus the previously unavailable terminal regions of the mouse genome can now be linked with the developing mouse STS YAC contig. Genetic markers such as D10Hgu1 allow the ends of the mouse genetic map to be defined, thus closing the map.

pileup.js: a JavaScript library for interactive and in-browser visualization of genomic data.

PubMed

Vanderkam, Dan; Aksoy, B Arman; Hodes, Isaac; Perrone, Jaclyn; Hammerbacher, Jeff

2016-08-01

P: ileup.js is a new browser-based genome viewer. It is designed to facilitate the investigation of evidence for genomic variants within larger web applications. It takes advantage of recent developments in the JavaScript ecosystem to provide a modular, reliable and easily embedded library. The code and documentation for pileup.js is publicly available at https://github.com/hammerlab/pileup.js under the Apache 2.0 license. correspondence@hammerlab.org. © The Author 2016. Published by Oxford University Press.

Theobroma cacao: A genetically integrated physical map and genome-scale comparative synteny analysis

USDA-ARS?s Scientific Manuscript database

A comprehensive integrated genomic framework is considered a centerpiece of genomic research. In collaboration with the USDA-ARS (SHRS) and Mars Inc., the Clemson University Genomics Institute (CUGI) has developed a genetically anchored physical map of the T. cacao genome. Three BAC libraries contai...

SVGenes: a library for rendering genomic features in scalable vector graphic format.

PubMed

Etherington, Graham J; MacLean, Daniel

2013-08-01

Drawing genomic features in attractive and informative ways is a key task in visualization of genomics data. Scalable Vector Graphics (SVG) format is a modern and flexible open standard that provides advanced features including modular graphic design, advanced web interactivity and animation within a suitable client. SVGs do not suffer from loss of image quality on re-scaling and provide the ability to edit individual elements of a graphic on the whole object level independent of the whole image. These features make SVG a potentially useful format for the preparation of publication quality figures including genomic objects such as genes or sequencing coverage and for web applications that require rich user-interaction with the graphical elements. SVGenes is a Ruby-language library that uses SVG primitives to render typical genomic glyphs through a simple and flexible Ruby interface. The library implements a simple Page object that spaces and contains horizontal Track objects that in turn style, colour and positions features within them. Tracks are the level at which visual information is supplied providing the full styling capability of the SVG standard. Genomic entities like genes, transcripts and histograms are modelled in Glyph objects that are attached to a track and take advantage of SVG primitives to render the genomic features in a track as any of a selection of defined glyphs. The feature model within SVGenes is simple but flexible and not dependent on particular existing gene feature formats meaning graphics for any existing datasets can easily be created without need for conversion. The library is provided as a Ruby Gem from https://rubygems.org/gems/bio-svgenes under the MIT license, and open source code is available at https://github.com/danmaclean/bioruby-svgenes also under the MIT License. dan.maclean@tsl.ac.uk.

Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening

PubMed Central

Joung, Julia; Konermann, Silvana; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Platt, Randall J.; Brigham, Mark D.; Sanjana, Neville E.; Zhang, Feng

2017-01-01

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks followed by 4–5 weeks of validation. PMID:28333914

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

PubMed

Joung, Julia; Konermann, Silvana; Gootenberg, Jonathan S; Abudayyeh, Omar O; Platt, Randall J; Brigham, Mark D; Sanjana, Neville E; Zhang, Feng

2017-04-01

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.

PubMed

Shi, Xue; Zeng, Haiyang; Xue, Yadong; Luo, Meizhong

2011-10-11

Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.

Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

USDA-ARS?s Scientific Manuscript database

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

PubMed Central

Shepherd, N S; Pfrogner, B D; Coulby, J N; Ackerman, S L; Vaidyanathan, G; Sauer, R H; Balkenhol, T C; Sternberg, N

1994-01-01

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. Images PMID:8146166

Final progress report, Construction of a genome-wide highly characterized clone resource for genome sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nierman, William C.

At TIGR, the human Bacterial Artificial Chromosome (BAC) end sequencing and trimming were with an overall sequencing success rate of 65%. CalTech human BAC libraries A, B, C and D as well as Roswell Park Cancer Institute's library RPCI-11 were used. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length {approx}460 bp for a total of 141 Mb covering {approx}4.7% of the genome. Over sixty percent of the clones have BAC end sequences (BESs) from both ends representing over five-fold coverage of the genome by the paired-end clones. The average phredmore » Q20 length is {approx}400 bp. This high accuracy makes our BESs match the human finished sequences with an average identity of 99% and a match length of 450 bp, and a frequency of one match per 12.8 kb contig sequence. Our sample tracking has ensured a clone tracking accuracy of >90%, which gives researchers a high confidence in (1) retrieving the right clone from the BA C libraries based on the sequence matches; and (2) building a minimum tiling path of sequence-ready clones across the genome and genome assembly scaffolds.« less

The SUPERFAMILY database in 2004: additions and improvements.

PubMed

Madera, Martin; Vogel, Christine; Kummerfeld, Sarah K; Chothia, Cyrus; Gough, Julian

2004-01-01

The SUPERFAMILY database provides structural assignments to protein sequences and a framework for analysis of the results. At the core of the database is a library of profile Hidden Markov Models that represent all proteins of known structure. The library is based on the SCOP classification of proteins: each model corresponds to a SCOP domain and aims to represent an entire superfamily. We have applied the library to predicted proteins from all completely sequenced genomes (currently 154), the Swiss-Prot and TrEMBL databases and other sequence collections. Close to 60% of all proteins have at least one match, and one half of all residues are covered by assignments. All models and full results are available for download and online browsing at http://supfam.org. Users can study the distribution of their superfamily of interest across all completely sequenced genomes, investigate with which other superfamilies it combines and retrieve proteins in which it occurs. Alternatively, concentrating on a particular genome as a whole, it is possible first, to find out its superfamily composition, and secondly, to compare it with that of other genomes to detect superfamilies that are over- or under-represented. In addition, the webserver provides the following standard services: sequence search; keyword search for genomes, superfamilies and sequence identifiers; and multiple alignment of genomic, PDB and custom sequences.

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

PubMed Central

Shimoda, Yoshikazu; Mitsui, Hisayuki; Kamimatsuse, Hiroko; Minamisawa, Kiwamu; Nishiyama, Eri; Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka; Shinpo, Sayaka; Watanabe, Akiko; Kohara, Mitsuyo; Yamada, Manabu; Nakamura, Yasukazu; Tabata, Satoshi; Sato, Shusei

2008-01-01

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology. PMID:18658183

BACCardI--a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison.

PubMed

Bartels, Daniela; Kespohl, Sebastian; Albaum, Stefan; Drüke, Tanja; Goesmann, Alexander; Herold, Julia; Kaiser, Olaf; Pühler, Alfred; Pfeiffer, Friedhelm; Raddatz, Günter; Stoye, Jens; Meyer, Folker; Schuster, Stephan C

2005-04-01

We provide the graphical tool BACCardI for the construction of virtual clone maps from standard assembler output files or BLAST based sequence comparisons. This new tool has been applied to numerous genome projects to solve various problems including (a) validation of whole genome shotgun assemblies, (b) support for contig ordering in the finishing phase of a genome project, and (c) intergenome comparison between related strains when only one of the strains has been sequenced and a large insert library is available for the other. The BACCardI software can seamlessly interact with various sequence assembly packages. Genomic assemblies generated from sequence information need to be validated by independent methods such as physical maps. The time-consuming task of building physical maps can be circumvented by virtual clone maps derived from read pair information of large insert libraries.

Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.

PubMed Central

Bookstein, R; Lee, E Y; To, H; Young, L J; Sery, T W; Hayes, R C; Friedmann, T; Lee, W H

1988-01-01

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families. Images PMID:2895471

Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14.

PubMed

Telke, Amar A; Rolain, Jean-Marc

2015-12-01

Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Genomic resources for songbird research and their use in characterizing gene expression during brain development

PubMed Central

Li, XiaoChing; Wang, Xiu-Jie; Tannenhauser, Jonathan; Podell, Sheila; Mukherjee, Piali; Hertel, Moritz; Biane, Jeremy; Masuda, Shoko; Nottebohm, Fernando; Gaasterland, Terry

2007-01-01

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3′ ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30–50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis. PMID:17426146

Comparative Genomics as a Foundation for Evo-Devo Studies in Birds.

PubMed

Grayson, Phil; Sin, Simon Y W; Sackton, Timothy B; Edwards, Scott V

2017-01-01

Developmental genomics is a rapidly growing field, and high-quality genomes are a useful foundation for comparative developmental studies. A high-quality genome forms an essential reference onto which the data from numerous assays and experiments, including ChIP-seq, ATAC-seq, and RNA-seq, can be mapped. A genome also streamlines and simplifies the development of primers used to amplify putative regulatory regions for enhancer screens, cDNA probes for in situ hybridization, microRNAs (miRNAs) or short hairpin RNAs (shRNA) for RNA interference (RNAi) knockdowns, mRNAs for misexpression studies, and even guide RNAs (gRNAs) for CRISPR knockouts. Finally, much can be gleaned from comparative genomics alone, including the identification of highly conserved putative regulatory regions. This chapter provides an overview of laboratory and bioinformatics protocols for DNA extraction, library preparation, library quantification, and genome assembly, from fresh or frozen tissue to a draft avian genome. Generating a high-quality draft genome can provide a developmental research group with excellent resources for their study organism, opening the doors to many additional assays and experiments.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

PubMed

Koo, Byoung-Mo; Kritikos, George; Farelli, Jeremiah D; Todor, Horia; Tong, Kenneth; Kimsey, Harvey; Wapinski, Ilan; Galardini, Marco; Cabal, Angelo; Peters, Jason M; Hachmann, Anna-Barbara; Rudner, David Z; Allen, Karen N; Typas, Athanasios; Gross, Carol A

2017-03-22

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genomic clones for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kott, M.; Venta, P.J.; Larsen, J.

1987-05-01

A human genomic library was prepared from peripheral white blood cells from a single donor by inserting an MboI partial digest into BamHI poly-linker sites of EMBL3. This library was screened using an oligolabeled human cholinesterase cDNA probe over 700 bp long. The latter probe was obtained from a human basal ganglia cDNA library. Of approximately 2 million clones screened with high stringency conditions several positive clones were identified; two have been plaque purified. One of these clones has been partially mapped using restriction enzymes known to cut within the coded region of the cDNA for human serum cholinesterase. Hybridizationmore » of the fragments and their sizes are as expected if the genomic clone is cholinesterase. Sequencing of the DNA fragments in M13 is in progress to verify the identify of the clone and the location of introns.« less

Comparative genome map of human and cattle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solinas-Toldo, S.; Fries, R.; Lengauer, C.

Chromosomal homologies between individual human chromosomes and the bovine karyotype have been established by using a new approach termed Zoo-FISH. Labeled DNA libraries from flow-sorted human chromosomes were used as probes for fluorescence in situ hybridization on cattle chromosomes. All human DNA libraries, except the Y chromosome library, hybridized to one or more cattle chromosomes, identifying and delineating 50 segments of homology, most of them corresponding to the regions of homology as identified by the previous mapping of individual conserved loci. However, Zoo-FISH refines the comparative maps constructed by molecular gene mapping of individual loci by providing information on themore » boundaries of conserved regions in the absence of obvious cytogenetic homologies of human and bovine chromosomes. It allows study of karyotypic evolution and opens new avenues for genomic analysis by facilitating the extrapolation of results from the human genome initiative. 50 refs., 3 figs., 1 tab.« less

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

PubMed

Olova, Nelly; Krueger, Felix; Andrews, Simon; Oxley, David; Berrens, Rebecca V; Branco, Miguel R; Reik, Wolf

2018-03-15

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

PubMed Central

2011-01-01

Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean. PMID:22118559

Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

PubMed

Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

2017-05-01

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.

BAC library development, and clone characterization for dormancy-responsive DREB4A, DAM, and FT from leafy spurge (Euphorbia esula L.) identifies differential splicing and conserved promoter motifs

USDA-ARS?s Scientific Manuscript database

We developed two leafy spurge BAC libraries that together represent approximately 5X coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the ...

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

PubMed Central

Ritschel, Patricia Silva; Lins, Tulio Cesar de Lima; Tristan, Rodrigo Lourenço; Buso, Gláucia Salles Cortopassi; Buso, José Amauri; Ferreira, Márcio Elias

2004-01-01

Background Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. Results Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. Conclusions Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon. PMID:15149552

Genome sequencing of a single tardigrade Hypsibius dujardini individual

PubMed Central

Arakawa, Kazuharu; Yoshida, Yuki; Tomita, Masaru

2016-01-01

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies. PMID:27529330

Genome sequencing of a single tardigrade Hypsibius dujardini individual.

PubMed

Arakawa, Kazuharu; Yoshida, Yuki; Tomita, Masaru

2016-08-16

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies.

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. Amore » minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.« less

Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library.

PubMed

Hulse-Kemp, Amanda M; Maheshwari, Shamoni; Stoffel, Kevin; Hill, Theresa A; Jaffe, David; Williams, Stephen R; Weisenfeld, Neil; Ramakrishnan, Srividya; Kumar, Vijay; Shah, Preyas; Schatz, Michael C; Church, Deanna M; Van Deynze, Allen

2018-01-01

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of human genomes, however, the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5-gigabase (Gb) diploid pepper ( Capsicum annuum ) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure that the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F 1 derived from a wide cross to assess the ability to derive both haplotypes and characterize a pungency gene with a large insertion/deletion. The Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Over 83% of the final assembly was anchored and oriented using four publicly available  de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5-Kb insertion/deletion haplotypes of the PUN1 locus in the F 1 sample that represents pungent and nonpungent peppers, as well as nearly full recovery of the BUSCO2 gene set within each of the two haplotypes. The most contiguous pepper genome assembly to date has been generated which demonstrates that Linked-Read library technology provides a tool to de novo assemble complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.

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.

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

PubMed Central

2010-01-01

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 indicated that both BAC libraries are valuable tools for molecular studies in chicory, one goal being the positional cloning of the S-locus in this Asteraceae species. PMID:20701751

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

PubMed

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 studies in chicory, one goal being the positional cloning of the S-locus in this Asteraceae species.

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

PubMed

Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

2008-09-01

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

A High-Quality Reference Genome for the Invasive Mosquitofish Gambusia affinis Using a Chicago Library.

PubMed

Hoffberg, Sandra L; Troendle, Nicholas J; Glenn, Travis C; Mahmud, Ousman; Louha, Swarnali; Chalopin, Domitille; Bennetzen, Jeffrey L; Mauricio, Rodney

2018-04-27

The western mosquitofish, Gambusia affinis, is a freshwater poecilid fish native to the southeastern United States but with a global distribution due to widespread human introduction. Gambusia affinis has been used as a model species for a broad range of evolutionary and ecological studies. We sequenced the genome of a male G. affinis to facilitate genetic studies in diverse fields including invasion biology and comparative genetics. We generated Illumina short read data from paired-end libraries and in vitro proximity-ligation libraries. We obtained 54.9× coverage, N50 contig length of 17.6 kb, and N50 scaffold length of 6.65 Mb. Compared to two other species in the Poeciliidae family, G. affinis has slightly fewer genes that have shorter total, exon, and intron length on average. Using a set of universal single-copy orthologs in fish genomes, we found 95.5% of these genes were complete in the G. affinis assembly. The number of transposable elements in the G. affinis assembly is similar to those of closely related species. The high-quality genome sequence and annotations we report will be valuable resources for scientists to map the genetic architecture of traits of interest in this species. Copyright © 2018, G3: Genes, Genomes, Genetics.

Next-generation libraries for robust RNA interference-based genome-wide screens

PubMed Central

Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

2015-01-01

Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

A Fast Solution to NGS Library Prep with Low Nanogram DNA Input

PubMed Central

Liu, Pingfang; Lohman, Gregory J.S.; Cantor, Eric; Langhorst, Bradley W.; Yigit, Erbay; Apone, Lynne M.; Munafo, Daniela B.; Stewart, Fiona J.; Evans, Thomas C.; Nichols, Nicole; Dimalanta, Eileen T.; Davis, Theodore B.; Sumner, Christine

2013-01-01

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

PubMed

Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

2013-01-01

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Begin at the beginning: A BAC-end view of the passion fruit (Passiflora) genome.

PubMed

Santos, Anselmo Azevedo; Penha, Helen Alves; Bellec, Arnaud; Munhoz, Carla de Freitas; Pedrosa-Harand, Andrea; Bergès, Hélène; Vieira, Maria Lucia Carneiro

2014-09-26

The passion fruit (Passiflora edulis) is a tropical crop of economic importance both for juice production and consumption as fresh fruit. The juice is also used in concentrate blends that are consumed worldwide. However, very little is known about the genome of the species. Therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. In this study, we have constructed a large-insert BAC library and provided the first view on the structure and content of the passion fruit genome, using BAC-end sequence (BES) data as a major resource. The library consisted of 82,944 clones and its levels of organellar DNA were very low. The library represents six haploid genome equivalents, and the average insert size was 108 kb. To check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight Passiflora gene sequences available in public databases. BACs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four BACs in three chromosomes (n=9). Then, we explored 10,000 BES and we identified reads likely to contain repetitive mobile elements (19.6% of all BES), simple sequence repeats and putative proteins, and to estimate the GC content (~42%) of the reads. Around 9.6% of all BES were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed (940). The vast majority of the top-hits made by our sequences were to Populus trichocarpa (24.8% of the total occurrences), Theobroma cacao (21.6%), Ricinus communis (14.3%), Vitis vinifera (6.5%) and Prunus persica (3.8%). We generated the first large-insert library for a member of Passifloraceae. This BAC library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. Remarkably, a number of BAC-end pair sequences could be mapped to intervals of the sequenced Arabidopsis thaliana, V. vinifera and P. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified.

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

PubMed Central

2017-01-01

ABSTRACT The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. PMID:29327641

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

PubMed Central

2011-01-01

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol. PMID:21205303

Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library.

PubMed

Sánchez, Cecilia Castaño; Smith, Timothy P L; Wiedmann, Ralph T; Vallejo, Roger L; Salem, Mohamed; Yao, Jianbo; Rexroad, Caird E

2009-11-25

To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts. The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

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

PubMed Central

2011-01-01

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 homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy. PMID:21767393

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

PubMed

Saski, Christopher A; Li, Zhigang; Feltus, Frank A; Luo, Hong

2011-07-18

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. 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. The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy.

A Glimpse into the Satellite DNA Library in Characidae Fish (Teleostei, Characiformes)

PubMed Central

Utsunomia, Ricardo; Ruiz-Ruano, Francisco J.; Silva, Duílio M. Z. A.; Serrano, Érica A.; Rosa, Ivana F.; Scudeler, Patrícia E. S.; Hashimoto, Diogo T.; Oliveira, Claudio; Camacho, Juan Pedro M.; Foresti, Fausto

2017-01-01

Satellite DNA (satDNA) is an abundant fraction of repetitive DNA in eukaryotic genomes and plays an important role in genome organization and evolution. In general, satDNA sequences follow a concerted evolutionary pattern through the intragenomic homogenization of different repeat units. In addition, the satDNA library hypothesis predicts that related species share a series of satDNA variants descended from a common ancestor species, with differential amplification of different satDNA variants. The finding of a same satDNA family in species belonging to different genera within Characidae fish provided the opportunity to test both concerted evolution and library hypotheses. For this purpose, we analyzed here sequence variation and abundance of this satDNA family in ten species, by a combination of next generation sequencing (NGS), PCR and Sanger sequencing, and fluorescence in situ hybridization (FISH). We found extensive between-species variation for the number and size of pericentromeric FISH signals. At genomic level, the analysis of 1000s of DNA sequences obtained by Illumina sequencing and PCR amplification allowed defining 150 haplotypes which were linked in a common minimum spanning tree, where different patterns of concerted evolution were apparent. This also provided a glimpse into the satDNA library of this group of species. In consistency with the library hypothesis, different variants for this satDNA showed high differences in abundance between species, from highly abundant to simply relictual variants. PMID:28855916

Engineering improved bio-jet fuel tolerance in Escherichia coli using a transgenic library from the hydrocarbon-degrader Marinobacter aquaeolei.

PubMed

Tomko, Timothy A; Dunlop, Mary J

2015-01-01

Recent metabolic engineering efforts have generated microorganisms that can produce biofuels, including bio-jet fuels, however these fuels are often toxic to cells, limiting production yields. There are natural examples of microorganisms that have evolved mechanisms for tolerating hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. Using genomic DNA from the hydrocarbon-degrading microbe Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy with a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. The genomes of hydrocarbon-tolerant microbes represent a rich resource for tolerance engineering. Using a transgenic library, we were able to identify a single gene that improves E. coli's tolerance to the bio-jet fuel precursor pinene.

BAC Libraries from Wheat Chromosome 7D – Efficient Tool for Positional Cloning of Aphid Resistance Genes

USDA-ARS?s Scientific Manuscript database

Positional cloning in bread wheat is a tedious task due to its huge genome size (~17 Gbp) and polyploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which make their screening very laborious. Here we pres...

Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.

PubMed

Song, Beng-Kah; Nadarajah, Kalaivani; Romanov, Michael N; Ratnam, Wickneswari

2005-01-01

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.

GenomeVista

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poliakov, Alexander; Couronne, Olivier

2002-11-04

Aligning large vertebrate genomes that are structurally complex poses a variety of problems not encountered on smaller scales. Such genomes are rich in repetitive elements and contain multiple segmental duplications, which increases the difficulty of identifying true orthologous SNA segments in alignments. The sizes of the sequences make many alignment algorithms designed for comparing single proteins extremely inefficient when processing large genomic intervals. We integrated both local and global alignment tools and developed a suite of programs for automatically aligning large vertebrate genomes and identifying conserved non-coding regions in the alignments. Our method uses the BLAT local alignment program tomore » find anchors on the base genome to identify regions of possible homology for a query sequence. These regions are postprocessed to find the best candidates which are then globally aligned using the AVID global alignment program. In the last step conserved non-coding segments are identified using VISTA. Our methods are fast and the resulting alignments exhibit a high degree of sensitivity, covering more than 90% of known coding exons in the human genome. The GenomeVISTA software is a suite of Perl programs that is built on a MySQL database platform. The scheduler gets control data from the database, builds a queve of jobs, and dispatches them to a PC cluster for execution. The main program, running on each node of the cluster, processes individual sequences. A Perl library acts as an interface between the database and the above programs. The use of a separate library allows the programs to function independently of the database schema. The library also improves on the standard Perl MySQL database interfere package by providing auto-reconnect functionality and improved error handling.« less

Scribl: an HTML5 Canvas-based graphics library for visualizing genomic data over the web.

PubMed

Miller, Chase A; Anthony, Jon; Meyer, Michelle M; Marth, Gabor

2013-02-01

High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported.

Bioprospecting for Genes that Confer Biofuel Tolerance to Escherichia Coli Using a Genomic Library Approach

NASA Astrophysics Data System (ADS)

Tomko, Timothy

Microorganisms are capable of producing advanced biofuels that can be used as 'drop-in' alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida, induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoD P. putida,, while others provide tolerance across a wide range of rpoDP. putida, levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

Genome-wide transposon insertion scanning of environmental survival functions in the polycyclic aromatic hydrocarbon degrading bacterium Sphingomonas wittichii RW1.

PubMed

Roggo, Clémence; Coronado, Edith; Moreno-Forero, Silvia K; Harshman, Keith; Weber, Johann; van der Meer, Jan Roelof

2013-10-01

Sphingomonas wittichii RW1 is a dibenzofuran and dibenzodioxin-degrading bacterium with potentially interesting properties for bioaugmentation of contaminated sites. In order to understand the capacity of the microorganism to survive in the environment we used a genome-wide transposon scanning approach. RW1 transposon libraries were generated with around 22,000 independent insertions. Libraries were grown for an average of 50 generations (five successive passages in batch liquid medium) with salicylate as sole carbon and energy source in presence or absence of salt stress at -1.5 MPa. Alternatively, libraries were grown in sand with salicylate, at 50% water holding capacity, for 4 and 10 days (equivalent to 7 generations). Library DNA was recovered from the different growth conditions and scanned by ultrahigh throughput sequencing for the positions and numbers of inserted transposed kanamycin resistance gene. No transposon reads were recovered in 579 genes (10% of all annotated genes in the RW1 genome) in any of the libraries, suggesting those to be essential for survival under the used conditions. Libraries recovered from sand differed strongly from those incubated in liquid batch medium. In particular, important functions for survival of cells in sand at the short term concerned nutrient scavenging, energy metabolism and motility. In contrast to this, fatty acid metabolism and oxidative stress response were essential for longer term survival of cells in sand. Comparison to transcriptome data suggested important functions in sand for flagellar movement, pili synthesis, trehalose and polysaccharide synthesis and putative cell surface antigen proteins. Interestingly, a variety of genes were also identified, interruption of which cause significant increase in fitness during growth on salicylate. One of these was an Lrp family transcription regulator and mutants in this gene covered more than 90% of the total library after 50 generations of growth on salicylate. Our results demonstrate the power of genome-wide transposon scanning approaches for analysis of complex traits. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Genomics of compositae weeds: EST libraries, microarrays, and evidence of introgression

USDA-ARS?s Scientific Manuscript database

• Premise of Study: Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we report on the development of genomic tools and resources for 11 weeds from the Compositae family that can serve ...

Genome-wide annotation of mutations in a phenotyped mutant library provides an efficient platform for discovery of casual gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant l...

78 FR 55752 - National Human Genome Research Institute; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-11

... applications. Place: National Human Genome Research Institute, 4th Floor Library, 5635 Fishers Lane, Rockville... Research Institute; Notice of Closed Meetings Pursuant to section 10(d) of the Federal Advisory Committee... clearly unwarranted invasion of personal privacy. Name of Committee: National Human Genome Research...

Modeling genome coverage in single-cell sequencing

PubMed Central

Daley, Timothy; Smith, Andrew D.

2014-01-01

Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Liao, Guochun; Brokstein, Peter

2002-08-12

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remainingmore » genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.« less

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity

PubMed Central

Hurst, Gregory D.D.

2017-01-01

High throughput (or ‘next generation’) sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and ‘contaminating’ material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these ‘contaminations’ provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee (Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo. We conclude that ‘contamination’ in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses. PMID:28717593

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity.

PubMed

Gerth, Michael; Hurst, Gregory D D

2017-01-01

High throughput (or 'next generation') sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and 'contaminating' material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these 'contaminations' provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee ( Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo . We conclude that 'contamination' in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses.

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices.

PubMed

Hoople, Gordon D; Richards, Andrew; Wu, Yan; Pisano, Albert P; Zhang, Kun

2018-03-26

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

PubMed Central

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

Global Genomic Analysis of Prostate, Breast and Pancreatic Cancer

DTIC Science & Technology

2012-10-01

fever virus (Lauck et al. 2011). The success of transposon-based genomic library construction for genomic analyses suggests that it should be possible...2011. Novel, divergent simian hemorrhagic Fever viruses in a wild ugandan red colobus Gertz et al. 140 Genome Research www.genome.org Cold Spring...2009. A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi. PLoS Genet 5: e1000569. doi: 10.1371

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

PubMed

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

PubMed

Park, Jeongbin; Bae, Sangsu

2018-03-15

Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time. Free access at http://www.rgenome.net/cpf1-database/. sangsubae@hanyang.ac.kr.

Genome-scale CRISPR-Cas9 knockout screening in human cells.

PubMed

Shalem, Ophir; Sanjana, Neville E; Hartenian, Ella; Shi, Xi; Scott, David A; Mikkelson, Tarjei; Heckl, Dirk; Ebert, Benjamin L; Root, David E; Doench, John G; Zhang, Feng

2014-01-03

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

Genome-wide mapping of autonomous promoter activity in human cells

PubMed Central

van Arensbergen, Joris; FitzPatrick, Vincent D.; de Haas, Marcel; Pagie, Ludo; Sluimer, Jasper; Bussemaker, Harmen J.; van Steensel, Bas

2017-01-01

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than 108 DNA fragments, each 0.2–2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, we show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites. PMID:28024146

Identification and characterization of dinucleotide repeat (CA)[sub n] markers for genetic mapping in dog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrander, E.A.; Sprague, G.F. Jr.; Rine, J.

1993-04-01

A large block of simple sequence repeat (SSR) polymorphisms for the dog genome has been isolated and characterized. Screening of primary libraries by conventional hybridization methods as well as by screening of enriched marker-selected libraries led to the isolation of a large number of genomic clones that contained (CA)[sub n] repeats. The sequences of 101 clones showed that the size and complexity of (CA)[sub n] repeats in the dog genome were similar to those reported for these markers in the human genome. Detailed analysis of a representative subset of these markers revealed that most markers were moderately to highly polymorphic,more » with PIC values exceeding 0.70 for 33% of the markers tested. An association between higher PIC values and markers containing longer (CA)[sub n] repeats was observed in these studies, as previously noted for similar markers in the human genome. A list of primer sequences that tag each characterized marker is provided, and a comprehensive system of nomenclature for the dog genome is suggested. 28 refs., 4 figs., 2 tabs.« less

Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yaung, Stephanie J.; Deng, Luxue; Li, Ning

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Populationmore » dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.« less

Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

DOE PAGES

Yaung, Stephanie J.; Deng, Luxue; Li, Ning; ...

2015-03-11

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Populationmore » dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.« less

Overview of hybridization and detection techniques.

PubMed

Hilario, Elena

2007-01-01

A misconception regarding the sensitivity of nonradioactive methods for screening genomic DNA libraries often hinders the establishment of these environmentally friendly techniques in molecular biology laboratories. Nonradioactive probes, properly prepared and quantified, can detect DNA target molecules to the femtomole range. However, appropriate hybridization techniques and detection methods should also be adopted for an efficient use of nonradioactive techniques. Detailed descriptions of genomic library handling before and during the nonradioactive hybridization and detection are often omitted from publications. This chapter aims to fill this void by providing a collection of technical tips on hybridization and detection techniques.

Multiplexed microsatellite recovery using massively parallel sequencing

USGS Publications Warehouse

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

Scribl: an HTML5 Canvas-based graphics library for visualizing genomic data over the web

PubMed Central

Miller, Chase A.; Anthony, Jon; Meyer, Michelle M.; Marth, Gabor

2013-01-01

Motivation: High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. Results: Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. Availability and implementation: Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported. Contact: gabor.marth@bc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23172864

Functional assessment of human enhancer activities using whole-genome STARR-sequencing.

PubMed

Liu, Yuwen; Yu, Shan; Dhiman, Vineet K; Brunetti, Tonya; Eckart, Heather; White, Kevin P

2017-11-20

Genome-wide quantification of enhancer activity in the human genome has proven to be a challenging problem. Recent efforts have led to the development of powerful tools for enhancer quantification. However, because of genome size and complexity, these tools have yet to be applied to the whole human genome.  In the current study, we use a human prostate cancer cell line, LNCaP as a model to perform whole human genome STARR-seq (WHG-STARR-seq) to reliably obtain an assessment of enhancer activity. This approach builds upon previously developed STARR-seq in the fly genome and CapSTARR-seq techniques in targeted human genomic regions. With an improved library preparation strategy, our approach greatly increases the library complexity per unit of starting material, which makes it feasible and cost-effective to explore the landscape of regulatory activity in the much larger human genome. In addition to our ability to identify active, accessible enhancers located in open chromatin regions, we can also detect sequences with the potential for enhancer activity that are located in inaccessible, closed chromatin regions. When treated with the histone deacetylase inhibitor, Trichostatin A, genes nearby this latter class of enhancers are up-regulated, demonstrating the potential for endogenous functionality of these regulatory elements. WHG-STARR-seq provides an improved approach to current pipelines for analysis of high complexity genomes to gain a better understanding of the intricacies of transcriptional regulation.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

ParallABEL: an R library for generalized parallelization of genome-wide association studies.

PubMed

Sangket, Unitsa; Mahasirimongkol, Surakameth; Chantratita, Wasun; Tandayya, Pichaya; Aulchenko, Yurii S

2010-04-29

Genome-Wide Association (GWA) analysis is a powerful method for identifying loci associated with complex traits and drug response. Parts of GWA analyses, especially those involving thousands of individuals and consuming hours to months, will benefit from parallel computation. It is arduous acquiring the necessary programming skills to correctly partition and distribute data, control and monitor tasks on clustered computers, and merge output files. Most components of GWA analysis can be divided into four groups based on the types of input data and statistical outputs. The first group contains statistics computed for a particular Single Nucleotide Polymorphism (SNP), or trait, such as SNP characterization statistics or association test statistics. The input data of this group includes the SNPs/traits. The second group concerns statistics characterizing an individual in a study, for example, the summary statistics of genotype quality for each sample. The input data of this group includes individuals. The third group consists of pair-wise statistics derived from analyses between each pair of individuals in the study, for example genome-wide identity-by-state or genomic kinship analyses. The input data of this group includes pairs of SNPs/traits. The final group concerns pair-wise statistics derived for pairs of SNPs, such as the linkage disequilibrium characterisation. The input data of this group includes pairs of individuals. We developed the ParallABEL library, which utilizes the Rmpi library, to parallelize these four types of computations. ParallABEL library is not only aimed at GenABEL, but may also be employed to parallelize various GWA packages in R. The data set from the North American Rheumatoid Arthritis Consortium (NARAC) includes 2,062 individuals with 545,080, SNPs' genotyping, was used to measure ParallABEL performance. Almost perfect speed-up was achieved for many types of analyses. For example, the computing time for the identity-by-state matrix was linearly reduced from approximately eight hours to one hour when ParallABEL employed eight processors. Executing genome-wide association analysis using the ParallABEL library on a computer cluster is an effective way to boost performance, and simplify the parallelization of GWA studies. ParallABEL is a user-friendly parallelization of GenABEL.

The Essential Genome of Escherichia coli K-12.

PubMed

Goodall, Emily C A; Robinson, Ashley; Johnston, Iain G; Jabbari, Sara; Turner, Keith A; Cunningham, Adam F; Lund, Peter A; Cole, Jeffrey A; Henderson, Ian R

2018-02-20

Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry. IMPORTANCE Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli , we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli , reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data. Copyright © 2018 Goodall et al.

Novel Approaches to Breast Cancer Prevention and Inhibition of Metastases

DTIC Science & Technology

2013-10-01

allow a functional characterization of human candidate breast cancer genes. The transgenic RNAi library is covering the whole Drosophila genome ...W81XWH-12-1-0093 / Penninger 15. SUBJECT TERMS Genome wide functional genetics, haploid stem cells, Drosophila cancer modeling...With the advent of modern genomics hundreds of candidate genes have been associated with breast cancer both in GWAS studies as well as by cancer genome

Adventures in the enormous: a 1.8 million clone BAC library for the 21.7 Gb genome of loblolly pine

Treesearch

Zenaida V. Magbanua; Seval Ozkan; Benjamin D. Bartlett; Philippe Chouvarine; Christopher A. Saski; Aaron Liston; Richard C. Cronn; C. Dana Nelson; Daniel G. Peterson

2011-01-01

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we...

Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina- and SMRT-based RNA-seq datasets

PubMed Central

Li, Yongping; Wei, Wei; Feng, Jia; Luo, Huifeng; Pi, Mengting; Liu, Zhongchi; Kang, Chunying

2018-01-01

Abstract The genome of the wild diploid strawberry species Fragaria vesca, an ideal model system of cultivated strawberry (Fragaria × ananassa, octoploid) and other Rosaceae family crops, was first published in 2011 and followed by a new assembly (Fvb). However, the annotation for Fvb mainly relied on ab initio predictions and included only predicted coding sequences, therefore an improved annotation is highly desirable. Here, a new annotation version named v2.0.a2 was created for the Fvb genome by a pipeline utilizing one PacBio library, 90 Illumina RNA-seq libraries, and 9 small RNA-seq libraries. Altogether, 18,641 genes (55.6% out of 33,538 genes) were augmented with information on the 5′ and/or 3′ UTRs, 13,168 (39.3%) protein-coding genes were modified or newly identified, and 7,370 genes were found to possess alternative isoforms. In addition, 1,938 long non-coding RNAs, 171 miRNAs, and 51,714 small RNA clusters were integrated into the annotation. This new annotation of F. vesca is substantially improved in both accuracy and integrity of gene predictions, beneficial to the gene functional studies in strawberry and to the comparative genomic analysis of other horticultural crops in Rosaceae family. PMID:29036429

Clone DB: an integrated NCBI resource for clone-associated data

PubMed Central

Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

2013-01-01

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

Clone and genomic repositories at the American Type Culture Collection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maglott, D.R.; Nierman, W.C.

1990-01-01

The American Type Culture Collection (ATCC) has a long history of characterizing, preserving, and distributing biological resource materials for the scientific community. Starting in 1925 as a repository for standard bacterial and fungal strains, its collections have diversified with technologic advances and in response to the requirements of its users. To serve the needs of the human genetics community, the National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), established an international Repository of Human DNA Probes and Libraries at the ATCC in 1985. This repository expanded the existing collections of recombinant clones and librariesmore » at the ATCC, with the specific purposes of (1) obtaining, amplifying, and distribution probes detecting restriction fragment length polymorphisms (RFLPs); (2) obtaining, amplifying, and distributing genomic and cDNA clones from known genes independent of RFLP detection; (3) distributing the chromosome-specific libraries generated by the National Laboratory Gene Library Project at the Lawrence Livermore and Los Alamos National Laboratories and (4) maintaining a public, online database describing the repository materials. Because it was recognized that animal models and comparative mapping can be crucial to genomic characterization, the scope of the repository was broadened in February 1989 to include probes from the mouse genome.« less

Reference quality assembly of the 3.5 Gb genome of Capsicum annuum form a single linked-read library

USDA-ARS?s Scientific Manuscript database

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Cap...

A second generation integrated map of the rainbow trout (Oncorhynchus mykiss) genome: analysis of synteny with model fish genomes

USDA-ARS?s Scientific Manuscript database

In this paper we generated DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is compose...

blend4php: a PHP API for galaxy

PubMed Central

Wytko, Connor; Soto, Brian; Ficklin, Stephen P.

2017-01-01

Galaxy is a popular framework for execution of complex analytical pipelines typically for large data sets, and is a commonly used for (but not limited to) genomic, genetic and related biological analysis. It provides a web front-end and integrates with high performance computing resources. Here we report the development of the blend4php library that wraps Galaxy’s RESTful API into a PHP-based library. PHP-based web applications can use blend4php to automate execution, monitoring and management of a remote Galaxy server, including its users, workflows, jobs and more. The blend4php library was specifically developed for the integration of Galaxy with Tripal, the open-source toolkit for the creation of online genomic and genetic web sites. However, it was designed as an independent library for use by any application, and is freely available under version 3 of the GNU Lesser General Public License (LPGL v3.0) at https://github.com/galaxyproject/blend4php. Database URL: https://github.com/galaxyproject/blend4php PMID:28077564

Reprogramming cell fate with a genome-scale library of artificial transcription factors.

PubMed

Eguchi, Asuka; Wleklinski, Matthew J; Spurgat, Mackenzie C; Heiderscheit, Evan A; Kropornicka, Anna S; Vu, Catherine K; Bhimsaria, Devesh; Swanson, Scott A; Stewart, Ron; Ramanathan, Parameswaran; Kamp, Timothy J; Slukvin, Igor; Thomson, James A; Dutton, James R; Ansari, Aseem Z

2016-12-20

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

PubMed Central

Lin, Jinke; Kudrna, Dave; Wing, Rod A.

2011-01-01

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers. PMID:21234344

Assembling short reads from jumping libraries with large insert sizes.

PubMed

Vasilinetc, Irina; Prjibelski, Andrey D; Gurevich, Alexey; Korobeynikov, Anton; Pevzner, Pavel A

2015-10-15

Advances in Next-Generation Sequencing technologies and sample preparation recently enabled generation of high-quality jumping libraries that have a potential to significantly improve short read assemblies. However, assembly algorithms have to catch up with experimental innovations to benefit from them and to produce high-quality assemblies. We present a new algorithm that extends recently described exSPAnder universal repeat resolution approach to enable its applications to several challenging data types, including jumping libraries generated by the recently developed Illumina Nextera Mate Pair protocol. We demonstrate that, with these improvements, bacterial genomes often can be assembled in a few contigs using only a single Nextera Mate Pair library of short reads. Described algorithms are implemented in C++ as a part of SPAdes genome assembler, which is freely available at bioinf.spbau.ru/en/spades. ap@bioinf.spbau.ru 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.

Reprogramming cell fate with a genome-scale library of artificial transcription factors

PubMed Central

Eguchi, Asuka; Wleklinski, Matthew J.; Spurgat, Mackenzie C.; Heiderscheit, Evan A.; Kropornicka, Anna S.; Vu, Catherine K.; Bhimsaria, Devesh; Swanson, Scott A.; Stewart, Ron; Ramanathan, Parameswaran; Kamp, Timothy J.; Slukvin, Igor; Thomson, James A.; Dutton, James R.; Ansari, Aseem Z.

2016-01-01

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices. PMID:27930301

Host-associated bacterial taxa from Chlorobi, Chloroflexi, GN02, Synergistetes, SR1, TM7, and WPS-2 Phyla/candidate divisions

PubMed Central

Camanocha, Anuj; Dewhirst, Floyd E.

2014-01-01

Background and objective In addition to the well-known phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, Fusobacteria, Tenericutes, and Chylamydiae, the oral microbiomes of mammals contain species from the lesser-known phyla or candidate divisions, including Synergistetes, TM7, Chlorobi, Chloroflexi, GN02, SR1, and WPS-2. The objectives of this study were to create phyla-selective 16S rDNA PCR primer pairs, create selective 16S rDNA clone libraries, identify novel oral taxa, and update canine and human oral microbiome databases. Design 16S rRNA gene sequences for members of the lesser-known phyla were downloaded from GenBank and Greengenes databases and aligned with sequences in our RNA databases. Primers with potential phylum level selectivity were designed heuristically with the goal of producing nearly full-length 16S rDNA amplicons. The specificity of primer pairs was examined by making clone libraries from PCR amplicons and determining phyla identity by BLASTN analysis. Results Phylum-selective primer pairs were identified that allowed construction of clone libraries with 96–100% specificity for each of the lesser-known phyla. From these clone libraries, seven human and two canine novel oral taxa were identified and added to their respective taxonomic databases. For each phylum, genome sequences closest to human oral taxa were identified and added to the Human Oral Microbiome Database to facilitate metagenomic, transcriptomic, and proteomic studies that involve tiling sequences to the most closely related taxon. While examining ribosomal operons in lesser-known phyla from single-cell genomes and metagenomes, we identified a novel rRNA operon order (23S-5S-16S) in three SR1 genomes and the splitting of the 23S rRNA gene by an I-CeuI-like homing endonuclease in a WPS-2 genome. Conclusions This study developed useful primer pairs for making phylum-selective 16S rRNA clone libraries. Phylum-specific libraries were shown to be useful for identifying previously unrecognized taxa in lesser-known phyla and would be useful for future environmental and host-associated studies. PMID:25317252

Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-gene Library

DTIC Science & Technology

2013-10-09

have desirable traits. We aim to enlarge the E. coli genome using Lactobacillusplantarum genes to build cells tolerant to EtOH and BT. L. plantarum is...chemicals III. Approach Objective 1 & la: Integrated heterologous (L. plantarum ) DNA into the E. coli chromosome and selected for insertions that...developed in combination with genes identified from screening L. plantarum libraries. Additionally, we have screened heterologous libraries for

Millstone: software for multiplex microbial genome analysis and engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. Here, we describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Millstone: software for multiplex microbial genome analysis and engineering.

PubMed

Goodman, Daniel B; Kuznetsov, Gleb; Lajoie, Marc J; Ahern, Brian W; Napolitano, Michael G; Chen, Kevin Y; Chen, Changping; Church, George M

2017-05-25

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Millstone: software for multiplex microbial genome analysis and engineering

DOE PAGES

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.; ...

2017-05-25

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. Here, we describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

PubMed

Tamborindeguy, C; Ben, C; Liboz, T; Gentzbittel, L

2004-04-01

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

Development of DNA-Free Sediment for Ecological Assays with Genomic Endpoints

EPA Science Inventory

Recent advances in genomics are currently being exploited to discern ecological changes that have conventionally been measured using laborious counting techniques. For example, next generation sequencing technologies can be used to create DNA libraries from benthic community ass...

First de novo whole genome sequencing and assembly of the pink-footed goose.

PubMed

Pujolar, J M; Dalén, L; Olsen, R A; Hansen, M M; Madsen, J

2018-03-01

Annotated genomes can provide new perspectives on the biology of species. We present the first de novo whole genome sequencing for the pink-footed goose. In order to obtain a high-quality de novo assembly the strategy used was to combine one short insert paired-end library with two mate-pair libraries. The pink-footed goose genome was assembled de novo using three different assemblers and an assembly evaluation was subsequently performed in order to choose the best assembler. For our data, ALLPATHS-LG performed the best, since the assembly produced covers most of the genome, while introducing the fewest errors. A total of 26,134 genes were annotated, with bird species accounting for virtually all BLAST hits. We also estimated the substitution rate in the pink-footed goose, which can be of use in future demographic studies, by using a comparative approach with the genome of the chicken, the mallard and the swan goose. A substitution rate of 1.38×10 -7 per nucleotide per generation was obtained when comparing the genomes of the two closely-related goose species (the pink-footed and the swan goose). Altogether, we provide a valuable tool for future genomic studies aiming at particular genes and regions of the pink-footed goose genome as well as other bird species. Copyright © 2017 Elsevier Inc. All rights reserved.

Genomics & Genetics | National Agricultural Library

Science.gov Websites

Skip to main content Home National Agricultural Library United States Department of Agriculture Ag agricultural and environmental settings. Deadpool proximal sensing cart docx xlsx 3x jpeg 5x pdf Data from Buytaert. NAL Home | USDA.gov | Agricultural Research Service | Plain Language | FOIA | Accessibility

Veterinary Pest Genomics Center | National Agricultural Library

Science.gov Websites

Skip to main content Home National Agricultural Library United States Department of Agriculture Ag Department of Agriculture's Agricultural Research Service (ARS). The vision for this initiative is to collaborator for the Bioinformatics Education in Agricultural Sciences (BEAS) project funded by the Hispanic

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

PubMed

Goya, Stephanie; Valinotto, Laura E; Tittarelli, Estefania; Rojo, Gabriel L; Nabaes Jodar, Mercedes S; Greninger, Alexander L; Zaiat, Jonathan J; Marti, Marcelo A; Mistchenko, Alicia S; Viegas, Mariana

2018-01-01

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

Capturing the 'ome': the expanding molecular toolbox for RNA and DNA library construction.

PubMed

Boone, Morgane; De Koker, Andries; Callewaert, Nico

2018-04-06

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application.

Defining the ABC of gene essentiality in streptococci.

PubMed

Charbonneau, Amelia R L; Forman, Oliver P; Cain, Amy K; Newland, Graham; Robinson, Carl; Boursnell, Mike; Parkhill, Julian; Leigh, James A; Maskell, Duncan J; Waller, Andrew S

2017-05-31

Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

PubMed Central

Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jakob; Gilchrist, Michael J; Panitz, Frank; Jørgensen, Claus; Scheibye-Knudsen, Karsten; Arvin, Troels; Lumholdt, Steen; Sawera, Milena; Green, Trine; Nielsen, Bente J; Havgaard, Jakob H; Rosenkilde, Carina; Wang, Jun; Li, Heng; Li, Ruiqiang; Liu, Bin; Hu, Songnian; Dong, Wei; Li, Wei; Yu, Jun; Wang, Jian; Stærfeldt, Hans-Henrik; Wernersson, Rasmus; Madsen, Lone B; Thomsen, Bo; Hornshøj, Henrik; Bujie, Zhan; Wang, Xuegang; Wang, Xuefei; Bolund, Lars; Brunak, Søren; Yang, Huanming; Bendixen, Christian; Fredholm, Merete

2007-01-01

Background Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. Results Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. Conclusion This EST collection, the largest to date in pig, represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies. PMID:17407547

Novel Approaches to Breast Cancer Prevention and Inhibition of Metastases

DTIC Science & Technology

2014-10-01

functional characterization of candidate breast cancer genes. The transgenic RNAi library is covering the whole Drosophila genome , giving us an...cancer prevention trials in BRCA1 carriers using RANKL blockade. Using Drosophila modeling of Ras-driven transformation, we performed a near- genome ... Genome wide functional genetics, haploid stem cells, Drosophila cancer modeling, breast cancer prevention, BRCA1 carriers 16. SECURITY

Short-Read Sequencing for Genomic Analysis of the Brown Rot Fungus Fibroporia radiculosa

Treesearch

J. D. Tang; A. D. Perkins; T. S. Sonstegard; S. G. Schroeder; S. C. Burgess; S. V. Diehl

2012-01-01

The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment...

High-Throughput SNP Discovery through Deep Resequencing of a Reduced Representation Library to Anchor and Orient Scaffolds in the Soybean Whole Genome Sequence

USDA-ARS?s Scientific Manuscript database

The soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy but only properly oriented 66% of the sequence scaffolds. To find additional single nucleotide polymorphism (SNP) markers for additiona...

A global assembly of cotton ESTs

PubMed Central

Udall, Joshua A.; Swanson, Jordan M.; Haller, Karl; Rapp, Ryan A.; Sparks, Michael E.; Hatfield, Jamie; Yu, Yeisoo; Wu, Yingru; Dowd, Caitriona; Arpat, Aladdin B.; Sickler, Brad A.; Wilkins, Thea A.; Guo, Jin Ying; Chen, Xiao Ya; Scheffler, Jodi; Taliercio, Earl; Turley, Ricky; McFadden, Helen; Payton, Paxton; Klueva, Natalya; Allen, Randell; Zhang, Deshui; Haigler, Candace; Wilkerson, Curtis; Suo, Jinfeng; Schulze, Stefan R.; Pierce, Margaret L.; Essenberg, Margaret; Kim, HyeRan; Llewellyn, Danny J.; Dennis, Elizabeth S.; Kudrna, David; Wing, Rod; Paterson, Andrew H.; Soderlund, Cari; Wendel, Jonathan F.

2006-01-01

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; AT and DT genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics. PMID:16478941

Development of DNA-Free Sediment for Ecological Assays with Genomic Endpoints (NAC SETAC)

EPA Science Inventory

Recent advances in genomics are currently being exploited to discern ecological changes that have conventionally been measured using laborious counting techniques. For example, next generation sequencing technologies can be used to create DNA libraries from benthic community ass...

From the ORFeome concept to highly comprehensive, full-genome screening libraries.

PubMed

Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

2013-02-01

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

Sandia National Laboratories: CRISPR genome-editing technology

Science.gov Websites

boom sets records, forges ties "Like a jar of jellybeans, a pooled CRISPR library is a complex like individually wrapped and labeled jellybeans. When you identify a particular CRISPR of interest arrayed libraries, the CRISPR of interest is already labeled, therefore hit identification is much easier

Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing.

PubMed

Cooper, James; Ding, Yi; Song, Jiuzhou; Zhao, Keji

2017-11-01

Increased chromatin accessibility is a feature of cell-type-specific cis-regulatory elements; therefore, mapping of DNase I hypersensitive sites (DHSs) enables the detection of active regulatory elements of transcription, including promoters, enhancers, insulators and locus-control regions. Single-cell DNase sequencing (scDNase-seq) is a method of detecting genome-wide DHSs when starting with either single cells or <1,000 cells from primary cell sources. This technique enables genome-wide mapping of hypersensitive sites in a wide range of cell populations that cannot be analyzed using conventional DNase I sequencing because of the requirement for millions of starting cells. Fresh cells, formaldehyde-cross-linked cells or cells recovered from formalin-fixed paraffin-embedded (FFPE) tissue slides are suitable for scDNase-seq assays. To generate scDNase-seq libraries, cells are lysed and then digested with DNase I. Circular carrier plasmid DNA is included during subsequent DNA purification and library preparation steps to prevent loss of the small quantity of DHS DNA. Libraries are generated for high-throughput sequencing on the Illumina platform using standard methods. Preparation of scDNase-seq libraries requires only 2 d. The materials and molecular biology techniques described in this protocol should be accessible to any general molecular biology laboratory. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

PubMed

Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

2016-05-01

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

Using relational databases for improved sequence similarity searching and large-scale genomic analyses.

PubMed

Mackey, Aaron J; Pearson, William R

2004-10-01

Relational databases are designed to integrate diverse types of information and manage large sets of search results, greatly simplifying genome-scale analyses. Relational databases are essential for management and analysis of large-scale sequence analyses, and can also be used to improve the statistical significance of similarity searches by focusing on subsets of sequence libraries most likely to contain homologs. This unit describes using relational databases to improve the efficiency of sequence similarity searching and to demonstrate various large-scale genomic analyses of homology-related data. This unit describes the installation and use of a simple protein sequence database, seqdb_demo, which is used as a basis for the other protocols. These include basic use of the database to generate a novel sequence library subset, how to extend and use seqdb_demo for the storage of sequence similarity search results and making use of various kinds of stored search results to address aspects of comparative genomic analysis.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR library

PubMed Central

Zhu, Shiyou; Li, Wei; Liu, Jingze; Chen, Chen-Hao; Liao, Qi; Xu, Ping; Xu, Han; Xiao, Tengfei; Cao, Zhongzheng; Peng, Jingyu; Yuan, Pengfei; Brown, Myles; Liu, Xiaole Shirley; Wei, Wensheng

2017-01-01

CRISPR/Cas9 screens have been widely adopted to analyse coding gene functions, but high throughput screening of non-coding elements using this method is more challenging, because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. Herein, we report a high throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We individually validated 9 lncRNAs using CRISPR/Cas9-mediated genomic deletion and functional rescue, CRISPR activation or inhibition, and gene expression profiling. Our high-throughput pgRNA genome deletion method should enable rapid identification of functional mammalian non-coding elements. PMID:27798563

Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shetty, Ameesha R.; de Gannes, Vidya; Obi, Chioma C.

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs inmore » two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Lastly, determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.« less

Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4

DOE PAGES

Shetty, Ameesha R.; de Gannes, Vidya; Obi, Chioma C.; ...

2015-08-15

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs inmore » two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Lastly, determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.« less

A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

PubMed

Bassett, Andrew R; Kong, Lesheng; Liu, Ji-Long

2015-06-20

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Interpreting a sequenced genome: toward a cosmid transgenic library of Caenorhabditis elegans.

PubMed

Janke, D L; Schein, J E; Ha, T; Franz, N W; O'Neil, N J; Vatcher, G P; Stewart, H I; Kuervers, L M; Baillie, D L; Rose, A M

1997-10-01

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.

Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

DOE PAGES

Bowers, Robert M.; Clum, Alicia; Tice, Hope; ...

2015-10-24

Background: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composi tion and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for largemore » initial DNA template quantities during library preparation. Results: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.« less

Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bowers, Robert M.; Clum, Alicia; Tice, Hope

Background: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composi tion and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for largemore » initial DNA template quantities during library preparation. Results: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.« less

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

PubMed

Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

2018-01-01

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes

DTIC Science & Technology

2007-02-01

genomic investigation would be the ability to perform genetic subtractive analysis with in vivo-derived genetic material originating from a...different DNA sequences present in one complimentary (31) or genomic (32) DNA library but absent in another. The advent of suppressive hybridization...of control specimens different from the native tissue for subtractive genomic analysis in some studies has created many inconclusive results. Cell

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

PubMed

Cannarozzi, Gina; Plaza-Wüthrich, Sonia; Esfeld, Korinna; Larti, Stéphanie; Wilson, Yi Song; Girma, Dejene; de Castro, Edouard; Chanyalew, Solomon; Blösch, Regula; Farinelli, Laurent; Lyons, Eric; Schneider, Michel; Falquet, Laurent; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

2014-07-09

Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Survey of microsatellite DNA in pine

Treesearch

Craig S. Echt; P. May-Marquardt

1997-01-01

A large insert genomic library from eastern white pine (Pinus strobus) was probed for the microsatellite motifs (AC)n and (AG)n, all 10 trinucleotide motifs, and 22 of the 33 possible tetranucleotide motifs. For comparison with a species from a different subgenus, a loblolly pine (Pinus taeda) genomic...

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2010-07-01

cloning them in phage display vectors. We are characterizing the libraries and trying to figure out how best to screen them. We ran into the problem...auto-antibody screening project onto glass slides for screening purposes. We have abandoned this aim in that we switched our approach to a phage ...display library which does not require glass slide. We made our first comprehensive library in a T7 display vector. Task 9 (Months 24-36) We will

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

A Computational Solution to Automatically Map Metabolite Libraries in the Context of Genome Scale Metabolic Networks.

PubMed

Merlet, Benjamin; Paulhe, Nils; Vinson, Florence; Frainay, Clément; Chazalviel, Maxime; Poupin, Nathalie; Gloaguen, Yoann; Giacomoni, Franck; Jourdan, Fabien

2016-01-01

This article describes a generic programmatic method for mapping chemical compound libraries on organism-specific metabolic networks from various databases (KEGG, BioCyc) and flat file formats (SBML and Matlab files). We show how this pipeline was successfully applied to decipher the coverage of chemical libraries set up by two metabolomics facilities MetaboHub (French National infrastructure for metabolomics and fluxomics) and Glasgow Polyomics (GP) on the metabolic networks available in the MetExplore web server. The present generic protocol is designed to formalize and reduce the volume of information transfer between the library and the network database. Matching of metabolites between libraries and metabolic networks is based on InChIs or InChIKeys and therefore requires that these identifiers are specified in both libraries and networks. In addition to providing covering statistics, this pipeline also allows the visualization of mapping results in the context of metabolic networks. In order to achieve this goal, we tackled issues on programmatic interaction between two servers, improvement of metabolite annotation in metabolic networks and automatic loading of a mapping in genome scale metabolic network analysis tool MetExplore. It is important to note that this mapping can also be performed on a single or a selection of organisms of interest and is thus not limited to large facilities.

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

blastjs: a BLAST+ wrapper for Node.js.

PubMed

Page, Martin; MacLean, Dan; Schudoma, Christian

2016-02-27

To cope with the ever-increasing amount of sequence data generated in the field of genomics, the demand for efficient and fast database searches that drive functional and structural annotation in both large- and small-scale genome projects is on the rise. The tools of the BLAST+ suite are the most widely employed bioinformatic method for these database searches. Recent trends in bioinformatics application development show an increasing number of JavaScript apps that are based on modern frameworks such as Node.js. Until now, there is no way of using database searches with the BLAST+ suite from a Node.js codebase. We developed blastjs, a Node.js library that wraps the search tools of the BLAST+ suite and thus allows to easily add significant functionality to any Node.js-based application. blastjs is a library that allows the incorporation of BLAST+ functionality into bioinformatics applications based on JavaScript and Node.js. The library was designed to be as user-friendly as possible and therefore requires only a minimal amount of code in the client application. The library is freely available under the MIT license at https://github.com/teammaclean/blastjs.

General M13 phage display: M13 phage display in identification and characterization of protein-protein interactions.

PubMed

Hertveldt, Kirsten; Beliën, Tim; Volckaert, Guido

2009-01-01

In M13 phage display, proteins and peptides are exposed on one of the surface proteins of filamentous phage particles and become accessible to affinity enrichment against a bait of interest. We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented "proteome" against protein baits. Gene fragment libraries allow a more in depth characterization of the protein-protein interaction site by identification of the protein region involved in the interaction.

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

PubMed

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite widespread use of E. coli to propagate foreign DNA in metagenomic libraries, the effects of in vivo transcriptional activity on clone stability are not well understood. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

ParallABEL: an R library for generalized parallelization of genome-wide association studies

PubMed Central

2010-01-01

Background Genome-Wide Association (GWA) analysis is a powerful method for identifying loci associated with complex traits and drug response. Parts of GWA analyses, especially those involving thousands of individuals and consuming hours to months, will benefit from parallel computation. It is arduous acquiring the necessary programming skills to correctly partition and distribute data, control and monitor tasks on clustered computers, and merge output files. Results Most components of GWA analysis can be divided into four groups based on the types of input data and statistical outputs. The first group contains statistics computed for a particular Single Nucleotide Polymorphism (SNP), or trait, such as SNP characterization statistics or association test statistics. The input data of this group includes the SNPs/traits. The second group concerns statistics characterizing an individual in a study, for example, the summary statistics of genotype quality for each sample. The input data of this group includes individuals. The third group consists of pair-wise statistics derived from analyses between each pair of individuals in the study, for example genome-wide identity-by-state or genomic kinship analyses. The input data of this group includes pairs of SNPs/traits. The final group concerns pair-wise statistics derived for pairs of SNPs, such as the linkage disequilibrium characterisation. The input data of this group includes pairs of individuals. We developed the ParallABEL library, which utilizes the Rmpi library, to parallelize these four types of computations. ParallABEL library is not only aimed at GenABEL, but may also be employed to parallelize various GWA packages in R. The data set from the North American Rheumatoid Arthritis Consortium (NARAC) includes 2,062 individuals with 545,080, SNPs' genotyping, was used to measure ParallABEL performance. Almost perfect speed-up was achieved for many types of analyses. For example, the computing time for the identity-by-state matrix was linearly reduced from approximately eight hours to one hour when ParallABEL employed eight processors. Conclusions Executing genome-wide association analysis using the ParallABEL library on a computer cluster is an effective way to boost performance, and simplify the parallelization of GWA studies. ParallABEL is a user-friendly parallelization of GenABEL. PMID:20429914

Capturing diversity of marine heterotrophic protists: one cell at a time

PubMed Central

Heywood, Jane L; Sieracki, Michael E; Bellows, Wendy; Poulton, Nicole J; Stepanauskas, Ramunas

2011-01-01

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups. PMID:20962875

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

PubMed

Lu, Fu-Hao; McKenzie, Neil; Kettleborough, George; Heavens, Darren; Clark, Matthew D; Bevan, Michael W

2018-05-01

The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

USDA-ARS?s Scientific Manuscript database

We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...

Characterizing the walnut genome through analyses of BAC end sequences

USDA-ARS?s Scientific Manuscript database

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots...

77 FR 6810 - National Human Genome Research Institute; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-09

... Research Institute; Notice of Closed Meetings Pursuant to section 10(d) of the Federal Advisory Committee... clearly unwarranted invasion of personal privacy. Name of Committee: National Human Genome Research... p.m. to 2 p.m. Agenda: To review and evaluate contract proposals. Place: NHGRI Twinbrook Library...

75 FR 8373 - National Human Genome Research Institute; Notice of Closed Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-24

... Research Institute; Notice of Closed Meeting Pursuant to section 10(d) of the Federal Advisory Committee... unwarranted invasion of personal privacy. Name of Committee: National Human Genome Research Institute Special..., NHGRI Twinbrook Library, 5635 Fishers Lane, Bethesda, MD 20892-9306. Contact Person: Keith McKenney, PhD...

77 FR 31863 - National Human Genome Research Institute; Notice of Closed Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-30

... Research Institute; Notice of Closed Meeting Pursuant to section 10(d) of the Federal Advisory Committee... unwarranted invasion of personal privacy. Name of Committee: National Human Genome Research Institute Special... review and evaluate grant applications. Place: NHGRI Twinbrook Library, 5635 Fishers Lane, Room 4076...

75 FR 10488 - National Human Genome Research Institute; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-08

... Research Institute; Notice of Closed Meetings Pursuant to section 10(d) of the Federal Advisory Committee... clearly unwarranted invasion of personal privacy. Name of Committee: National Human Genome Research...: 11 a.m. to 1 p.m. Agenda: To review and evaluate grant applications. Place: NHGRI Twinrook Library...

Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes

DTIC Science & Technology

2006-02-01

have been conducted with in vitro-derived genetic materials from both mammalian and nonmammalian systems (27-29), a major leap in functional genomic ...sequences present in one complimentary (31) or genomic (32) DNA library but absent in another. The advent of suppressive hybridization technique...control specimens different from the native tissue for subtractive genomic analysis in some studies has created many inconclusive results. Cell to cell

A strategy for rapid production and screening of yeast artificial chromosome libraries.

PubMed

Strauss, W M; Jaenisch, E; Jaenisch, R

1992-01-01

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

GBParsy: a GenBank flatfile parser library with high speed.

PubMed

Lee, Tae-Ho; Kim, Yeon-Ki; Nahm, Baek Hie

2008-07-25

GenBank flatfile (GBF) format is one of the most popular sequence file formats because of its detailed sequence features and ease of readability. To use the data in the file by a computer, a parsing process is required and is performed according to a given grammar for the sequence and the description in a GBF. Currently, several parser libraries for the GBF have been developed. However, with the accumulation of DNA sequence information from eukaryotic chromosomes, parsing a eukaryotic genome sequence with these libraries inevitably takes a long time, due to the large GBF file and its correspondingly large genomic nucleotide sequence and related feature information. Thus, there is significant need to develop a parsing program with high speed and efficient use of system memory. We developed a library, GBParsy, which was C language-based and parses GBF files. The parsing speed was maximized by using content-specified functions in place of regular expressions that are flexible but slow. In addition, we optimized an algorithm related to memory usage so that it also increased parsing performance and efficiency of memory usage. GBParsy is at least 5-100x faster than current parsers in benchmark tests. GBParsy is estimated to extract annotated information from almost 100 Mb of a GenBank flatfile for chromosomal sequence information within a second. Thus, it should be used for a variety of applications such as on-time visualization of a genome at a web site.

Construction of high quality Gateway™ entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon.

PubMed

Cao, Shuanghe; Siriwardana, Chamindika L; Kumimoto, Roderick W; Holt, Ben F

2011-05-19

Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings), developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium) is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H) libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark) and long day (LD, 20/4-h light/dark) grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each) and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user-defined purposes and the derived Y2H libraries can be immediately applied to large scale screening and discovery of novel protein-protein interactions. All libraries are freely available for distribution to the research community.

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea.

PubMed

Martin, Stanton L; Blackmon, Barbara P; Rajagopalan, Ravi; Houfek, Thomas D; Sceeles, Robert G; Denn, Sheila O; Mitchell, Thomas K; Brown, Douglas E; Wing, Rod A; Dean, Ralph A

2002-01-01

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.

Analysis of genomic regions of Trichoderma harzianum IOC-3844 related to biomass degradation.

PubMed

Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

2015-01-01

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.

Analysis of Genomic Regions of Trichoderma harzianum IOC-3844 Related to Biomass Degradation

PubMed Central

Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

2015-01-01

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes. PMID:25836973

Construction, database integration, and application of an Oenothera EST library.

PubMed

Mrácek, Jaroslav; Greiner, Stephan; Cho, Won Kyong; Rauwolf, Uwe; Braun, Martha; Umate, Pavan; Altstätter, Johannes; Stoppel, Rhea; Mlcochová, Lada; Silber, Martina V; Volz, Stefanie M; White, Sarah; Selmeier, Renate; Rudd, Stephen; Herrmann, Reinhold G; Meurer, Jörg

2006-09-01

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

Genome sequence of the olive tree, Olea europaea.

PubMed

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.

Next generation sequencing technology: a powerful tool for the genome characterization of sugarcane mosaic virus from Sorghum almum

USDA-ARS?s Scientific Manuscript database

Next generation sequencing (NGS) technology was used to analyze the occurrence of viruses in Sorghum almum plants in Florida exhibiting mosaic symptoms. Total RNA was extracted from symptomatic leaves and used as a template for cDNA library preparation. The resulting library was sequenced on an Illu...

Genome-Wide Analysis of Differentially Expressed Genes Relevant to Rhizome Formation in Lotus Root (Nelumbo nucifera Gaertn)

PubMed Central

Yin, Jingjing; Li, Liangjun; Chen, Xuehao

2013-01-01

Lotus root is a popular wetland vegetable which produces edible rhizome. At the molecular level, the regulation of rhizome formation is very complex, which has not been sufficiently addressed in research. In this study, to identify differentially expressed genes (DEGs) in lotus root, four libraries (L1 library: stolon stage, L2 library: initial swelling stage, L3 library: middle swelling stage, L4: later swelling stage) were constructed from the rhizome development stages. High-throughput tag-sequencing technique was used which is based on Solexa Genome Analyzer Platform. Approximately 5.0 million tags were sequenced, and 4542104, 4474755, 4777919, and 4750348 clean tags including 151282, 137476, 215872, and 166005 distinct tags were obtained after removal of low quality tags from each library respectively. More than 43% distinct tags were unambiguous tags mapping to the reference genes, and 40% were unambiguous tag-mapped genes. From L1, L2, L3, and L4, total 20471, 18785, 23448, and 21778 genes were annotated, after mapping their functions in existing databases. Profiling of gene expression in L1/L2, L2/L3, and L3/L4 libraries were different among most of the selected 20 DEGs. Most of the DEGs in L1/L2 libraries were relevant to fiber development and stress response, while in L2/L3 and L3/L4 libraries, major of the DEGs were involved in metabolism of energy and storage. All up-regulated transcriptional factors in four libraries and 14 important rhizome formation-related genes in four libraries were also identified. In addition, the expression of 9 genes from identified DEGs was performed by qRT-PCR method. In a summary, this study provides a comprehensive understanding of gene expression during the rhizome formation in lotus root. PMID:23840598

Evaluation of microsatellite loci from libraries derived from the wild diploid 'Calcutta 4' and 'Ouro' banana cultivars.

PubMed

Silva, P R O; Jesus, O N J; Creste, S; Figueira, A; Amorim, E P; Ferreira, C F

2015-09-25

Microsatellite markers have been widely used in the quantification of genetic variability and for genetic breeding in Musa spp. The objective of the present study was to evaluate the discriminatory power of microsatellite markers derived from 'Calcutta 4' and 'Ouro' genomic libraries, and to analyze the genetic variability among 30 banana accessions. Thirty-eight markers were used: 15 from the 'Ouro' library and 23 from the 'Calcutta 4' library. Genetic diversity was evaluated by considering SSR markers as both dominant markers because of the presence of triploid accessions, and co-dominant markers. For the dominant analysis, polymorphism information content (PIC) values for 44 polymorphic markers ranged from 0.063 to 0.533, with a mean value of 0.24. A dendrogram analysis separated the BGB-Banana accessions into 4 groups: the 'Ouro' and 'Muísa Tia' accessions were the most dissimilar (93% dissimilarity), while the most similar accessions were 'Pacovan' and 'Walha'. The mean genetic distance between samples was 0.74. For the analysis considering SSR markers as co-dominants, using only diploid accessions, two groups were separated based on their genome contents (A and B). The PIC values for the markers from the 'Calcutta 4' library varied from 0.4836 to 0.7886, whereas those from the 'Ouro' library ranged from 0.3800 to 0.7521. Given the high PIC values, the markers from both the libraries showed high discriminatory power, and can therefore be widely applied for analysis of genetic diversity, population structures, and linkage mapping in Musa spp.

Undermethylated DNA as a source of microsatellites from a conifer genome.

PubMed

Zhou, Y; Bui, T; Auckland, L D; Williams, C G

2002-02-01

Developing microsatellites from the large, highly duplicated conifer genome requires special tools. To improve the efficiency of developing Pinus taeda L. microsatellites, undermethylated (UM) DNA fragments were used to construct a microsatellite-enriched copy library. A methylation-sensitive restriction enzyme, McrBC, was used to enrich for UM DNA before library construction. Digested DNA fragments larger than 9 kb were then excised and digested with RsaI and used to construct nine dinucleotide and trinucleotide libraries. A total of 1016 microsatellite-positive clones were detected among 11 904 clones and 620 of these were unique. Of 245 primer sets that produced a PCR product, 113 could be developed as UM microsatellite markers and 70 were polymorphic. Inheritance and marker informativeness were tested for a random sample of 36 polymorphic markers using a three-generation outbred pedigree. Thirty-one microsatellites (86%) had single-locus inheritance despite the highly duplicated nature of the P. taeda genome. Nineteen UM microsatellites had highly informative intercross mating type configurations. Allele number and frequency were estimated for eleven UM microsatellites using a population survey. Allele numbers for these UM microsatellites ranged from 3 to 12 with an average of 5.7 alleles/locus. Frequencies for the 63 alleles were mostly in the low-common range; only 14 of the 63 were in the rare allele (q < 0.05) class. Enriching for UM DNA was an efficient method for developing polymorphic microsatellites from a large plant genome.

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

PubMed Central

Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

2014-01-01

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

A physical map of the bovine genome

PubMed Central

Snelling, Warren M; Chiu, Readman; Schein, Jacqueline E; Hobbs, Matthew; Abbey, Colette A; Adelson, David L; Aerts, Jan; Bennett, Gary L; Bosdet, Ian E; Boussaha, Mekki; Brauning, Rudiger; Caetano, Alexandre R; Costa, Marcos M; Crawford, Allan M; Dalrymple, Brian P; Eggen, André; Everts-van der Wind, Annelie; Floriot, Sandrine; Gautier, Mathieu; Gill, Clare A; Green, Ronnie D; Holt, Robert; Jann, Oliver; Jones, Steven JM; Kappes, Steven M; Keele, John W; de Jong, Pieter J; Larkin, Denis M; Lewin, Harris A; McEwan, John C; McKay, Stephanie; Marra, Marco A; Mathewson, Carrie A; Matukumalli, Lakshmi K; Moore, Stephen S; Murdoch, Brenda; Nicholas, Frank W; Osoegawa, Kazutoyo; Roy, Alice; Salih, Hanni; Schibler, Laurent; Schnabel, Robert D; Silveri, Licia; Skow, Loren C; Smith, Timothy PL; Sonstegard, Tad S; Taylor, Jeremy F; Tellam, Ross; Van Tassell, Curtis P; Williams, John L; Womack, James E; Wye, Natasja H; Yang, George; Zhao, Shaying

2007-01-01

Background Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. Results A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. Conclusion Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans. PMID:17697342

Sequencing, Annotation and Analysis of the Syrian Hamster (Mesocricetus auratus) Transcriptome

PubMed Central

Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L.; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F.; Feldmann, Heinz

2014-01-01

Background The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. Results A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. Conclusions This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional genomics studies. PMID:25398096

Genome-Wide Mutagenesis in Borrelia burgdorferi.

PubMed

Lin, Tao; Gao, Lihui

2018-01-01

Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector. STM procedure combined with Luminex Flex ® Map™ technology and next-generation sequencing (e.g., Tn-seq) are the powerful high-throughput tools for the determination of Borrelia burgdorferi virulence determinants. The assessment of multiple tissue sites and two DNA resources at two different time points using Luminex Flex ® Map™ technology provides a robust data set. B. burgdorferi transposon mutant screening indicates that a high proportion of genes are the novel virulence determinants that are required for mouse and tick infection. In this protocol, an effective signature-tagged Himar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectious B. burgdorferi B31 clone. In STM, signature-tagged suicide vectors are constructed by inserting unique DNA sequences (tags) into the transposable elements. The signature-tagged transposon mutants are generated when transposon suicide vectors are transformed into an infectious B. burgdorferi clone, and the transposable element is transposed into the 5'-TA-3' sequence in the B. burgdorferi genome with the signature tag. The transposon library is created and consists of many sub-libraries, each sub-library has several hundreds of mutants with same tags. A group of mice or ticks are infected with a mixed population of mutants with different tags, after recovered from different tissues of infected mice and ticks, mutants from output pool and input pool are detected using high-throughput, semi-quantitative Luminex ® FLEXMAP™ or next-generation sequencing (Tn-seq) technologies. Thus far, we have created a high-density, sequence-defined transposon library of over 6600 STM mutants for the efficient genome-wide investigation of genes and gene products required for wild-type pathogenesis, host-pathogen interactions, in vitro growth, in vivo survival, physiology, morphology, chemotaxis, motility, structure, metabolism, gene regulation, plasmid maintenance and replication, etc. The insertion sites of 4480 transposon mutants have been determined. About 800 predicted protein-encoding genes in the genome were disrupted in the STM transposon library. The infectivity and some functions of 800 mutants in 500 genes have been determined. Analysis of these transposon mutants has yielded valuable information regarding the genes and gene products important in the pathogenesis and biology of B. burgdorferi and its tick vectors.

Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes

DTIC Science & Technology

2008-02-01

systems (27-29), a major leap in functional genomic investigation would be the ability to perform genetic subtractive analysis with in vivo-derived...been designed to detect and isolate different DNA sequences present in one complimentary (31) or genomic (32) DNA library but absent in another. The...many disorders if applied correctly, the use of control specimens different from the native tissue for subtractive genomic analysis in some studies has

Phased genotyping-by-sequencing enhances analysis of genetic diversity and reveals divergent copy number variants in maize

USDA-ARS?s Scientific Manuscript database

High-throughput sequencing of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken fr...

Animal selection for whole genome sequencing by quantifying the unique contribution of homozygous haplotypes sequenced

USDA-ARS?s Scientific Manuscript database

Major whole genome sequencing projects promise to identify rare and causal variants within livestock species; however, the efficient selection of animals for sequencing remains a major problem within these surveys. The goal of this project was to develop a library of high accuracy genetic variants f...

i5k | National Agricultural Library

Science.gov Websites

genome browser, and the Apollo manual curation service. Over 50 arthropod genomes are now part of the i5k (done by Dan Hughes at Baylor) with manual annotations by the research community (done via Web Apollo with manual annotations by the research community (via the Apollo manual annotation software). insects

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM)

PubMed Central

Skinnider, Michael A.; Dejong, Chris A.; Rees, Philip N.; Johnston, Chad W.; Li, Haoxin; Webster, Andrew L. H.; Wyatt, Morgan A.; Magarvey, Nathan A.

2015-01-01

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/. PMID:26442528

Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides

DOE PAGES

Burger, Brian T.; Imam, Saheed; Scarborough, Matthew J.; ...

2017-06-06

Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosyntheticmore » growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species.« less

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

PubMed Central

2011-01-01

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. PMID:21794110

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

PubMed

Feltus, Frank A; Saski, Christopher A; Mockaitis, Keithanne; Haiminen, Niina; Parida, Laxmi; Smith, Zachary; Ford, James; Staton, Margaret E; Ficklin, Stephen P; Blackmon, Barbara P; Cheng, Chun-Huai; Schnell, Raymond J; Kuhn, David N; Motamayor, Juan-Carlos

2011-07-27

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. 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. 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.

Improving prokaryotic transposable elements identification using a combination of de novo and profile HMM methods.

PubMed

Kamoun, Choumouss; Payen, Thibaut; Hua-Van, Aurélie; Filée, Jonathan

2013-10-11

Insertion Sequences (ISs) and their non-autonomous derivatives (MITEs) are important components of prokaryotic genomes inducing duplication, deletion, rearrangement or lateral gene transfers. Although ISs and MITEs are relatively simple and basic genetic elements, their detection remains a difficult task due to their remarkable sequence diversity. With the advent of high-throughput genome and metagenome sequencing technologies, the development of fast, reliable and sensitive methods of ISs and MITEs detection become an important challenge. So far, almost all studies dealing with prokaryotic transposons have used classical BLAST-based detection methods against reference libraries. Here we introduce alternative methods of detection either taking advantages of the structural properties of the elements (de novo methods) or using an additional library-based method using profile HMM searches. In this study, we have developed three different work flows dedicated to ISs and MITEs detection: the first two use de novo methods detecting either repeated sequences or presence of Inverted Repeats; the third one use 28 in-house transposase alignment profiles with HMM search methods. We have compared the respective performances of each method using a reference dataset of 30 archaeal and 30 bacterial genomes in addition to simulated and real metagenomes. Compared to a BLAST-based method using ISFinder as library, de novo methods significantly improve ISs and MITEs detection. For example, in the 30 archaeal genomes, we discovered 30 new elements (+20%) in addition to the 141 multi-copies elements already detected by the BLAST approach. Many of the new elements correspond to ISs belonging to unknown or highly divergent families. The total number of MITEs has even doubled with the discovery of elements displaying very limited sequence similarities with their respective autonomous partners (mainly in the Inverted Repeats of the elements). Concerning metagenomes, with the exception of short reads data (<300 bp) for which both techniques seem equally limited, profile HMM searches considerably ameliorate the detection of transposase encoding genes (up to +50%) generating low level of false positives compare to BLAST-based methods. Compared to classical BLAST-based methods, the sensitivity of de novo and profile HMM methods developed in this study allow a better and more reliable detection of transposons in prokaryotic genomes and metagenomes. We believed that future studies implying ISs and MITEs identification in genomic data should combine at least one de novo and one library-based method, with optimal results obtained by running the two de novo methods in addition to a library-based search. For metagenomic data, profile HMM search should be favored, a BLAST-based step is only useful to the final annotation into groups and families.

blend4php: a PHP API for galaxy.

PubMed

Wytko, Connor; Soto, Brian; Ficklin, Stephen P

2017-01-01

Galaxy is a popular framework for execution of complex analytical pipelines typically for large data sets, and is a commonly used for (but not limited to) genomic, genetic and related biological analysis. It provides a web front-end and integrates with high performance computing resources. Here we report the development of the blend4php library that wraps Galaxy's RESTful API into a PHP-based library. PHP-based web applications can use blend4php to automate execution, monitoring and management of a remote Galaxy server, including its users, workflows, jobs and more. The blend4php library was specifically developed for the integration of Galaxy with Tripal, the open-source toolkit for the creation of online genomic and genetic web sites. However, it was designed as an independent library for use by any application, and is freely available under version 3 of the GNU Lesser General Public License (LPGL v3.0) at https://github.com/galaxyproject/blend4phpDatabase URL: https://github.com/galaxyproject/blend4php. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The ‘thousand-dollar genome': an ethical exploration

PubMed Central

Dondorp, Wybo J; de Wert, Guido M W R

2013-01-01

Sequencing an individual's complete genome is expected to be possible for a relatively low sum ‘one thousand dollars' within a few years. Sequencing refers to determining the order of base pairs that make up the genome. The result is a library of three billion letter combinations. Cheap whole-genome sequencing is of greatest importance to medical scientific research. Comparing individual complete genomes will lead to a better understanding of the contribution genetic variation makes to health and disease. As knowledge increases, the ‘thousand-dollar genome' will also become increasingly important to healthcare. The applications that come within reach raise a number of ethical questions. This monitoring report addresses the issue. PMID:23677179

Genome-Wide Association Study of a Validated Case Definition of Gulf War Illness in a Population-Representative Sample

DTIC Science & Technology

2013-09-01

sequence dataset. All procedures were performed by personnel in the IIMT UT Southwestern Genomics and Microarray Core using standard protocols. More... sequencing run, samples were demultiplexed using standard algorithms in the Genomics and Microarray Core and processed into individual sample Illumina single... Sequencing (RNA-Seq), using Illumina’s multiplexing mRNA-Seq to generate full sequence libraries from the poly-A tailed RNA to a read depth of 30

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

PubMed Central

Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

2015-01-01

A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

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

PubMed

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.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

PubMed

Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

2014-05-22

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Kmerind: A Flexible Parallel Library for K-mer Indexing of Biological Sequences on Distributed Memory Systems.

PubMed

Pan, Tony; Flick, Patrick; Jain, Chirag; Liu, Yongchao; Aluru, Srinivas

2017-10-09

Counting and indexing fixed length substrings, or k-mers, in biological sequences is a key step in many bioinformatics tasks including genome alignment and mapping, genome assembly, and error correction. While advances in next generation sequencing technologies have dramatically reduced the cost and improved latency and throughput, few bioinformatics tools can efficiently process the datasets at the current generation rate of 1.8 terabases every 3 days. We present Kmerind, a high performance parallel k-mer indexing library for distributed memory environments. The Kmerind library provides a set of simple and consistent APIs with sequential semantics and parallel implementations that are designed to be flexible and extensible. Kmerind's k-mer counter performs similarly or better than the best existing k-mer counting tools even on shared memory systems. In a distributed memory environment, Kmerind counts k-mers in a 120 GB sequence read dataset in less than 13 seconds on 1024 Xeon CPU cores, and fully indexes their positions in approximately 17 seconds. Querying for 1% of the k-mers in these indices can be completed in 0.23 seconds and 28 seconds, respectively. Kmerind is the first k-mer indexing library for distributed memory environments, and the first extensible library for general k-mer indexing and counting. Kmerind is available at https://github.com/ParBLiSS/kmerind.

Len Gen: The international lentil genome sequencing project

USDA-ARS?s Scientific Manuscript database

We have been sequencing CDC Redberry using NGS of paired-end and mate-pair libraries over a wide range of sizes and technologies. The most recent draft (v0.7) of approximately 150x coverage produced scaffolds covering over half the genome (2.7 Gb of the expected 4.3 Gb). Long reads from PacBio sequ...

A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses

USDA-ARS?s Scientific Manuscript database

Several biosafety level (BSL)-3/4 pathogens are high consequence, single-stranded RNA viruses and their genomes, when introduced into permissive cells, are infectious. Moreover many of these viruses are Select Agents (SAs), and their genomes are also considered SAs. For this reason cDNAs and/or th...

The use of PacBio and Hi-C data in denovo assembly of the goat genome

USDA-ARS?s Scientific Manuscript database

Generating de novo reference genome assemblies for non-model organisms is a laborious task that often requires a large amount of data from several sequencing platforms and cytogenetic surveys. By using PacBio sequence data and new library creation techniques, we present a de novo, high quality refer...

A framework linkage map of perennial ryegrass based on SSR markers

Treesearch

G.P. Gill; P.L. Wilcox; D.J. Whittaker; R.A. Winz; P. Bickerstaff; Craig E. Echt; J. Kent; M.O. Humphreys; K.M. Elborough; R.C. Gardner

2006-01-01

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third ( 124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci...

Simplifier: a web tool to eliminate redundant NGS contigs.

PubMed

Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Azevedo, Vasco; Schneider, Maria Paula; Barh, Debmalya; Silva, Artur

2012-01-01

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms. Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.

VariantSpark: population scale clustering of genotype information.

PubMed

O'Brien, Aidan R; Saunders, Neil F W; Guo, Yi; Buske, Fabian A; Scott, Rodney J; Bauer, Denis C

2015-12-10

Genomic information is increasingly used in medical practice giving rise to the need for efficient analysis methodology able to cope with thousands of individuals and millions of variants. The widely used Hadoop MapReduce architecture and associated machine learning library, Mahout, provide the means for tackling computationally challenging tasks. However, many genomic analyses do not fit the Map-Reduce paradigm. We therefore utilise the recently developed SPARK engine, along with its associated machine learning library, MLlib, which offers more flexibility in the parallelisation of population-scale bioinformatics tasks. The resulting tool, VARIANTSPARK provides an interface from MLlib to the standard variant format (VCF), offers seamless genome-wide sampling of variants and provides a pipeline for visualising results. To demonstrate the capabilities of VARIANTSPARK, we clustered more than 3,000 individuals with 80 Million variants each to determine the population structure in the dataset. VARIANTSPARK is 80 % faster than the SPARK-based genome clustering approach, ADAM, the comparable implementation using Hadoop/Mahout, as well as ADMIXTURE, a commonly used tool for determining individual ancestries. It is over 90 % faster than traditional implementations using R and Python. The benefits of speed, resource consumption and scalability enables VARIANTSPARK to open up the usage of advanced, efficient machine learning algorithms to genomic data.

Company profile: Complete Genomics Inc.

PubMed

Reid, Clifford

2011-02-01

Complete Genomics Inc. is a life sciences company that focuses on complete human genome sequencing. It is taking a completely different approach to DNA sequencing than other companies in the industry. Rather than building a general-purpose platform for sequencing all organisms and all applications, it has focused on a single application - complete human genome sequencing. The company's Complete Genomics Analysis Platform (CGA™ Platform) comprises an integrated package of biochemistry, instrumentation and software that sequences human genomes at the highest quality, lowest cost and largest scale available. Complete Genomics offers a turnkey service that enables customers to outsource their human genome sequencing to the company's genome sequencing center in Mountain View, CA, USA. Customers send in their DNA samples, the company does all the library preparation, DNA sequencing, assembly and variant analysis, and customers receive research-ready data that they can use for biological discovery.

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

PubMed Central

Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

2011-01-01

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 conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation. PMID:21909358

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

The Genome of the Beluga Whale (Delphinapterus leucas)

PubMed Central

Taylor, Gregory A.; Chan, Simon; Warren, René L.; Hammond, S. Austin; Bilobram, Steven; Mordecai, Gideon; Miller, Kristina M.; Schulze, Angela; Chan, Amy M.; Jones, Samantha J.; Tse, Kane; Li, Irene; Cheung, Dorothy; Mungall, Karen L.; Choo, Caleb; Ally, Adrian; Dhalla, Noreen; Tam, Angela K. Y.; Troussard, Armelle; Kirk, Heather; Pandoh, Pawan; Paulino, Daniel; Coope, Robin J. N.; Moore, Richard; Zhao, Yongjun; Birol, Inanc; Ma, Yussanne; Marra, Marco; Haulena, Martin

2017-01-01

The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus. The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue. Assembled sequence and all of the underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the Bioproject accession number PRJNA360851A. PMID:29232881

The Genome of the Beluga Whale (Delphinapterus leucas).

PubMed

Jones, Steven J M; Taylor, Gregory A; Chan, Simon; Warren, René L; Hammond, S Austin; Bilobram, Steven; Mordecai, Gideon; Suttle, Curtis A; Miller, Kristina M; Schulze, Angela; Chan, Amy M; Jones, Samantha J; Tse, Kane; Li, Irene; Cheung, Dorothy; Mungall, Karen L; Choo, Caleb; Ally, Adrian; Dhalla, Noreen; Tam, Angela K Y; Troussard, Armelle; Kirk, Heather; Pandoh, Pawan; Paulino, Daniel; Coope, Robin J N; Mungall, Andrew J; Moore, Richard; Zhao, Yongjun; Birol, Inanc; Ma, Yussanne; Marra, Marco; Haulena, Martin

2017-12-11

The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus . The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue. Assembled sequence and all of the underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the Bioproject accession number PRJNA360851A.

Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

PubMed

Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

2015-01-27

Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

Isolation of a DNA Probe for Lactobacillus curvatus

PubMed Central

Petrick, Hendrik A. R.; Ambrosio, Riccardo E.; Holzapfel, Wilhelm H.

1988-01-01

A genomic library of Lactobacillus curvatus DSM 20019 was constructed in bacteriophage λ gt11. A 1.2-kilobase DNA probe specific for L. curvatus was isolated from this library. When this probe was hybridized to DNA from Lactobacillus isolates from different sources classified by conventional techniques, differing degrees of hybridization were obtained. This could imply that these isolates may have been incorrectly classified. Images PMID:16347554

High yield of functional metagenomic library from mangroves constructed in fosmid vector.

PubMed

Gonçalves, A C S; dos Santos, A C F; dos Santos, T F; Pessoa, T B A; Dias, J C T; Rezende, R P

2015-10-02

In the present study, metagenomic technique and fosmid vectors were used to construct a library of clones for exploring the biotechnological potential of mangrove soils by isolation of functional genes encoding hydrolytic enzymes. The library was built with genomic DNA from the soil samples of mangrove sediments and the functional screening of 1824 clones (~64 Mbp) was performed to detect the hydrolytic activity specific for cellulases, amylases (at acidic, neutral and basic pH), lipases/esterases, proteases, and nitrilases. Significant numbers of clones, positive for the tested enzyme activities were obtained. Our results indicate the importance and biotechnological potential of mangrove soils especially when compared to those obtained using other soil metagenomic libraries.

Observing copepods through a genomic lens

PubMed Central

2011-01-01

Background Copepods outnumber every other multicellular animal group. They are critical components of the world's freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited. Discussion The extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for copepods. Summary Genomics research on copepods is needed to extend our exploration and characterization of their fundamental biological traits, so that we can better understand how copepods function and interact in diverse environments. Availability of large scale genomics resources will also open doors to a wide range of systems biology type studies that view the organism as the fundamental system in which to address key questions in ecology and evolution. PMID:21933388

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

PubMed

Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

2018-01-08

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

PubMed

Koike-Yusa, Hiroko; Li, Yilong; Tan, E-Pien; Velasco-Herrera, Martin Del Castillo; Yusa, Kosuke

2014-03-01

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

High-throughput automated microfluidic sample preparation for accurate microbial genomics

PubMed Central

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B.; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P.; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C.

2017-01-01

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications. PMID:28128213

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burger, Brian T.; Imam, Saheed; Scarborough, Matthew J.

Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosyntheticmore » growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species.« less

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

BAC-end sequence-based SNP mining in Allotetraploid Cotton (Gossypium) utilizing re-sequencing data, phylogenetic inferences and perspectives for genetic mapping

USDA-ARS?s Scientific Manuscript database

A bacterial artificial chromosome (BAC) library and BAC-end sequences for Gossypium hirsutum L. have recently been developed. Here we report on genomic-based genome-wide SNP mining utilizing re-sequencing data with a BAC-end sequence reference for twelve G. hirsutum L. lines, one G. barbadense L. li...

Dr. Janie Merkel is interviewed by Ryan Blum and Janice Friend.

PubMed

Merkel, Janie

2007-12-01

Dr. Janie Merkel is the director of Yale's Chemical Genomics Screening Facility, a high-throughput screening laboratory that is part of the Yale University Center for Genomics and Proteomics. The Screening Facility connects Yale researchers with industry-quality robotic machinery and a diverse group of compound libraries, which have been used successfully to link therapeutic targets with potential therapies.

Mutagenesis of diploid mammalian genes by gene entrapment

PubMed Central

Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

2006-01-01

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM).

PubMed

Skinnider, Michael A; Dejong, Chris A; Rees, Philip N; Johnston, Chad W; Li, Haoxin; Webster, Andrew L H; Wyatt, Morgan A; Magarvey, Nathan A

2015-11-16

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A computational search for box C/D snoRNA genes in the Drosophila melanogaster genome.

PubMed

Accardo, M C; Giordano, E; Riccardo, S; Digilio, F A; Iazzetti, G; Calogero, R A; Furia, M

2004-12-12

In eukaryotes, the family of non-coding RNA genes includes a number of genes encoding small nucleolar RNAs (mainly C/D and H/ACA snoRNAs), which act as guides in the maturation or post-transcriptional modifications of target RNA molecules. Since in Drosophila melanogaster (Dm) only few examples of snoRNAs have been identified so far by cDNA libraries screening, integration of the molecular data with in silico identification of these types of genes could throw light on their organization in the Dm genome. We have performed a computational screening of the Dm genome for C/D snoRNA genes, followed by experimental validation of the putative candidates. Few of the 26 confirmed snoRNAs had been recognized by cDNA library analysis. Organization of the Dm genome was also found to be more variegated than previously suspected, with snoRNA genes nested in both the introns and exons of protein-coding genes. This finding suggests that the presence of additional mechanisms of snoRNA biogenesis based on the alternative production of overlapping mRNA/snoRNA molecules. Additional information is available at http://www.bioinformatica.unito.it/bioinformatics/snoRNAs.

Comparative genomic data of the Avian Phylogenomics Project.

PubMed

Zhang, Guojie; Li, Bo; Li, Cai; Gilbert, M Thomas P; Jarvis, Erich D; Wang, Jun

2014-01-01

The evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics. The 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses. Here we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.

Physical mapping of complex genomes

DOEpatents

Evans, G.A.

1993-06-15

A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.

Transcriptomic Changes of Drought-Tolerant and Sensitive Banana Cultivars Exposed to Drought Stress

PubMed Central

Muthusamy, Muthusamy; Uma, Subbaraya; Backiyarani, Suthanthiram; Saraswathi, Marimuthu Somasundaram; Chandrasekar, Arumugam

2016-01-01

In banana, drought responsive gene expression profiles of drought-tolerant and sensitive genotypes remain largely unexplored. In this research, the transcriptome of drought-tolerant banana cultivar (Saba, ABB genome) and sensitive cultivar (Grand Naine, AAA genome) was monitored using mRNA-Seq under control and drought stress condition. A total of 162.36 million reads from tolerant and 126.58 million reads from sensitive libraries were produced and mapped onto the Musa acuminata genome sequence and assembled into 23,096 and 23,079 unigenes. Differential gene expression between two conditions (control and drought) showed that at least 2268 and 2963 statistically significant, functionally known, non-redundant differentially expressed genes (DEGs) from tolerant and sensitive libraries. Drought has up-regulated 991 and 1378 DEGs and down-regulated 1104 and 1585 DEGs respectively in tolerant and sensitive libraries. Among DEGs, 15.9% are coding for transcription factors (TFs) comprising 46 families and 9.5% of DEGs are constituted by protein kinases from 82 families. Most enriched DEGs are mainly involved in protein modifications, lipid metabolism, alkaloid biosynthesis, carbohydrate degradation, glycan metabolism, and biosynthesis of amino acid, cofactor, nucleotide-sugar, hormone, terpenoids and other secondary metabolites. Several, specific genotype-dependent gene expression pattern was observed for drought stress in both cultivars. A subset of 9 DEGs was confirmed using quantitative reverse transcription-PCR. These results will provide necessary information for developing drought-resilient banana plants. PMID:27867388

Gene discovery in an invasive tephritid model pest species, the Mediterranean fruit fly, Ceratitis capitata

PubMed Central

Gomulski, Ludvik M; Dimopoulos, George; Xi, Zhiyong; Soares, Marcelo B; Bonaldo, Maria F; Malacrida, Anna R; Gasperi, Giuliano

2008-01-01

Background The medfly, Ceratitis capitata, is a highly invasive agricultural pest that has become a model insect for the development of biological control programs. Despite research into the behavior and classical and population genetics of this organism, the quantity of sequence data available is limited. We have utilized an expressed sequence tag (EST) approach to obtain detailed information on transcriptome signatures that relate to a variety of physiological systems in the medfly; this information emphasizes on reproduction, sex determination, and chemosensory perception, since the study was based on normalized cDNA libraries from embryos and adult heads. Results A total of 21,253 high-quality ESTs were obtained from the embryo and head libraries. Clustering analyses performed separately for each library resulted in 5201 embryo and 6684 head transcripts. Considering an estimated 19% overlap in the transcriptomes of the two libraries, they represent about 9614 unique transcripts involved in a wide range of biological processes and molecular functions. Of particular interest are the sequences that share homology with Drosophila genes involved in sex determination, olfaction, and reproductive behavior. The medfly transformer2 (tra2) homolog was identified among the embryonic sequences, and its genomic organization and expression were characterized. Conclusion The sequences obtained in this study represent the first major dataset of expressed genes in a tephritid species of agricultural importance. This resource provides essential information to support the investigation of numerous questions regarding the biology of the medfly and other related species and also constitutes an invaluable tool for the annotation of complete genome sequences. Our study has revealed intriguing findings regarding the transcript regulation of tra2 and other sex determination genes, as well as insights into the comparative genomics of genes implicated in chemosensory reception and reproduction. PMID:18500975

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Draft genome and reference transcriptomic resources for the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera: Notodontidae).

PubMed

Gschloessl, B; Dorkeld, F; Berges, H; Beydon, G; Bouchez, O; Branco, M; Bretaudeau, A; Burban, C; Dubois, E; Gauthier, P; Lhuillier, E; Nichols, J; Nidelet, S; Rocha, S; Sauné, L; Streiff, R; Gautier, M; Kerdelhué, C

2018-05-01

The pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae) is the main pine defoliator in the Mediterranean region. Its urticating larvae cause severe human and animal health concerns in the invaded areas. This species shows a high phenotypic variability for various traits, such as phenology, fecundity and tolerance to extreme temperatures. This study presents the construction and analysis of extensive genomic and transcriptomic resources, which are an obligate prerequisite to understand their underlying genetic architecture. Using a well-studied population from Portugal with peculiar phenological characteristics, the karyotype was first determined and a first draft genome of 537 Mb total length was assembled into 68,292 scaffolds (N50 = 164 kb). From this genome assembly, 29,415 coding genes were predicted. To circumvent some limitations for fine-scale physical mapping of genomic regions of interest, a 3X coverage BAC library was also developed. In particular, 11 BACs from this library were individually sequenced to assess the assembly quality. Additionally, de novo transcriptomic resources were generated from various developmental stages sequenced with HiSeq and MiSeq Illumina technologies. The reads were de novo assembled into 62,376 and 63,175 transcripts, respectively. Then, a robust subset of the genome-predicted coding genes, the de novo transcriptome assemblies and previously published 454/Sanger data were clustered to obtain a high-quality and comprehensive reference transcriptome consisting of 29,701 bona fide unigenes. These sequences covered 99% of the cegma and 88% of the busco highly conserved eukaryotic genes and 84% of the busco arthropod gene set. Moreover, 90% of these transcripts could be localized on the draft genome. The described information is available via a genome annotation portal (http://bipaa.genouest.org/sp/thaumetopoea_pityocampa/). © 2018 John Wiley & Sons Ltd.

Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

PubMed Central

Legault, Boris A; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R Thane

2006-01-01

Background Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities. PMID:16820057

Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

PubMed Central

Devirgiliis, Chiara; Barile, Simona; Perozzi, Giuditta

2014-01-01

Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest. PMID:25243126

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

PubMed

Ludgate, Jackie L; Wright, James; Stockwell, Peter A; Morison, Ian M; Eccles, Michael R; Chatterjee, Aniruddha

2017-08-31

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

Informative genomic microsatellite markers for efficient genotyping applications in sugarcane.

PubMed

Parida, Swarup K; Kalia, Sanjay K; Kaul, Sunita; Dalal, Vivek; Hemaprabha, G; Selvi, Athiappan; Pandit, Awadhesh; Singh, Archana; Gaikwad, Kishor; Sharma, Tilak R; Srivastava, Prem Shankar; Singh, Nagendra K; Mohapatra, Trilochan

2009-01-01

Genomic microsatellite markers are capable of revealing high degree of polymorphism. Sugarcane (Saccharum sp.), having a complex polyploid genome requires more number of such informative markers for various applications in genetics and breeding. With the objective of generating a large set of microsatellite markers designated as Sugarcane Enriched Genomic MicroSatellite (SEGMS), 6,318 clones from genomic libraries of two hybrid sugarcane cultivars enriched with 18 different microsatellite repeat-motifs were sequenced to generate 4.16 Mb high-quality sequences. Microsatellites were identified in 1,261 of the 5,742 non-redundant clones that accounted for 22% enrichment of the libraries. Retro-transposon association was observed for 23.1% of the identified microsatellites. The utility of the microsatellite containing genomic sequences were demonstrated by higher primer designing potential (90%) and PCR amplification efficiency (87.4%). A total of 1,315 markers including 567 class I microsatellite markers were designed and placed in the public domain for unrestricted use. The level of polymorphism detected by these markers among sugarcane species, genera, and varieties was 88.6%, while cross-transferability rate was 93.2% within Saccharum complex and 25% to cereals. Cloning and sequencing of size variant amplicons revealed that the variation in the number of repeat-units was the main source of SEGMS fragment length polymorphism. High level of polymorphism and wide range of genetic diversity (0.16-0.82 with an average of 0.44) assayed with the SEGMS markers suggested their usefulness in various genotyping applications in sugarcane.

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

PubMed

Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

2017-08-01

To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

Database Resources of the BIG Data Center in 2018

PubMed Central

Xu, Xingjian; Hao, Lili; Zhu, Junwei; Tang, Bixia; Zhou, Qing; Song, Fuhai; Chen, Tingting; Zhang, Sisi; Dong, Lili; Lan, Li; Wang, Yanqing; Sang, Jian; Hao, Lili; Liang, Fang; Cao, Jiabao; Liu, Fang; Liu, Lin; Wang, Fan; Ma, Yingke; Xu, Xingjian; Zhang, Lijuan; Chen, Meili; Tian, Dongmei; Li, Cuiping; Dong, Lili; Du, Zhenglin; Yuan, Na; Zeng, Jingyao; Zhang, Zhewen; Wang, Jinyue; Shi, Shuo; Zhang, Yadong; Pan, Mengyu; Tang, Bixia; Zou, Dong; Song, Shuhui; Sang, Jian; Xia, Lin; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Zhang, Yang; Sheng, Xin; Lu, Mingming; Wang, Qi; Xiao, Jingfa; Zou, Dong; Wang, Fan; Hao, Lili; Liang, Fang; Li, Mengwei; Sun, Shixiang; Zou, Dong; Li, Rujiao; Yu, Chunlei; Wang, Guangyu; Sang, Jian; Liu, Lin; Li, Mengwei; Li, Man; Niu, Guangyi; Cao, Jiabao; Sun, Shixiang; Xia, Lin; Yin, Hongyan; Zou, Dong; Xu, Xingjian; Ma, Lina; Chen, Huanxin; Sun, Yubin; Yu, Lei; Zhai, Shuang; Sun, Mingyuan; Zhang, Zhang; Zhao, Wenming; Xiao, Jingfa; Bao, Yiming; Song, Shuhui; Hao, Lili; Li, Rujiao; Ma, Lina; Sang, Jian; Wang, Yanqing; Tang, Bixia; Zou, Dong; Wang, Fan

2018-01-01

Abstract The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. PMID:29036542

Construction of CRISPR Libraries for Functional Screening.

PubMed

Carstens, Carsten P; Felts, Katherine A; Johns, Sarah E

2018-01-01

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

Economical analysis of saturation mutagenesis experiments

PubMed Central

Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

2015-01-01

Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

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

PubMed Central

2012-01-01

Background Bread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. Results The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29%) were 3A-specific and compared to 17 (18%) for 96 SSRs. Conclusion This work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping. PMID:22559868

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

PubMed Central

2011-01-01

Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns. PMID:21599934

Enhancing genome assemblies by integrating non-sequence based data

PubMed Central

2011-01-01

Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses. PMID:21554765

Enhancing genome assemblies by integrating non-sequence based data.

PubMed

Heider, Thomas N; Lindsay, James; Wang, Chenwei; O'Neill, Rachel J; Pask, Andrew J

2011-05-28

Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

Flexible CRISPR library construction using parallel oligonucleotide retrieval

PubMed Central

Read, Abigail; Gao, Shaojian; Batchelor, Eric

2017-01-01

Abstract CRISPR/Cas9-based gene knockout libraries have emerged as a powerful tool for functional screens. We present here a set of pre-designed human and mouse sgRNA sequences that are optimized for both high on-target potency and low off-target effect. To maximize the chance of target gene inactivation, sgRNAs were curated to target both 5΄ constitutive exons and exons that encode conserved protein domains. We describe here a robust and cost-effective method to construct multiple small sized CRISPR library from a single oligo pool generated by array synthesis using parallel oligonucleotide retrieval. Together, these resources provide a convenient means for individual labs to generate customized CRISPR libraries of variable size and coverage depth for functional genomics application. PMID:28334828

Discovery of new antimalarial chemotypes through chemical methodology and library development.

PubMed

Brown, Lauren E; Chih-Chien Cheng, Ken; Wei, Wan-Guo; Yuan, Pingwei; Dai, Peng; Trilles, Richard; Ni, Feng; Yuan, Jing; MacArthur, Ryan; Guha, Rajarshi; Johnson, Ronald L; Su, Xin-zhuan; Dominguez, Melissa M; Snyder, John K; Beeler, Aaron B; Schaus, Scott E; Inglese, James; Porco, John A

2011-04-26

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

PubMed

Hall, Thomas E; Martel, Nick; Lo, Harriet P; Xiong, Zherui; Parton, Robert G

2017-01-01

The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

PubMed

Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

2014-06-01

The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. © 2014 The Royal Entomological Society.

Los Alamos Science: The Human Genome Project. Number 20, 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, N G; Shea, N

1992-01-01

This article provides a broad overview of the Human Genome Project, with particular emphasis on work being done at Los Alamos. It tries to emphasize the scientific aspects of the project, compared to the more speculative information presented in the popular press. There is a brief introduction to modern genetics, including a review of classic work. There is a broad overview of the Genome Project, describing what the project is, what are some of its major five-year goals, what are major technological challenges ahead of the project, and what can the field of biology, as well as society expect tomore » see as benefits from this project. Specific results on the efforts directed at mapping chromosomes 16 and 5 are discussed. A brief introduction to DNA libraries is presented, bearing in mind that Los Alamos has housed such libraries for many years prior to the Genome Project. Information on efforts to do applied computational work related to the project are discussed, as well as experimental efforts to do rapid DNA sequencing by means of single-molecule detection using applied spectroscopic methods. The article introduces the Los Alamos staff which are working on the Genome Project, and concludes with brief discussions on ethical, legal, and social implications of this work; a brief glimpse of genetics as it may be practiced in the next century; and a glossary of relevant terms.« less

Los Alamos Science: The Human Genome Project. Number 20, 1992

DOE R&D Accomplishments Database

Cooper, N. G.; Shea, N. eds.

1992-01-01

This document provides a broad overview of the Human Genome Project, with particular emphasis on work being done at Los Alamos. It tries to emphasize the scientific aspects of the project, compared to the more speculative information presented in the popular press. There is a brief introduction to modern genetics, including a review of classic work. There is a broad overview of the Genome Project, describing what the project is, what are some of its major five-year goals, what are major technological challenges ahead of the project, and what can the field of biology, as well as society expect to see as benefits from this project. Specific results on the efforts directed at mapping chromosomes 16 and 5 are discussed. A brief introduction to DNA libraries is presented, bearing in mind that Los Alamos has housed such libraries for many years prior to the Genome Project. Information on efforts to do applied computational work related to the project are discussed, as well as experimental efforts to do rapid DNA sequencing by means of single-molecule detection using applied spectroscopic methods. The article introduces the Los Alamos staff which are working on the Genome Project, and concludes with brief discussions on ethical, legal, and social implications of this work; a brief glimpse of genetics as it may be practiced in the next century; and a glossary of relevant terms.

Construction of a transfer vector for a clonal isolate of LdNPV

Treesearch

Shivanand T. Hiremath; Martha Fikes; Audrey Ichida

1991-01-01

Deoxyribonucleic acid from a clonal isolate of LdNPV (CI A2-1), obtained by in vivo cloning procedures, was used to construct genomic libraries in phage (lamda Gem 11) and cosmid (pHC79) vectors. Overlapping clones were selected to generate a restriction enzyme map. The restriction enzyme map, covering about 85% of the CI A2-1 genome, was determined...

Genome Sequence of the Verrucomicrobium Opitutus terrae PB90-1, an Abundant Inhabitant of Rice Paddy Soil Ecosystems▿

PubMed Central

van Passel, Mark W. J.; Kant, Ravi; Palva, Airi; Copeland, Alex; Lucas, Susan; Lapidus, Alla; Glavina del Rio, Tijana; Pitluck, Sam; Goltsman, Eugene; Clum, Alicia; Sun, Hui; Schmutz, Jeremy; Larimer, Frank W.; Land, Miriam L.; Hauser, Loren; Kyrpides, Nikolaos; Mikhailova, Natalia; Richardson, P. Paul; Janssen, Peter H.; de Vos, Willem M.; Smidt, Hauke

2011-01-01

Bacteria of the deeply branching phylum Verrucomicrobia are rarely cultured yet commonly detected in metagenomic libraries from aquatic, terrestrial, and intestinal environments. We have sequenced the genome of Opitutus terrae PB90-1, a fermentative anaerobe within this phylum, isolated from rice paddy soil and capable of propionate production from plant-derived polysaccharides. PMID:21398538

De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

PubMed Central

Muñoz-Mérida, Antonio; González-Plaza, Juan José; Cañada, Andrés; Blanco, Ana María; García-López, Maria del Carmen; Rodríguez, José Manuel; Pedrola, Laia; Sicardo, M. Dolores; Hernández, M. Luisa; De la Rosa, Raúl; Belaj, Angjelina; Gil-Borja, Mayte; Luque, Francisco; Martínez-Rivas, José Manuel; Pisano, David G.; Trelles, Oswaldo; Valpuesta, Victoriano; Beuzón, Carmen R.

2013-01-01

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation. PMID:23297299

Jannovar: a java library for exome annotation.

PubMed

Jäger, Marten; Wang, Kai; Bauer, Sebastian; Smedley, Damian; Krawitz, Peter; Robinson, Peter N

2014-05-01

Transcript-based annotation and pedigree analysis are two basic steps in the computational analysis of whole-exome sequencing experiments in genetic diagnostics and disease-gene discovery projects. Here, we present Jannovar, a stand-alone Java application as well as a Java library designed to be used in larger software frameworks for exome and genome analysis. Jannovar uses an interval tree to identify all transcripts affected by a given variant, and provides Human Genome Variation Society-compliant annotations both for variants affecting coding sequences and splice junctions as well as untranslated regions and noncoding RNA transcripts. Jannovar can also perform family-based pedigree analysis with Variant Call Format (VCF) files with data from members of a family segregating a Mendelian disorder. Using a desktop computer, Jannovar requires a few seconds to annotate a typical VCF file with exome data. Jannovar is freely available under the BSD2 license. Source code as well as the Java application and library file can be downloaded from http://compbio.charite.de (with tutorial) and https://github.com/charite/jannovar. © 2014 WILEY PERIODICALS, INC.

Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

PubMed Central

Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

2016-01-01

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

Construction of a general human chromosome jumping library, with application to cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, F.S.; Drumm, M.L.; Cole, J.L.

1987-02-27

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as reverse genetics, in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of itsmore » usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should be applicable to any genetic locus for which a closely linked DNA marker is available.« less

Update of the Diatom EST Database: a new tool for digital transcriptomics

PubMed Central

Maheswari, Uma; Mock, Thomas; Armbrust, E. Virginia; Bowler, Chris

2009-01-01

The Diatom Expressed Sequence Tag (EST) Database was constructed to provide integral access to ESTs from these ecologically and evolutionarily interesting microalgae. It has now been updated with 130 000 Phaeodactylum tricornutum ESTs from 16 cDNA libraries and 77 000 Thalassiosira pseudonana ESTs from seven libraries, derived from cells grown in different nutrient and stress regimes. The updated relational database incorporates results from statistical analyses such as log-likelihood ratios and hierarchical clustering, which help to identify differentially expressed genes under different conditions, and allow similarities in gene expression in different libraries to be investigated in a functional context. The database also incorporates links to the recently sequenced genomes of P. tricornutum and T. pseudonana, enabling an easy cross-talk between the expression pattern of diatom orthologs and the genome browsers. These improvements will facilitate exploration of diatom responses to conditions of ecological relevance and will aid gene function identification of diatom-specific genes and in silico gene prediction in this largely unexplored class of eukaryotes. The updated Diatom EST Database is available at http://www.biologie.ens.fr/diatomics/EST3. PMID:19029140

A database of annotated tentative orthologs from crop abiotic stress transcripts.

PubMed

Balaji, Jayashree; Crouch, Jonathan H; Petite, Prasad V N S; Hoisington, David A

2006-10-07

A minimal requirement to initiate a comparative genomics study on plant responses to abiotic stresses is a dataset of orthologous sequences. The availability of a large amount of sequence information, including those derived from stress cDNA libraries allow for the identification of stress related genes and orthologs associated with the stress response. Orthologous sequences serve as tools to explore genes and their relationships across species. For this purpose, ESTs from stress cDNA libraries across 16 crop species including 6 important cereal crops and 10 dicots were systematically collated and subjected to bioinformatics analysis such as clustering, grouping of tentative orthologous sets, identification of protein motifs/patterns in the predicted protein sequence, and annotation with stress conditions, tissue/library source and putative function. All data are available to the scientific community at http://intranet.icrisat.org/gt1/tog/homepage.htm. We believe that the availability of annotated plant abiotic stress ortholog sets will be a valuable resource for researchers studying the biology of environmental stresses in plant systems, molecular evolution and genomics.

An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity.

PubMed

Zhang, Wenli; Fu, Jun; Liu, Jing; Wang, Hailong; Schiwon, Maren; Janz, Sebastian; Schaffarczyk, Lukas; von der Goltz, Lukas; Ehrke-Schulz, Eric; Dörner, Johannes; Solanki, Manish; Boehme, Philip; Bergmann, Thorsten; Lieber, Andre; Lauber, Chris; Dahl, Andreas; Petzold, Andreas; Zhang, Youming; Stewart, A Francis; Ehrhardt, Anja

2017-05-23

Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Giardia genome project database.

PubMed

McArthur, A G; Morrison, H G; Nixon, J E; Passamaneck, N Q; Kim, U; Hinkle, G; Crocker, M K; Holder, M E; Farr, R; Reich, C I; Olsen, G E; Aley, S B; Adam, R D; Gillin, F D; Sogin, M L

2000-08-15

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simon, M. I.; Kim, U.-J.

We developed a great deal of expertise in building large BAC libraries from a variety of DNA sources including humans, mice, corn, microorganisms, worms, and Arabidopsis. We greatly improved the technology for screening these libraries rapidly and for selecting appropriate BACs and mapping BACs to develop large overlapping contigs. We became involved in supplying BACs and BAC contigs to a variety of sequencing and mapping projects and we began to collaborate with Drs. Adams and Venter at TIGR and with Dr. Leroy Hood and his group at University of Washington to provide BACs for end sequencing and for mapping andmore » sequencing of large fragments of chromosome 16. Together with Dr. Ian Dunham and his co-workers at the Sanger Center we completed the mapping and they completed the sequencing of the first human chromosome, chromosome 22. This was published in Nature in 1999 and our BAC contigs made a major contribution to this sequencing effort. Drs. Shizuya and Ding invented an automated highly accurate BAC mapping technique. We also developed long-term collaborations with Dr. Uli Weier at UCSF in the design of BAC probes for characterization of human tumors and specific chromosome deletions and breakpoints. Finally the contribution of our work to the human genome project has been recognized in the publication both by the international consortium and the NIH of a draft sequence of the human genome in Nature last year. Dr. Shizuya was acknowledged in the authorship of that landmark paper. Dr. Simon was also an author on the Venter/Adams Celera project sequencing the human genome that was published in Science last year.« less

Multiple Myeloma Genomics: A Systematic Review.

PubMed

Weaver, Casey J; Tariman, Joseph D

2017-08-01

This integrative review describes the genomic variants that have been found to be associated with poor prognosis in patients diagnosed with multiple myeloma (MM). Second, it identifies MM genetic and genomic changes using next-generation sequencing, specifically whole-genome sequencing or exome sequencing. A search for peer-reviewed articles through PubMed, EBSCOhost, and DePaul WorldCat Libraries Worldwide yielded 33 articles that were included in the final analysis. The most commonly reported genetic changes were KRAS, NRAS, TP53, FAM46C, BRAF, DIS3, ATM, and CCND1. These genetic changes play a role in the pathogenesis of MM, prognostication, and therapeutic targets for novel therapies. MM genetics and genomics are expanding rapidly; oncology nurse clinicians must have basic competencies in genetics and genomics to help patients understand the complexities of genetic and genomic alterations and be able to refer patients to appropriate genomic professionals if needed. Copyright © 2017 Elsevier Inc. All rights reserved.

Diverse circovirus-like genome architectures revealed by environmental metagenomics.

PubMed

Rosario, Karyna; Duffy, Siobain; Breitbart, Mya

2009-10-01

Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.

Multiplexed genome engineering and genotyping methods applications for synthetic biology and metabolic engineering.

PubMed

Wang, Harris H; Church, George M

2011-01-01

Engineering at the scale of whole genomes requires fundamentally new molecular biology tools. Recent advances in recombineering using synthetic oligonucleotides enable the rapid generation of mutants at high efficiency and specificity and can be implemented at the genome scale. With these techniques, libraries of mutants can be generated, from which individuals with functionally useful phenotypes can be isolated. Furthermore, populations of cells can be evolved in situ by directed evolution using complex pools of oligonucleotides. Here, we discuss ways to utilize these multiplexed genome engineering methods, with special emphasis on experimental design and implementation. Copyright © 2011 Elsevier Inc. All rights reserved.

A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL

PubMed Central

Meyer, Matthias; Kircher, Martin; Gansauge, Marie-Theres; Li, Heng; Racimo, Fernando; Mallick, Swapan; Schraiber, Joshua G.; Jay, Flora; Prüfer, Kay; de Filippo, Cesare; Sudmant, Peter H.; Alkan, Can; Fu, Qiaomei; Do, Ron; Rohland, Nadin; Tandon, Arti; Siebauer, Michael; Green, Richard E.; Bryc, Katarzyna; Briggs, Adrian W.; Stenzel, Udo; Dabney, Jesse; Shendure, Jay; Kitzman, Jacob; Hammer, Michael F.; Shunkov, Michael V.; Derevianko, Anatoli P.; Patterson, Nick; Andrés, Aida M.; Eichler, Evan E.; Slatkin, Montgomery; Reich, David; Kelso, Janet; Pääbo, Svante

2013-01-01

We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans. PMID:22936568

De novo Transcriptome Assembly of Common Wild Rice (Oryza rufipogon Griff.) and Discovery of Drought-Response Genes in Root Tissue Based on Transcriptomic Data.

PubMed

Tian, Xin-Jie; Long, Yan; Wang, Jiao; Zhang, Jing-Wen; Wang, Yan-Yan; Li, Wei-Min; Peng, Yu-Fa; Yuan, Qian-Hua; Pei, Xin-Wu

2015-01-01

The perennial O. rufipogon (common wild rice), which is considered to be the ancestor of Asian cultivated rice species, contains many useful genetic resources, including drought resistance genes. However, few studies have identified the drought resistance and tissue-specific genes in common wild rice. In this study, transcriptome sequencing libraries were constructed, including drought-treated roots (DR) and control leaves (CL) and roots (CR). Using Illumina sequencing technology, we generated 16.75 million bases of high-quality sequence data for common wild rice and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into 119,332 unigenes with an average length of 715 bp. A total of 88,813 distinct sequences (74.42% of unigenes) significantly matched known genes in the NCBI NT database. Differentially expressed gene (DEG) analysis showed that 3617 genes were up-regulated and 4171 genes were down-regulated in the CR library compared with the CL library. Among the DEGs, 535 genes were expressed in roots but not in shoots. A similar comparison between the DR and CR libraries showed that 1393 genes were up-regulated and 315 genes were down-regulated in the DR library compared with the CR library. Finally, 37 genes that were specifically expressed in roots were screened after comparing the DEGs identified in the above-described analyses. This study provides a transcriptome sequence resource for common wild rice plants and establishes a digital gene expression profile of wild rice plants under drought conditions using the assembled transcriptome data as a reference. Several tissue-specific and drought-stress-related candidate genes were identified, representing a fully characterized transcriptome and providing a valuable resource for genetic and genomic studies in plants.

Automated multiplex genome-scale engineering in yeast

PubMed Central

Si, Tong; Chao, Ran; Min, Yuhao; Wu, Yuying; Ren, Wen; Zhao, Huimin

2017-01-01

Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast. PMID:28469255

Genetic and Functional Heterogeneity of Tumors in Neurofibromatosis 2

DTIC Science & Technology

2016-07-01

mapped to the human genome build 37 (hg19) through BWA v. 0.5.9 [1] with parameters -q 5 -l 32 -k 2 –o 1. The resulting alignments were further sorted...see Table 2). Table 2 – Isogenic human arachnoidal cell (AC) clones with NF2 (exon 8) inactivating mutations generated by CRISPR/Cas genome editing...libraries were aligned to the human genome (GrCH37, Ensembl build 71) using Gsnap [19] version 2014_12_19. Expression levels of genes in the units of count

Peroxisome Mini-Libraries: Systematic Approaches to Study Peroxisomes Made Easy.

PubMed

Dahan, Noa; Schuldiner, Maya; Zalckvar, Einat

2017-01-01

High-throughput methodologies have been extensively used in the budding yeast, Saccharomyces cerevisiae, to uncover fundamental principles of cell biology. Over the years, several collections of yeast strains (libraries) were built to enable systematic exploration of cellular functions. However, using these libraries experimentally is often labor intensive and restricted to laboratories that hold high throughput platforms. Utilizing the available full genome libraries we handpicked a subset of strains that represent all known and predicted peroxisomal proteins as well as proteins that have central roles in peroxisome biology. These smaller collections of strains, mini-libraries, can be rapidly and easily used for complicated screens by any lab. Since one of the libraries is built such that it can be easily modified in the tag, promoter and selection, we also discuss how these collections form the basis for creating a diversity of new peroxisomal libraries for future studies. Using manual tools, available in any yeast lab, coupled with few simple genetic approaches, we will show how these libraries can be "mixed and matched" to create tailor made libraries for screening. These yeast collections may now be exploited to study uncharted territories in the biology of peroxisomes by anyone, anywhere.

Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

PubMed Central

Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

2009-01-01

Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA libraries generated by SGP represent a valuable cCDS FLIc source. The conservation of 7-mers in 3'UTRs indicates that these motifs are functionally important. Identity between some of these 7-mers and miRNA target sequences suggests that they are miRNA targets in Salmo salar transcripts as well. PMID:19878547

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

PubMed

de Andrade, Roberto R S; Vaslin, Maite F S

2014-03-07

Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data

PubMed Central

2014-01-01

Background Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. Methods In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. Results The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. Conclusions SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. Availability and implementation SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/. PMID:24607237

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Rongming; Liang, Liya; Garst, Andrew D.

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)Hmore » metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.« less

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

DOE PAGES

Liu, Rongming; Liang, Liya; Garst, Andrew D.; ...

2018-03-29

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)Hmore » metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.« less

Composition and applications of focus libraries to phenotypic assays

PubMed Central

Wassermann, Anne Mai; Camargo, Luiz M.; Auld, Douglas S.

2014-01-01

The wealth of bioactivity information now available on low-molecular weight compounds has enabled a paradigm shift in chemical biology and early phase drug discovery efforts. Traditionally chemical libraries have been most commonly employed in screening approaches where a bioassay is used to characterize a chemical library in a random search for active samples. However, robust curating of bioassay data, establishment of ontologies enabling mining of large chemical biology datasets, and a wealth of public chemical biology information has made possible the establishment of highly annotated compound collections. Such annotated chemical libraries can now be used to build a pathway/target hypothesis and have led to a new view where chemical libraries are used to characterize a bioassay. In this article we discuss the types of compounds in these annotated libraries composed of tools, probes, and drugs. As well, we provide rationale and a few examples for how such libraries can enable phenotypic/forward chemical genomic approaches. As with any approach, there are several pitfalls that need to be considered and we also outline some strategies to avoid these. PMID:25104937

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent. | Office of Cancer Genomics

Cancer.gov

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling.

Emory University: High-Throughput Protein-Protein Interaction Dataset for Lung Cancer-Associated Genes | Office of Cancer Genomics

Cancer.gov

To discover novel PPI signaling hubs for lung cancer, CTD2 Center at Emory utilized large-scale genomics datasets and literature to compile a set of lung cancer-associated genes. A library of expression vectors were generated for these genes and utilized for detecting pairwise PPIs with cell lysate-based TR-FRET assays in high-throughput screening format. Read the abstract.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

PubMed Central

Michlewski, Gracjan; Finnegan, David J.; Elfick, Alistair; Rosser, Susan J.

2017-01-01

Abstract Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. PMID:28204586

Methods comparison for microsatellite marker development: Different isolation methods, different yield efficiency

NASA Astrophysics Data System (ADS)

Zhan, Aibin; Bao, Zhenmin; Hu, Xiaoli; Lu, Wei; Hu, Jingjie

2009-06-01

Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop ( Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the microsatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

An optimized library for reference-based deconvolution of whole-blood biospecimens assayed using the Illumina HumanMethylationEPIC BeadArray.

PubMed

Salas, Lucas A; Koestler, Devin C; Butler, Rondi A; Hansen, Helen M; Wiencke, John K; Kelsey, Karl T; Christensen, Brock C

2018-05-29

Genome-wide methylation arrays are powerful tools for assessing cell composition of complex mixtures. We compare three approaches to select reference libraries for deconvoluting neutrophil, monocyte, B-lymphocyte, natural killer, and CD4+ and CD8+ T-cell fractions based on blood-derived DNA methylation signatures assayed using the Illumina HumanMethylationEPIC array. The IDOL algorithm identifies a library of 450 CpGs, resulting in an average R 2  = 99.2 across cell types when applied to EPIC methylation data collected on artificial mixtures constructed from the above cell types. Of the 450 CpGs, 69% are unique to EPIC. This library has the potential to reduce unintended technical differences across array platforms.

Database Resources of the BIG Data Center in 2018.

PubMed

2018-01-04

The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing

PubMed Central

Xiao, Yongli; Sheng, Zong-Mei; Taubenberger, Jeffery K.

2015-01-01

The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus at high coverage was determined in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. PMID:26344216

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

PubMed

Han, Guomin; Shao, Qian; Li, Cuiping; Zhao, Kai; Jiang, Li; Fan, Jun; Jiang, Haiyang; Tao, Fang

2018-05-01

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 10 6 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

PubMed Central

Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

PubMed

Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: a genomic resource for studying agricultural pests.

PubMed

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-03-03

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest.

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

PubMed Central

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T.; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries. PMID:24205269

Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

NASA Astrophysics Data System (ADS)

Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

2014-09-01

Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

Global mapping of transposon location.

PubMed

Gabriel, Abram; Dapprich, Johannes; Kunkel, Mark; Gresham, David; Pratt, Stephen C; Dunham, Maitreya J

2006-12-15

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

The first report of a Pelecaniformes defensin cluster: Characterization of β-defensin genes in the crested ibis based on BAC libraries

PubMed Central

Lan, Hong; Chen, Hui; Chen, Li-Cheng; Wang, Bei-Bing; Sun, Li; Ma, Mei-Ying; Fang, Sheng-Guo; Wan, Qiu-Hong

2014-01-01

Defensins play a key role in the innate immunity of various organisms. Detailed genomic studies of the defensin cluster have only been reported in a limited number of birds. Herein, we present the first characterization of defensins in a Pelecaniformes species, the crested ibis (Nipponia nippon), which is one of the most endangered birds in the world. We constructed bacterial artificial chromosome libraries, including a 4D-PCR library and a reverse-4D library, which provide at least 40 equivalents of this rare bird's genome. A cluster including 14 β-defensin loci within 129 kb was assigned to chromosome 3 by FISH, and one gene duplication of AvBD1 was found. The ibis defensin genes are characterized by multiform gene organization ranging from two to four exons through extensive exon fusion. Splicing signal variations and alternative splice variants were also found. Comparative analysis of four bird species identified one common and multiple species-specific duplications, which might be associated with high GC content. Evolutionary analysis revealed birth-and-death mode and purifying selection for avian defensin evolution, resulting in different defensin gene numbers among bird species and functional conservation within orthologous genes, respectively. Additionally, we propose various directions for further research on genetic conservation in the crested ibis. PMID:25372018

MISSION LentiPlex pooled shRNA library screening in mammalian cells.

PubMed

Coussens, Matthew J; Corman, Courtney; Fischer, Ashley L; Sago, Jack; Swarthout, John

2011-12-21

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies. There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library. The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10(8) TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification. Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Genome improvement of the acarbose producer Actinoplanes sp. SE50/110 and annotation refinement based on RNA-seq analysis.

PubMed

Wolf, Timo; Schneiker-Bekel, Susanne; Neshat, Armin; Ortseifen, Vera; Wibberg, Daniel; Zemke, Till; Pühler, Alfred; Kalinowski, Jörn

2017-06-10

Actinoplanes sp. SE50/110 is the natural producer of acarbose, which is used in the treatment of diabetes mellitus type II. However, until now the transcriptional organization and regulation of the acarbose biosynthesis are only understood rudimentarily. The genome sequence of Actinoplanes sp. SE50/110 was known before, but was resequenced in this study to remove assembly artifacts and incorrect base callings. The annotation of the genome was refined in a multi-step approach, including modern bioinformatic pipelines, transcriptome and proteome data. A whole transcriptome RNA-seq library as well as an RNA-seq library enriched for primary 5'-ends were used for the detection of transcription start sites, to correct tRNA predictions, to identify novel transcripts like small RNAs and to improve the annotation through the correction of falsely annotated translation start sites. The transcriptome data sets were also applied to identify 31 cis-regulatory RNA structures, such as riboswitches or RNA thermometers as well as three leaderless transcribed short peptides found in putative attenuators upstream of genes for amino acid biosynthesis. The transcriptional organization of the acarbose biosynthetic gene cluster was elucidated in detail and fourteen novel biosynthetic gene clusters were suggested. The accurate genome sequence and precise annotation of the Actinoplanes sp. SE50/110 genome will be the foundation for future genetic engineering and systems biology studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

PubMed

Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A

2016-01-01

The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

PubMed

Danhorn, Thomas; Young, Curtis R; DeLong, Edward F

2012-11-01

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics1

PubMed Central

Weitemier, Kevin; Straub, Shannon C. K.; Cronn, Richard C.; Fishbein, Mark; Schmickl, Roswitha; McDonnell, Angela; Liston, Aaron

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. PMID:25225629

University of Texas Southwestern Medical Center: U01 Natural Products Screening | Office of Cancer Genomics

Cancer.gov

The goal of this project was to enlarge the chemical space probed by Project 1 (High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer Cell Line Panel) by screening an expanded natural products library (~40,000) in an effort to further define vulnerabilities and therapeutic targets in non-small cell lung cancer. This new library is derived from a diverse collection of marine bacteria (prepared by Dr. John MacMillan, University of Texas Southwestern).

University of Texas Southwestern Medical Center (UTSW): U01 Natural Products Screening | Office of Cancer Genomics

Cancer.gov

The goal of this project was to enlarge the chemical space probed by Project 1 (High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer Cell Line Panel) by screening an expanded natural products library (~40,000) in an effort to further define vulnerabilities and therapeutic targets in non-small cell lung cancer. This new library is derived from a diverse collection of marine bacteria (prepared by Dr. John MacMillan, University of Texas Southwestern).

A Genome-Wide Knockout Screen to Identify Genes Involved in Acquired Carboplatin Resistance

DTIC Science & Technology

2016-07-01

library screen to identify genes that when knocked out render human ovarian cells > 2.5-fold resistant to CBDCA; 2) Validate the ability of...a GeCKOv2 library screen to identify genes that when knocked out render human ovarian cells > 2.5-fold resistant to CBDCA; 2) validate the ability of...resistance in either cell lines or clinical samples. The CRIPSR-cas9 technology now provides us with a major new tool to introduce knock out mutations

A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles | Office of Cancer Genomics

Cancer.gov

Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells.

Bacterial Artificial Chromosome Libraries of Pulse Crops: Characteristics and Applications

PubMed Central

Yu, Kangfu

2012-01-01

Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops. PMID:21811383

Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics

PubMed Central

2013-01-01

Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that not only was the gene nucleotide sequence conserved between soybean and lupin GRRs, but the order and orientation of particular genes in syntenic blocks was homologous, as well. These findings will be valuable to the forthcoming sequencing of the lupin genome. PMID:23379841

[Artificial Intelligence in Drug Discovery].

PubMed

Fujiwara, Takeshi; Kamada, Mayumi; Okuno, Yasushi

2018-04-01

According to the increase of data generated from analytical instruments, application of artificial intelligence(AI)technology in medical field is indispensable. In particular, practical application of AI technology is strongly required in "genomic medicine" and "genomic drug discovery" that conduct medical practice and novel drug development based on individual genomic information. In our laboratory, we have been developing a database to integrate genome data and clinical information obtained by clinical genome analysis and a computational support system for clinical interpretation of variants using AI. In addition, with the aim of creating new therapeutic targets in genomic drug discovery, we have been also working on the development of a binding affinity prediction system for mutated proteins and drugs by molecular dynamics simulation using supercomputer "Kei". We also have tackled for problems in a drug virtual screening. Our developed AI technology has successfully generated virtual compound library, and deep learning method has enabled us to predict interaction between compound and target protein.

Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

PubMed Central

Curson, Andrew R. J.; Burns, Oliver J.; Voget, Sonja; Daniel, Rolf; Todd, Jonathan D.; McInnis, Kathryn; Wexler, Margaret; Johnston, Andrew W. B.

2014-01-01

Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA. PMID:24848004

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies.

PubMed

Frentiu, Francesca D; Adamski, Marcin; McGraw, Elizabeth A; Blows, Mark W; Chenoweth, Stephen F

2009-01-21

The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup. A normalized cDNA library was constructed from whole fly bodies at several developmental stages, including larvae and adults. Assembly of 11,616 clones sequenced from the 3' end allowed us to identify 6,607 unique contigs, of which at least 90% encoded peptides. Partial transcripts were discovered from a variety of genes of evolutionary interest by BLASTing contigs against the 12 Drosophila genomes currently sequenced. By incorporating into the cDNA library multiple individuals from populations spanning a large portion of the geographical range of D. serrata, we were able to identify 11,057 putative single nucleotide polymorphisms (SNPs), with 278 different contigs having at least one "double hit" SNP that is highly likely to be a real polymorphism. At least 394 EST-associated microsatellite markers, representing 355 different contigs, were also found, providing an additional set of genetic markers. The assembled EST library is available online at http://www.chenowethlab.org/serrata/index.cgi. We have provided the first gene collection and largest set of polymorphic genetic markers, to date, for the fly D. serrata. The EST collection will provide much needed genomic resources for this model species and facilitate comparative evolutionary studies within the montium subgroup of the D. melanogaster lineage.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

PubMed Central

Ramsey, John S; Wilson, Alex CC; de Vos, Martin; Sun, Qi; Tamborindeguy, Cecilia; Winfield, Agnese; Malloch, Gaynor; Smith, Dawn M; Fenton, Brian; Gray, Stewart M; Jander, Georg

2007-01-01

Background The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects. PMID:18021414

Defining Translational Reprogramming in Tuberous Sclerosis Complex

DTIC Science & Technology

2015-07-01

Genomics & Development, Cornell University, Ithaca, New York, USA. 3Department of Chemistry, The Scripps Research Inst tute, Jupiter , Florida, USA...4Molecular Therapeutics and Natural Products Library Initiative, The Scripps Research Institute, Jupiter , Florida, USA. 5Present address

Postgenomic strategies in antibacterial drug discovery.

PubMed

Brötz-Oesterhelt, Heike; Sass, Peter

2010-10-01

During the last decade the field of antibacterial drug discovery has changed in many aspects including bacterial organisms of primary interest, discovery strategies applied and pharmaceutical companies involved. Target-based high-throughput screening had been disappointingly unsuccessful for antibiotic research. Understanding of this lack of success has increased substantially and the lessons learned refer to characteristics of targets, screening libraries and screening strategies. The 'genomics' approach was replaced by a diverse array of discovery strategies, for example, searching for new natural product leads among previously abandoned compounds or new microbial sources, screening for synthetic inhibitors by targeted approaches including structure-based design and analyses of focused libraries and designing resistance-breaking properties into antibiotics of established classes. Furthermore, alternative treatment options are being pursued including anti-virulence strategies and immunotherapeutic approaches. This article summarizes the lessons learned from the genomics era and describes discovery strategies resulting from that knowledge.

Physical mapping of complex genomes

DOEpatents

Evans, Glen A.

1993-01-01

Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.

''After the Genome 5 Conference'' to be held October 6-10, 1999 in Jackson Hole, Wyoming

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roger Brent

OAK B139 The postgenomic era is arriving faster than anyone had imagined--sometime during 2000 we'll have a large fraction of the human genome sequence. Heretofore, our understanding of function has come from non-industrial experiments whose conclusions were largely framed in human language. The advent of large amounts of sequence data, and of ''functional genomic'' data types such as mRNA expression data, have changed this picture. These data share the feature that individual observations and measurements are typically relatively low value adding. Such data is now being generated so rapidly that the amount of information contained in it will surpass themore » amount of biological information collected by traditional means. It is tantalizing to envision using genomic information to create a quantitative biology with a very strong data component. Unfortunately, we are very early in our understanding of how to ''compute on'' genomic information so as to extract biological knowledge from i t. In fact, some current efforts to come to grips with genomic information often resemble a computer savvy library science, where the most important issues concern categories, classification schemes, and information retrieval. When exploring new libraries, a measure of cataloging and inventory is surely inevitable. However, at some point we will need to move from library science to scholarship.We would like to achieve a quantitative and predictive understanding of biological function. We realize that making the bridge from knowledge of systems to the sets of abstractions that constitute computable entities is not easy. The After the Genome meetings were started in 1995 to help the biological community think about and prepare for the changes in biological research in the face of the oncoming flow of genomic information. The term ''After the Genome'' refers to a future in which complete inventories of the gene products of entire organisms become available.Since then, many more biologists have become cognizant of the issues raised by this future, and, in response, the organizers intend to distinguish this meeting from other ''postgenomic'' meetings by bringing together intellectuals from subject fields far outside of conventional biology with the expectation that this will help focus thinking beyond the immediate future. To this end, After the Genome 5 will bring together industrial and university researchers, including: (1) Physicists, chemists, and engineers who are devising and using new data gathering techniques, such as microarrays, protein mass spectrometry, and single molecule measurements (2) Computer scientists from fields as diverse as geology and wargames, who have experience moving from broad knowledge of systems to analysis that results in models and simulations (3) Neurobiologists and computer scientists who combine physiological experimentation and computer modeling to understand single cells and small networks of cells (4) Biologists who are trying to model genetic networks (5) All-around visionary thinkers (6) policy makers, to suggest how to convey any good ideas to organizations that can commit resources to them.« less

"After the Genome 5, Conference to be held October 6-10, 1999, Jackson Hole, Wyoming"

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brent, Roger

The postgenomic era is arriving faster than anyone had imagined-- sometime during 2000 we'll have a large fraction of the human genome sequence. Heretofore, our understanding of function has come from non-industrial experiments whose conclusions were largely framed in human language. The advent of large amounts of sequence data, and of "functional genomic" data types such as mRNA expression data, have changed this picture. These data share the feature that individual observations and measurements are typically relatively low value adding. Such data is now being generated so rapidly that the amount of information contained in it will surpass the amountmore » of biological information collected by traditional means. It is tantalizing to envision using genomic information to create a quantitative biology with a very strong data component. Unfortunately, we are very early in our understanding of how to "compute on" genomic information so as to extract biological knowledge from it. In fact, some current efforts to come to grips with genomic information often resemble a computer savvy library science, where the most important issues concern categories, classification schemes, and information retrieval. When exploring new libraries, a measure of cataloging and inventory is surely inevitable. However, at some point we will need to move from library science to scholarship. We would like to achieve a quantitative and predictive understanding of biological function. We realize that making the bridge from knowledge of systems to the sets of abstractions that constitute computable entities is not easy. The After the Genome meetings were started in 1995 to help the biological community think about and prepare for the changes in biological research in the face of the oncoming flow of genomic information. The term "After the Genome" refers to a future in which complete inventories of the gene products of entire organisms become available. Since then, many more biologists have become cognizant of the issues raised by this future, and, in response, the organizers intend to distinguish this meeting from other "postgenomic" meetings by bringing together intellectuals from subject fields far outside of conventional biology with the expectation that this will help focus thinking beyond the immediate future. To this end, After the Genome 5 will bring together industrial and university researchers, including: 1) Physicists, chemists, and engineers who are devising and using new data gathering techniques, such as microarrays, protein mass spectrometry, and single molecule measurements 2) Computer scientists from fields as diverse as geology and wargames, who have experience moving from broad knowledge of systems to analysis that results in models and simulations 3) Neurobiologists and computer scientists who combine physiological experimentation and computer modeling to understand single cells and small networks of cells 4) Biologists who are trying to model genetic networks 5) All- around visionary thinkers 7) policy makers, to suggest how to convey any good ideas to organizations that can commit resources to them.« less

Genomic Characterization of a Novel Phage Found in Black Abalone (Haliotis cracherodii) Infected with Withering Syndrome

NASA Astrophysics Data System (ADS)

Closek, C. J.; Langevin, S.; Burge, C. A.; Crosson, L.; White, S.; Friedman, C. S.

2016-02-01

Withering syndrome (WS), caused by the bacterium Candidatus Xenohaliotis californiensis, a Rickettsia-like organism (RLO), infects many species of abalone. Black abalone (Haliotis cracherodii), one of two endangered species of abalone, has experienced high population losses along the California coast due to WS. Recently, we observed reduced pathogenicity and mortality events in RLO-infected abalone when a novel bacteriophage (phage) was also present. To better understand phage-bacterium dynamics and develop more informative diagnostic tools, we sequenced the genome of the novel phage associated with the RLO responsible for WS. Metagenomic sequencing libraries were prepared with extracted genomic DNA from two experimentally infected H. cracherodii and phage sequences were enriched using hydroxyapatite chromatography normalization. Normalized libraries were individually barcoded and sequenced with Illumina MiSeq. Raw sequence reads were processed using VIrominer and de novo assembly produced one single phage-like contig (35.7Kb) from the experimentally infected abalone. This highly divergent genome had closest homology with a virus associated with abalone shriveling syndrome (SS). Of the 34 predicted ORFs, overlapping homology with the SS virus ranged from 20-72%, demonstrating the phage sequenced is genetically distinct from any known phage. The phage-like sequences represented a significant portion of the total reads sequenced ( 2 million of the 12 million paired-end reads; 17%) and we obtained 94,000X coverage across the novel phage genome. Beyond characterization of this novel phage, which appears to reduce pathogenicity of the RLO, the genome enabled us to develop quantitative PCR and in situ hybridization assays as diagnostic tools. These tools allow us to detect and quantify this phage in the endangered H. cracherodii.

PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

PubMed

Pauthenier, Cyrille; Faulon, Jean-Loup

2014-07-01

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

PubMed Central

Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.

2013-01-01

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. PMID:23686272

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

SPOCS: software for predicting and visualizing orthology/paralogy relationships among genomes.

PubMed

Curtis, Darren S; Phillips, Aaron R; Callister, Stephen J; Conlan, Sean; McCue, Lee Ann

2013-10-15

At the rate that prokaryotic genomes can now be generated, comparative genomics studies require a flexible method for quickly and accurately predicting orthologs among the rapidly changing set of genomes available. SPOCS implements a graph-based ortholog prediction method to generate a simple tab-delimited table of orthologs and in addition, html files that provide a visualization of the predicted ortholog/paralog relationships to which gene/protein expression metadata may be overlaid. A SPOCS web application is freely available at http://cbb.pnnl.gov/portal/tools/spocs.html. Source code for Linux systems is also freely available under an open source license at http://cbb.pnnl.gov/portal/software/spocs.html; the Boost C++ libraries and BLAST are required.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma. | Office of Cancer Genomics

Cancer.gov

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target.

Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.

PubMed

Zhang, Huimin; He, Hongkui; Yu, Xiujuan; Xu, Zhaohui; Zhang, Zhizhou

2016-11-01

It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

PubMed Central

Meng, Jia; Kanzaki, Gregory; Meas, Diane; Lam, Christopher K.; Crummer, Heather; Tain, Justina; Xu, H. Howard

2013-01-01

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250,000 library transformants for conditional growth-inhibitory recombinant clones from two shot-gun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer-sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes while 18 originated from non-essential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12 fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression. PMID:22268863

A new estimator of the discovery probability.

PubMed

Favaro, Stefano; Lijoi, Antonio; Prünster, Igor

2012-12-01

Species sampling problems have a long history in ecological and biological studies and a number of issues, including the evaluation of species richness, the design of sampling experiments, and the estimation of rare species variety, are to be addressed. Such inferential problems have recently emerged also in genomic applications, however, exhibiting some peculiar features that make them more challenging: specifically, one has to deal with very large populations (genomic libraries) containing a huge number of distinct species (genes) and only a small portion of the library has been sampled (sequenced). These aspects motivate the Bayesian nonparametric approach we undertake, since it allows to achieve the degree of flexibility typically needed in this framework. Based on an observed sample of size n, focus will be on prediction of a key aspect of the outcome from an additional sample of size m, namely, the so-called discovery probability. In particular, conditionally on an observed basic sample of size n, we derive a novel estimator of the probability of detecting, at the (n+m+1)th observation, species that have been observed with any given frequency in the enlarged sample of size n+m. Such an estimator admits a closed-form expression that can be exactly evaluated. The result we obtain allows us to quantify both the rate at which rare species are detected and the achieved sample coverage of abundant species, as m increases. Natural applications are represented by the estimation of the probability of discovering rare genes within genomic libraries and the results are illustrated by means of two expressed sequence tags datasets. © 2012, The International Biometric Society.

A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis.

PubMed

Namouchi, Amine; Mardassi, Helmi

2006-11-01

Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.

Molecular mapping of chromosomes 17 and X

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, D.F.

1991-01-15

Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition ofmore » new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping clones from a larger genome.« less

Molecular mapping of chromosomes 17 and X. Progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, D.F.

1991-01-15

Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition ofmore » new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping@ clones from a larger genome.« less

Genome-Wide Profiling of RNA–Protein Interactions Using CLIP-Seq

PubMed Central

Stork, Cheryl; Zheng, Sika

2017-01-01

UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein–RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein–epitope tag is then used to immunoprecipitate the protein–RNA complexes. After stringent washing and gel separation the RBP–RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP. PMID:26965263

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: A genomic resource for studying agricultural pests

PubMed Central

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-01-01

Background The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. Results More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. Conclusion The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest. PMID:18315884

Rapid storage and retrieval of genomic intervals from a relational database system using nested containment lists

PubMed Central

Wiley, Laura K.; Sivley, R. Michael; Bush, William S.

2013-01-01

Efficient storage and retrieval of genomic annotations based on range intervals is necessary, given the amount of data produced by next-generation sequencing studies. The indexing strategies of relational database systems (such as MySQL) greatly inhibit their use in genomic annotation tasks. This has led to the development of stand-alone applications that are dependent on flat-file libraries. In this work, we introduce MyNCList, an implementation of the NCList data structure within a MySQL database. MyNCList enables the storage, update and rapid retrieval of genomic annotations from the convenience of a relational database system. Range-based annotations of 1 million variants are retrieved in under a minute, making this approach feasible for whole-genome annotation tasks. Database URL: https://github.com/bushlab/mynclist PMID:23894185

Rapid storage and retrieval of genomic intervals from a relational database system using nested containment lists.

PubMed

Wiley, Laura K; Sivley, R Michael; Bush, William S

2013-01-01

Efficient storage and retrieval of genomic annotations based on range intervals is necessary, given the amount of data produced by next-generation sequencing studies. The indexing strategies of relational database systems (such as MySQL) greatly inhibit their use in genomic annotation tasks. This has led to the development of stand-alone applications that are dependent on flat-file libraries. In this work, we introduce MyNCList, an implementation of the NCList data structure within a MySQL database. MyNCList enables the storage, update and rapid retrieval of genomic annotations from the convenience of a relational database system. Range-based annotations of 1 million variants are retrieved in under a minute, making this approach feasible for whole-genome annotation tasks. Database URL: https://github.com/bushlab/mynclist.

Development and Application of a Salmonid EST Database and cDNA Microarray: Data Mining and Interspecific Hybridization Characteristics

PubMed Central

Rise, Matthew L.; von Schalburg, Kristian R.; Brown, Gordon D.; Mawer, Melanie A.; Devlin, Robert H.; Kuipers, Nathanael; Busby, Maura; Beetz-Sargent, Marianne; Alberto, Roberto; Gibbs, A. Ross; Hunt, Peter; Shukin, Robert; Zeznik, Jeffrey A.; Nelson, Colleen; Jones, Simon R.M.; Smailus, Duane E.; Jones, Steven J.M.; Schein, Jacqueline E.; Marra, Marco A.; Butterfield, Yaron S.N.; Stott, Jeff M.; Ng, Siemon H.S.; Davidson, William S.; Koop, Ben F.

2004-01-01

We report 80,388 ESTs from 23 Atlantic salmon (Salmo salar) cDNA libraries (61,819 ESTs), 6 rainbow trout (Oncorhynchus mykiss) cDNA libraries (14,544 ESTs), 2 chinook salmon (Oncorhynchus tshawytscha) cDNA libraries (1317 ESTs), 2 sockeye salmon (Oncorhynchus nerka) cDNA libraries (1243 ESTs), and 2 lake whitefish (Coregonus clupeaformis) cDNA libraries (1465 ESTs). The majority of these are 3′ sequences, allowing discrimination between paralogs arising from a recent genome duplication in the salmonid lineage. Sequence assembly reveals 28,710 different S. salar, 8981 O. mykiss, 1085 O. tshawytscha, 520 O. nerka, and 1176 C. clupeaformis putative transcripts. We annotate the submitted portion of our EST database by molecular function. Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher-molecular weight libraries. Pyloric caecum library group annotations indicate this organ may function in redox control and as a barrier against systemic uptake of xenobiotics. A microarray is described, containing 7356 salmonid elements representing 3557 different cDNAs. Analyses of cross-species hybridizations to this cDNA microarray indicate that this resource may be used for studies involving all salmonids. PMID:14962987

UPIC + GO: Zeroing in on informative markers

USDA-ARS?s Scientific Manuscript database

Microsatellites/SSRs (simple sequence repeats) have become a powerful tool in genomic biology because of their broad range of applications and availability. An efficient method recently developed to generate microsatellite-enriched libraries used in combination with high throughput DNA pyrosequencin...

Primer-Free Aptamer Selection Using A Random DNA Library

PubMed Central

Pan, Weihua; Xin, Ping; Patrick, Susan; Dean, Stacey; Keating, Christine; Clawson, Gary

2010-01-01

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10. We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format. PMID:20689511

Transcriptome Sequencing and Characterization of Japanese Scallop Patinopecten yessoensis from Different Shell Color Lines

PubMed Central

Chang, Yaqing; Zhao, Wenming; Du, Zhenlin; Hao, Zhenlin

2015-01-01

Shell color is an important trait that is used in breeding the Japanese scallop Patinopecten yessoensis, the most economically important scallop species in China. We constructed four transcriptome libraries from different shell color lines of P. yessoensis: the left and right shell mantles of ordinary strains of P. yessoensis and the left shell mantles of the ‘Ivory’ and ‘Maple’ strains. These four libraries were paired-end sequenced using the Illumina HiSeq 2000 platform and contained 54,802,692 sequences, 40,798,962 sequences, 74,019,262 sequences, and 44,466,166 sequences, respectively. A total of 214,087,082 expressed sequence tags were assembled into 73,522 unigenes with an average size of 1,163 bp. When the data were compared against the public Nr and Swiss-Prot databases using BlastX, nearly 30.55% (22,458) of the unigenes were significantly matched to known unique proteins. Gene Ontology annotation and pathway mapping analysis using the Kyoto Encyclopedia of Genes and Genomes categorized unigenes according to their diverse biological functions and processes and identified candidate genes that were potentially involved in growth, pigmentation, metal transcription, and immunity. Expression profile analysis was performed on all four libraries and many differentially expressed genes were identified. In addition, 5,772 simple sequence repeats were obtained from the P. yessoensis transcriptomes, and 464,197, 395,646, and 310,649 single nucleotide polymorphisms were revealed in the ordinary strains, the ‘Ivory’ strain, and the ‘Maple’ strain, respectively. These results provide valuable information for future genomic studies on P. yessoensis and improve our understanding of the molecular mechanisms involved in the growth, immunity, shell coloring, and shell biomineralization of this species. These resources also may be used in a variety of applications, such as trait mapping, marker-assisted breeding, studies of population genetics and genomics, and work on functional genomics. PMID:25680107

Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers

PubMed Central

2010-01-01

Background Expressed Sequence Tag (EST) has been a cost-effective tool in molecular biology and represents an abundant valuable resource for genome annotation, gene expression, and comparative genomics in plants. Results In this study, we constructed a cDNA library of Prunus mume flower and fruit, sequenced 10,123 clones of the library, and obtained 8,656 expressed sequence tag (EST) sequences with high quality. The ESTs were assembled into 4,473 unigenes composed of 1,492 contigs and 2,981 singletons and that have been deposited in NCBI (accession IDs: GW868575 - GW873047), among which 1,294 unique ESTs were with known or putative functions. Furthermore, we found 1,233 putative simple sequence repeats (SSRs) in the P. mume unigene dataset. We randomly tested 42 pairs of PCR primers flanking potential SSRs, and 14 pairs were identified as true-to-type SSR loci and could amplify polymorphic bands from 20 individual plants of P. mume. We further used the 14 EST-SSR primer pairs to test the transferability on peach and plum. The result showed that nearly 89% of the primer pairs produced target PCR bands in the two species. A high level of marker polymorphism was observed in the plum species (65%) and low in the peach (46%), and the clustering analysis of the three species indicated that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the Prunus species. Conclusions We have constructed the first cDNA library of P. mume flower and fruit, and our data provide sets of molecular biology resources for P. mume and other Prunus species. These resources will be useful for further study such as genome annotation, new gene discovery, gene functional analysis, molecular breeding, evolution and comparative genomics between Prunus species. PMID:20626882

Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of Gossypium hirsutum L

PubMed Central

Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

2013-01-01

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species. PMID:24146870

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

PubMed

Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

2015-11-01

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

The complete genomic sequence of egg drop syndrome virus strain AAV-2.

PubMed

Jin, Q; Zeng, L; Yang, F; Li, M; Hou, Y

1999-12-01

In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restriction endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the complete genome nucleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on genomic structure and the distribution of open reading frame (ORF). There are no clear E1, E3 and E4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding ElA, pV and pIX, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group III Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus and will also help the search of all Adenoviruses.

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

PubMed Central

Narayanan, Ajay M.; Ramsey, Matthew M.

2017-01-01

ABSTRACT Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans. One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising >1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen. IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen. PMID:28476775

Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2.

PubMed

Dong, Lanlan; Zhou, Simin; He, Yuan; Jia, Yan; Bai, Qunhua; Deng, Peng; Gao, Jieying; Li, Yingli; Xiao, Hong

2018-05-01

This study is to investigate the genome sequence of Serratia sp. S2. The genomic DNA of Serratia sp. S2 was extracted and the sequencing library was constructed. The sequencing was carried out by Illumina 2000 and complete genomic sequences were obtained. Gene function annotation and bioinformatics analysis were performed by comparing with the known databases. The genome size of Serratia sp. S2 was 5,604,115 bp and the G+C content was 57.61%. There were 5373 protein coding genes, and 3732, 3614, and 3942 genes were respectively annotated into the GO, KEGG, and COG databases. There were 12 genes related to chromium metabolism in the Serratia sp. S2 genome. The whole genome sequence of Serratia sp. S2 is submitted to the GenBank database with gene accession number of LNRP00000000. Our findings may provide theoretical basis for the subsequent development of new biotechnology to repair environmental chromium pollution.

DNA modification and functional delivery into human cells using Escherichia coli DH10B

PubMed Central

Narayanan, Kumaran; Warburton, Peter E.

2003-01-01

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ∼200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies. PMID:12711696

Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes

PubMed Central

Tartar, Aurélien; Wheeler, Marsha M; Zhou, Xuguo; Coy, Monique R; Boucias, Drion G; Scharf, Michael E

2009-01-01

Background Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing. Results Over 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding. Conclusion To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a single termite species. This sequence database represents an important new genomic resource for use in further studies of collaborative host-symbiont termite digestion, as well as development of coevolved host and symbiont-derived biocatalysts for use in industrial biomass-to-bioethanol applications. Additionally, this study demonstrates that: (i) phenoloxidase activities are prominent in the R. flavipes gut and are not symbiont derived, (ii) expands the known number of host and symbiont glycosyl hydrolase families in Reticulitermes, and (iii) supports previous models of lignin degradation and host-symbiont collaboration in cellulose/hemicellulose digestion in the termite gut. All sequences in this paper are available publicly with the accession numbers FL634956-FL640828 (Termite Gut library) and FL641015-FL645753 (Symbiont library). PMID:19832970

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.

PubMed

Gibbs, M; Dawson, D A; McCamley, C; Wardle, A F; Armour, J A; Burke, T

1997-12-01

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.

MicroRNA-focused CRISPR-Cas9 Library Screen Reveals Fitness-Associated miRNAs.

PubMed

Kurata, Jessica S; Lin, Ren-Jang

2018-05-02

MicroRNAs (miRNAs) are posttranscriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however few libraries have included miRNA genes. Here we constructed a miRNA-focused CRISPR-Cas9 library that targets 1,594 (85%) annotated human miRNA stem-loops. The sgRNAs in our LX-miR library are designed to have high on-target and low off-target activity, and each miRNA is targeted by 4-5 sgRNAs. We used this sgRNA library to screen for miRNAs that affect cell fitness of HeLa or NCI-N87 cells by monitoring the change in frequency of each sgRNA over time. By considering the expression in the tested cells and the dysregulation of the miRNAs in cancer specimens, we identified five HeLa pro-fitness and cervical cancer up-regulated miRNAs (miR-31-5p, miR-92b-3p, miR-146b-5p, miR-151a-3p, and miR-194-5p). Similarly, we identified six NCI-N87 pro-fitness and gastric cancer up-regulated miRNAs (miR-95-3p, miR-181a-5p, miR-188-5p, miR-196b-5p, miR-584-5p, and miR-1304-3p), as well as three anti-fitness and down-regulated miRNAs (let-7a-3p, miR-100-5p, and miR-149-5p). Some of those miRNAs are known to be oncogenic or tumor-suppressive, but others are novel. Taken together, the LX-miR library is useful for genome-wide unbiased screening to identify miRNAs important for cellular fitness and likely to be useful for other functional screens. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome.

PubMed

Nicholson, Matthew J; Theodorou, Michael K; Brookman, Jayne L

2005-01-01

The anaerobic gut fungi occupy a unique niche in the intestinal tract of large herbivorous animals and are thought to act as primary colonizers of plant material during digestion. They are the only known obligately anaerobic fungi but molecular analysis of this group has been hampered by difficulties in their culture and manipulation, and by their extremely high A+T nucleotide content. This study begins to answer some of the fundamental questions about the structure and organization of the anaerobic gut fungal genome. Directed plasmid libraries using genomic DNA digested with highly or moderately rich AT-specific restriction enzymes (VspI and EcoRI) were prepared from a polycentric Orpinomyces isolate. Clones were sequenced from these libraries and the breadth of genomic inserts, both genic and intergenic, was characterized. Genes encoding numerous functions not previously characterized for these fungi were identified, including cytoskeletal, secretory pathway and transporter genes. A peptidase gene with no introns and having sequence similarity to a gene encoding a bacterial peptidase was also identified, extending the range of metabolic enzymes resulting from apparent trans-kingdom transfer from bacteria to fungi, as previously characterized largely for genes encoding plant-degrading enzymes. This paper presents the first thorough analysis of the genic, intergenic and rDNA regions of a variety of genomic segments from an anaerobic gut fungus and provides observations on rules governing intron boundaries, the codon biases observed with different types of genes, and the sequence of only the second anaerobic gut fungal promoter reported. Large numbers of retrotransposon sequences of different types were found and the authors speculate on the possible consequences of any such transposon activity in the genome. The coding sequences identified included several orphan gene sequences, including one with regions strongly suggestive of structural proteins such as collagens and lampirin. This gene was present as a single copy in Orpinomyces, was expressed during vegetative growth and was also detected in genomes from another gut fungal genus, Neocallimastix.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2007-07-01

The T7Select 10-3b system of lytic phage display is a mid-copy vector that displays between 5-15 copies on the surface of the T7 capsid. The natural... Phage are amplified on a bacterial host that carries an ampicillin-resistant plasmid expressing additional 10A capsid protein from a T7 promoter. We... phage display library of coding fragments encompassing all open reading frames of the human genome. We designed approximately 467,000 overlapping

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa.

PubMed

van Belkum, Alex; Soriaga, Leah B; LaFave, Matthew C; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S; Richardson, Toby H; Peterson, Todd C; Hubby, Bolyn; Cady, Kyle C

2015-11-24

Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance. Copyright © 2015 van Belkum et al.

Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank

PubMed Central

Simpanya, Mukoma F.; Wistow, Graeme; Gao, James; David, Larry L.; Giblin, Frank J.

2008-01-01

Purpose To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags. Methods RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). Results Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of γD-, γE-, and γF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the ‘rest-of-eye’ library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. Conclusions Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of γD-, γE-, and γF-crystallin were found in EST, proteomic, or the current guinea pig genome data. PMID:19104676

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms.

PubMed

Milnthorpe, Andrew T; Soloviev, Mikhail

2011-04-15

The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries. We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter. Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used.

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms

PubMed Central

2011-01-01

Background The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries. Results We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter. Conclusion Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used. PMID:21496233

Ocean biogeochemistry modeled with emergent trait-based genomics

NASA Astrophysics Data System (ADS)

Coles, V. J.; Stukel, M. R.; Brooks, M. T.; Burd, A.; Crump, B. C.; Moran, M. A.; Paul, J. H.; Satinsky, B. M.; Yager, P. L.; Zielinski, B. L.; Hood, R. R.

2017-12-01

Marine ecosystem models have advanced to incorporate metabolic pathways discovered with genomic sequencing, but direct comparisons between models and “omics” data are lacking. We developed a model that directly simulates metagenomes and metatranscriptomes for comparison with observations. Model microbes were randomly assigned genes for specialized functions, and communities of 68 species were simulated in the Atlantic Ocean. Unfit organisms were replaced, and the model self-organized to develop community genomes and transcriptomes. Emergent communities from simulations that were initialized with different cohorts of randomly generated microbes all produced realistic vertical and horizontal ocean nutrient, genome, and transcriptome gradients. Thus, the library of gene functions available to the community, rather than the distribution of functions among specific organisms, drove community assembly and biogeochemical gradients in the model ocean.

Computational functional genomics-based approaches in analgesic drug discovery and repurposing.

PubMed

Lippmann, Catharina; Kringel, Dario; Ultsch, Alfred; Lötsch, Jörn

2018-06-01

Persistent pain is a major healthcare problem affecting a fifth of adults worldwide with still limited treatment options. The search for new analgesics increasingly includes the novel research area of functional genomics, which combines data derived from various processes related to DNA sequence, gene expression or protein function and uses advanced methods of data mining and knowledge discovery with the goal of understanding the relationship between the genome and the phenotype. Its use in drug discovery and repurposing for analgesic indications has so far been performed using knowledge discovery in gene function and drug target-related databases; next-generation sequencing; and functional proteomics-based approaches. Here, we discuss recent efforts in functional genomics-based approaches to analgesic drug discovery and repurposing and highlight the potential of computational functional genomics in this field including a demonstration of the workflow using a novel R library 'dbtORA'.

Analysis of Illumina Microbial Assemblies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clum, Alicia; Foster, Brian; Froula, Jeff

2010-05-28

Since the emerging of second generation sequencing technologies, the evaluation of different sequencing approaches and their assembly strategies for different types of genomes has become an important undertaken. Next generation sequencing technologies dramatically increase sequence throughput while decreasing cost, making them an attractive tool for whole genome shotgun sequencing. To compare different approaches for de-novo whole genome assembly, appropriate tools and a solid understanding of both quantity and quality of the underlying sequence data are crucial. Here, we performed an in-depth analysis of short-read Illumina sequence assembly strategies for bacterial and archaeal genomes. Different types of Illumina libraries as wellmore » as different trim parameters and assemblers were evaluated. Results of the comparative analysis and sequencing platforms will be presented. The goal of this analysis is to develop a cost-effective approach for the increased throughput of the generation of high quality microbial genomes.« less

Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling.

PubMed

Wang, Shenghai; Duan, Mengjie; Liu, Yalan; Fan, Sen; Lin, Xiaoshan; Zhang, Yi

2017-03-01

To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling. A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180 U g -1 , approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 U g -1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201. Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

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

PubMed

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.

Mapping 3D genome architecture through in situ DNase Hi-C.

PubMed

Ramani, Vijay; Cusanovich, Darren A; Hause, Ronald J; Ma, Wenxiu; Qiu, Ruolan; Deng, Xinxian; Blau, C Anthony; Disteche, Christine M; Noble, William S; Shendure, Jay; Duan, Zhijun

2016-11-01

With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves ∼4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.

Microsatellite primers for red drum (Sciaenops ocellatus)

USDA-ARS?s Scientific Manuscript database

In this note, we document polymerase-chain-reaction (PCR) primer pairs for 101, nuclear-encoded microsatellites designed and developed from a red drum (Sciaenops ocellatus) genomic library. The 101 microsatellites (Genbank Accession Numbers EU015882-EU015982) were amplified successfully and used to...

20180312 - Mechanistic Modeling of Developmental Defects through Computational Embryology (SOT)

EPA Science Inventory

Significant advances in the genome sciences, in automated high-throughput screening (HTS), and in alternative methods for testing enable rapid profiling of chemical libraries for quantitative effects on diverse cellular activities. While a surfeit of HTS data and information is n...

Customizing the Connectivity Map Approach for Functional Evaluation in Toxicogenomics Studies (SOT)

EPA Science Inventory

Evaluating effects on the transcriptome can provide insight on putative chemical-specific mechanisms of action (MOAs). With whole genome transcriptomics technologies becoming more amenable to high-throughput screening, libraries of chemicals can be evaluated in vitro to produce l...

Genome shuffling of Lactobacillus plantarum C88 improves adhesion.

PubMed

Zhao, Yujuan; Duan, Cuicui; Gao, Lei; Yu, Xue; Niu, Chunhua; Li, Shengyu

2017-01-01

Genome shuffling is an important method for rapid improvement in microbial strains for desired phenotypes. In this study, ultraviolet irradiation and nitrosoguanidine were used as mutagens to enhance the adhesion of the wild-type Lactobacillus plantarum C88. Four strains with better property were screened after mutagenesis to develop a library of parent strains for three rounds of genome shuffling. Fusants F3-1, F3-2, F3-3, and F3-4 were screened as the improved strains. The in vivo and in vitro tests results indicated that the population after three rounds of genome shuffling exhibited improved adhesive property. Random Amplified Polymorphic DNA results showed significant differences between the parent strain and recombinant strains at DNA level. These results suggest that the adhesive property of L. plantarum C88 can be significantly improved by genome shuffling. Improvement in the adhesive property of bacterial cells by genome shuffling enhances the colonization of probiotic strains which further benefits to exist probiotic function.

An Annotated Draft Genome for Radix auricularia (Gastropoda, Mollusca)

PubMed Central

Feldmeyer, Barbara; Schmidt, Hanno; Greshake, Bastian; Tills, Oliver; Truebano, Manuela; Rundle, Simon D.; Paule, Juraj; Ebersberger, Ingo; Pfenninger, Markus

2017-01-01

Molluscs are the second most species-rich phylum in the animal kingdom, yet only 11 genomes of this group have been published so far. Here, we present the draft genome sequence of the pulmonate freshwater snail Radix auricularia. Six whole genome shotgun libraries with different layouts were sequenced. The resulting assembly comprises 4,823 scaffolds with a cumulative length of 910 Mb and an overall read coverage of 72×. The assembly contains 94.6% of a metazoan core gene collection, indicating an almost complete coverage of the coding fraction. The discrepancy of ∼690 Mb compared with the estimated genome size of R. auricularia (1.6 Gb) results from a high repeat content of 70% mainly comprising DNA transposons. The annotation of 17,338 protein coding genes was supported by the use of publicly available transcriptome data. This draft will serve as starting point for further genomic and population genetic research in this scientifically important phylum. PMID:28204581

Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina.

PubMed

Valles, Steven M; Porter, Sanford D; Calcaterra, Luis A

2018-01-01

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.

Genetic Control of Plant Root Colonization by the Biocontrol agent, Pseudomonas fluorescens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cole, Benjamin J.; Fletcher, Meghan; Waters, Jordan

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguouslymore » identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.« less

Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina

PubMed Central

Porter, Sanford D.; Calcaterra, Luis A.

2018-01-01

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations. PMID:29466388

Allele Identification for Transcriptome-Based Population Genomics in the Invasive Plant Centaurea solstitialis

PubMed Central

Dlugosch, Katrina M.; Lai, Zhao; Bonin, Aurélie; Hierro, José; Rieseberg, Loren H.

2013-01-01

Transcriptome sequences are becoming more broadly available for multiple individuals of the same species, providing opportunities to derive population genomic information from these datasets. Using the 454 Life Science Genome Sequencer FLX and FLX-Titanium next-generation platforms, we generated 11−430 Mbp of sequence for normalized cDNA for 40 wild genotypes of the invasive plant Centaurea solstitialis, yellow starthistle, from across its worldwide distribution. We examined the impact of sequencing effort on transcriptome recovery and overlap among individuals. To do this, we developed two novel publicly available software pipelines: SnoWhite for read cleaning before assembly, and AllelePipe for clustering of loci and allele identification in assembled datasets with or without a reference genome. AllelePipe is designed specifically for cases in which read depth information is not appropriate or available to assist with disentangling closely related paralogs from allelic variation, as in transcriptome or previously assembled libraries. We find that modest applications of sequencing effort recover most of the novel sequences present in the transcriptome of this species, including single-copy loci and a representative distribution of functional groups. In contrast, the coverage of variable sites, observation of heterozygosity, and overlap among different libraries are all highly dependent on sequencing effort. Nevertheless, the information gained from overlapping regions was informative regarding coarse population structure and variation across our small number of population samples, providing the first genetic evidence in support of hypothesized invasion scenarios. PMID:23390612

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes.

PubMed

Meng, Jia; Kanzaki, Gregory; Meas, Diane; Lam, Christopher K; Crummer, Heather; Tain, Justina; Xu, H Howard

2012-04-01

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250 000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

PubMed

Nair, Shalima S; Luu, Phuc-Loi; Qu, Wenjia; Maddugoda, Madhavi; Huschtscha, Lily; Reddel, Roger; Chenevix-Trench, Georgia; Toso, Martina; Kench, James G; Horvath, Lisa G; Hayes, Vanessa M; Stricker, Phillip D; Hughes, Timothy P; White, Deborah L; Rasko, John E J; Wong, Justin J-L; Clark, Susan J

2018-05-28

Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

Identification of the genomic locus for the human Rieske Fe-S Protein gene on Chromosome 19q12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pennacchio, L.A.

1994-05-06

We have identified the chromosomal location of the human Rieske Iron-Sulfur Protein (UQCRFS1) gene. Mapping by hybridization to a panel of monochromosomal hybrid cell lines indicated that the gene was either on chromosome 19 or 22. By screening a human chromosome 19 specific genomic cosmid library with an oligonucleotide probe made from the published Rieske cDNA sequence, we identified a corresponding cosmid. Portions of this cosmid were sequenced directly. The exon, exon:intron junction, and flanking sequences verified that this cosmid contains the genomic locus. Fluorescent in situ hybridization (FISH) was performed to localize this cosmid to chromosome band 19q12.

Complete Genome Sequence of Escherichia coli ER1821R, a Laboratory K-12 Derivative Engineered To Be Deficient in All Methylcytosine and Methyladenine Restriction Systems.

PubMed

Jobling, Michael G; Raleigh, Elisabeth A; Frank, Daniel N

2016-08-11

We present here the complete genomic sequence of a rifampin-resistant derivative of the Escherichia coli K-12 laboratory strain ER1821, engineered to be deficient in all known restriction systems, making it suitable for generating unbiased libraries from organisms with non-K-12 methylation patterns. The ER1821R genome is most closely related to that of DH1, another popular cloning strain (both derived from MM294), but is deleted for the e14 prophage (McrA(-)) and the immigration control (McrBC(-) EcoKI R(-) M(-) Mrr(-)) loci. Copyright © 2016 Jobling et al.

Single-cell genomic sequencing using Multiple Displacement Amplification.

PubMed

Lasken, Roger S

2007-10-01

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).

A Ruby API to query the Ensembl database for genomic features.

PubMed

Strozzi, Francesco; Aerts, Jan

2011-04-01

The Ensembl database makes genomic features available via its Genome Browser. It is also possible to access the underlying data through a Perl API for advanced querying. We have developed a full-featured Ruby API to the Ensembl databases, providing the same functionality as the Perl interface with additional features. A single Ruby API is used to access different releases of the Ensembl databases and is also able to query multi-species databases. Most functionality of the API is provided using the ActiveRecord pattern. The library depends on introspection to make it release independent. The API is available through the Rubygem system and can be installed with the command gem install ruby-ensembl-api.

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

PubMed

Schreiber, T; Tissier, A

2016-01-01

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs.

PubMed

Yuan, Xiao-Long; Zhang, Zhe; Pan, Rong-Yang; Gao, Ning; Deng, Xi; Li, Bin; Zhang, Hao; Sangild, Per Torp; Li, Jia-Qi

2017-01-01

Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome. We found that the Msp I-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110-220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40-110 bp, 110-220 bp and 40-220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40-220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions. In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.

Effects of methylation-sensitive enzymes on the enrichment of genic SNPs and the degree of genome complexity reduction in a two-enzyme genotyping-by-sequencing (GBS) approach: a case study in oil palm (Elaeis guineensis).

PubMed

Pootakham, Wirulda; Sonthirod, Chutima; Naktang, Chaiwat; Jomchai, Nukoon; Sangsrakru, Duangjai; Tangphatsornruang, Sithichoke

2016-01-01

Advances in next generation sequencing have facilitated a large-scale single nucleotide polymorphism (SNP) discovery in many crop species. Genotyping-by-sequencing (GBS) approach couples next generation sequencing with genome complexity reduction techniques to simultaneously identify and genotype SNPs. Choice of enzymes used in GBS library preparation depends on several factors including the number of markers required, the desired level of multiplexing, and whether the enrichment of genic SNP is preferred. We evaluated various combinations of methylation-sensitive ( Aat II, Pst I, Msp I) and methylation-insensitive ( Sph I, Mse I) enzymes for their effectiveness in genome complexity reduction and enrichment of genic SNPs. We discovered that the use of two methylation-sensitive enzymes effectively reduced genome complexity and did not require a size selection step. On the contrary, the genome coverage of libraries constructed with methylation-insensitive enzymes was quite high, and the additional size selection step may be required to increase the overall read depth. We also demonstrated the effectiveness of methylation-sensitive enzymes in enriching for SNPs located in genic regions. When two methylation-insensitive enzymes were used, only 16% of SNPs identified were located in genes and 18% in the vicinity (± 5 kb) of the genic regions, while most SNPs resided in the intergenic regions. In contrast, a remarkable degree of enrichment was observed when two methylation-sensitive enzymes were employed. Almost two thirds of the SNPs were located either inside (32-36%) or in the vicinity (28-31%) of the genic regions. These results provide useful information to help researchers choose appropriate GBS enzymes in oil palm and other crop species.

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

NASA Technical Reports Server (NTRS)

Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

2002-01-01

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology

PubMed Central

Gao, Ge; Smith, David I.

2015-01-01

DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients. PMID:26262638

Microsatellite Markers for Raspberries and Blackberries

USDA-ARS?s Scientific Manuscript database

Twelve microsatellites were isolated from SSR-enriched genomic libraries of Rubus idaeus L.‘Meeker’ red raspberry (diploid) and R. loganobaccus L. H. Bailey ‘Marion’ blackberry-raspberry hybrid (hexaploid). These primer pairs, with the addition of one developed from a GenBank R. idaeus sequence, w...

Protein-Protein Interactions (PPI) reagents: | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below.

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

PubMed

Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-09-24

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

PubMed Central

Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-01-01

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp. PMID:24019490

Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic.

PubMed

Santiago, Marina; Lee, Wonsik; Fayad, Antoine Abou; Coe, Kathryn A; Rajagopal, Mithila; Do, Truc; Hennessen, Fabienne; Srisuknimit, Veerasak; Müller, Rolf; Meredith, Timothy C; Walker, Suzanne

2018-06-01

Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Genotype Specification Language.

PubMed

Wilson, Erin H; Sagawa, Shiori; Weis, James W; Schubert, Max G; Bissell, Michael; Hawthorne, Brian; Reeves, Christopher D; Dean, Jed; Platt, Darren

2016-06-17

We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

Kullback Leibler divergence in complete bacterial and phage genomes

PubMed Central

Akhter, Sajia; Kashef, Mona T.; Ibrahim, Eslam S.; Bailey, Barbara

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback–Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses. PMID:29204318

Kullback Leibler divergence in complete bacterial and phage genomes.

PubMed

Akhter, Sajia; Aziz, Ramy K; Kashef, Mona T; Ibrahim, Eslam S; Bailey, Barbara; Edwards, Robert A

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback-Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

PubMed

Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Successful development of microsatellite markers in a challenging species: the horizontal borer Austroplatypus incompertus (Coleoptera: Curculionidae).

PubMed

Smith, S; Joss, T; Stow, A

2011-10-01

The analysis of microsatellite loci has allowed significant advances in evolutionary biology and pest management. However, until very recently, the potential benefits have been compromised by the high costs of developing these neutral markers. High-throughput sequencing provides a solution to this problem. We describe the development of 13 microsatellite markers for the eusocial ambrosia beetle, Austroplatypus incompertus, a significant pest of forests in southeast Australia. The frequency of microsatellite repeats in the genome of A. incompertus was determined to be low, and previous attempts at microsatellite isolation using a traditional genomic library were problematic. Here, we utilised two protocols, microsatellite-enriched genomic library construction and high-throughput 454 sequencing and characterised 13 loci which were polymorphic among 32 individuals. Numbers of alleles per locus ranged from 2 to 17, and observed and expected heterozygosities from 0.344 to 0.767 and from 0.507 to 0.860, respectively. These microsatellites have the resolution required to analyse fine-scale colony and population genetic structure. Our work demonstrates the utility of next-generation 454 sequencing as a method for rapid and cost-effective acquisition of microsatellites where other techniques have failed, or for taxa where marker development has historically been both complicated and expensive.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Optimization and quality control of genome-wide Hi-C library preparation.

PubMed

Zhang, Xiang-Yuan; He, Chao; Ye, Bing-Yu; Xie, De-Jian; Shi, Ming-Lei; Zhang, Yan; Shen, Wen-Long; Li, Ping; Zhao, Zhi-Hu

2017-09-20

Highest-throughput chromosome conformation capture (Hi-C) is one of the key assays for genome- wide chromatin interaction studies. It is a time-consuming process that involves many steps and many different kinds of reagents, consumables, and equipments. At present, the reproducibility is unsatisfactory. By optimizing the key steps of the Hi-C experiment, such as crosslinking, pretreatment of digestion, inactivation of restriction enzyme, and in situ ligation etc., we established a robust Hi-C procedure and prepared two biological replicates of Hi-C libraries from the GM12878 cells. After preliminary quality control by Sanger sequencing, the two replicates were high-throughput sequenced. The bioinformatics analysis of the raw sequencing data revealed the mapping-ability and pair-mate rate of the raw data were around 90% and 72%, respectively. Additionally, after removal of self-circular ligations and dangling-end products, more than 96% of the valid pairs were reached. Genome-wide interactome profiling shows clear topological associated domains (TADs), which is consistent with previous reports. Further correlation analysis showed that the two biological replicates strongly correlate with each other in terms of both bin coverage and all bin pairs. All these results indicated that the optimized Hi-C procedure is robust and stable, which will be very helpful for the wide applications of the Hi-C assay.

Genetic screens in human cells using the CRISPR-Cas9 system.

PubMed

Wang, Tim; Wei, Jenny J; Sabatini, David M; Lander, Eric S

2014-01-03

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

On the Power and the Systematic Biases of the Detection of Chromosomal Inversions by Paired-End Genome Sequencing

PubMed Central

Lucas Lledó, José Ignacio; Cáceres, Mario

2013-01-01

One of the most used techniques to study structural variation at a genome level is paired-end mapping (PEM). PEM has the advantage of being able to detect balanced events, such as inversions and translocations. However, inversions are still quite difficult to predict reliably, especially from high-throughput sequencing data. We simulated realistic PEM experiments with different combinations of read and library fragment lengths, including sequencing errors and meaningful base-qualities, to quantify and track down the origin of false positives and negatives along sequencing, mapping, and downstream analysis. We show that PEM is very appropriate to detect a wide range of inversions, even with low coverage data. However, % of inversions located between segmental duplications are expected to go undetected by the most common sequencing strategies. In general, longer DNA libraries improve the detectability of inversions far better than increments of the coverage depth or the read length. Finally, we review the performance of three algorithms to detect inversions —SVDetect, GRIAL, and VariationHunter—, identify common pitfalls, and reveal important differences in their breakpoint precisions. These results stress the importance of the sequencing strategy for the detection of structural variants, especially inversions, and offer guidelines for the design of future genome sequencing projects. PMID:23637806

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

PubMed

Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

2017-03-01

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

PubMed

Wu, Wells W; Phue, Je-Nie; Lee, Chun-Ting; Lin, Changyi; Xu, Lai; Wang, Rong; Zhang, Yaqin; Shen, Rong-Fong

2018-05-04

Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

In silico Analysis of 2085 Clones from a Normalized Rat Vestibular Periphery 3′ cDNA Library

PubMed Central

Roche, Joseph P.; Cioffi, Joseph A.; Kwitek, Anne E.; Erbe, Christy B.; Popper, Paul

2005-01-01

The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3′ cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa's ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5′ end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead to the identification of genes with vestibular-specific functions. Continued analysis of the rat vestibular periphery transcriptome should provide new insights into vestibular function and generate new hypotheses. Physiological studies are necessary to further elucidate the roles of the identified genes and novel sequences in vestibular function. PMID:16103642

Molecular Mechanisms of Cytopathogenicity of Primate Lymphotropic Retroviruses: Relevance to Treatment and Vaccine for AIDS

DTIC Science & Technology

1989-03-10

fragment of the HIV-1 genome was isolated from XBH10 and inserted into an M13 phage vector. Mutations were introduced by use of 25-mer oligonucleotides which... M13 by Eco RI and religated into the corresponding position of pHXB2gpt. The mutant AS was prepared directly from pHXB2gpt by digestion with Nde I and...detect immunocomplexes. Molecular Cloning and Sequencing of Proviral DNA A X phage library was constructed from the genomic DNA isolated from Hut78 cells

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

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.

PubMed

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.

Online Bioinformatics Tutorials | Office of Cancer Genomics

Cancer.gov

Bioinformatics is a scientific discipline that applies computer science and information technology to help understand biological processes. The NIH provides a list of free online bioinformatics tutorials, either generated by the NIH Library or other institutes, which includes introductory lectures and "how to" videos on using various tools.

Survey of microsatellite DNA in pine

Treesearch

C. S. Echt; P. May-Marquardt

1997-01-01

A large insert genomic library from eastern white pine (Pinus strobus) was probed for the microsatellite motifs (AC)n and (AG)n, all 10 trinucleotide motifs, and 22 of the 33 possible tetranucleotide motifs. For comparison with a species from a different subgenus, a loblolly pine (Pinus taeda...

Micro computed tomography (CT) scanned anatomical gateway to insect pest bioinformatics

USDA-ARS?s Scientific Manuscript database

An international collaboration to establish an interactive Digital Video Library for a Systems Biology Approach to study the Asian citrus Psyllid and psyllid genomics/proteomics interactions is demonstrated. Advances in micro-CT, digital computed tomography (CT) scan uses X-rays to make detailed pic...

ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

EPA Science Inventory

We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

USDA-ARS?s Scientific Manuscript database

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

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

PubMed

Pedersen, Carsten; Wu, Boqian; Giese, Henriette

2002-11-01

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 contig-building difficult. To identify overlapping clones, several strategies were used: colony hybridisation, PCR screening, fingerprinting techniques and the use of single-copy expressed sequence tags. The latter proved to be the most efficient method for identification of overlapping clones. Two 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 and Aspergillus fumigatus. The library provides an invaluable tool for the isolation of avirulence genes from B. graminis and for the study of gene synteny between this fungus and other fungi.

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

The draft genome of MD-2 pineapple using hybrid error correction of long reads

PubMed Central

Redwan, Raimi M.; Saidin, Akzam; Kumar, S. Vijay

2016-01-01

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. PMID:27374615

The i5k Workspace@NAL—enabling genomic data access, visualization and curation of arthropod genomes

PubMed Central

Poelchau, Monica; Childers, Christopher; Moore, Gary; Tsavatapalli, Vijaya; Evans, Jay; Lee, Chien-Yueh; Lin, Han; Lin, Jun-Wei; Hackett, Kevin

2015-01-01

The 5000 arthropod genomes initiative (i5k) has tasked itself with coordinating the sequencing of 5000 insect or related arthropod genomes. The resulting influx of data, mostly from small research groups or communities with little bioinformatics experience, will require visualization, dissemination and curation, preferably from a centralized platform. The National Agricultural Library (NAL) has implemented the i5k Workspace@NAL (http://i5k.nal.usda.gov/) to help meet the i5k initiative's genome hosting needs. Any i5k member is encouraged to contact the i5k Workspace with their genome project details. Once submitted, new content will be accessible via organism pages, genome browsers and BLAST search engines, which are implemented via the open-source Tripal framework, a web interface for the underlying Chado database schema. We also implement the Web Apollo software for groups that choose to curate gene models. New content will add to the existing body of 35 arthropod species, which include species relevant for many aspects of arthropod genomic research, including agriculture, invasion biology, systematics, ecology and evolution, and developmental research. PMID:25332403

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

PubMed Central

Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

2010-01-01

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

PubMed Central

Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

2012-01-01

To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

PubMed Central

Pietras, D F; Bennett, K L; Siracusa, L D; Woodworth-Gutai, M; Chapman, V M; Gross, K W; Kane-Haas, C; Hastie, N D

1983-01-01

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species. Images PMID:6314268

ViDiT-CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications.

PubMed

Verhoeven, Joost Theo Petra; Canuti, Marta; Munro, Hannah J; Dufour, Suzanne C; Lang, Andrew S

2018-04-19

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (<5 US dollars per sample) and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing and microbiome profiling. ViDiT-CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal-cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT-CACTUS demonstrated its validity in a wide range of microbiology applications and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

DOE PAGES

Vetting, Matthew W.; Al-Obaidi, Nawar; Zhao, Suwen; ...

2014-12-25

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. Here in this paper, we describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of themore » library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of D-Ala-D-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.« less

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

PubMed

Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

2016-11-03

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Targeting LSD1 Epigenetic Signature in Castration-Recurrent Prostate Cancer

DTIC Science & Technology

2014-10-01

Sequencing libraries have been prepared and samples are currently queued at the Genomic Core facility at Roswell Park and results will be available...results to various meetings and seminars at Roswell Park, allowing me to get useful feedback from both clinicians and researchers. Furthermore, a brief

Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

USDA-ARS?s Scientific Manuscript database

We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

High-throughput illumina strand-specific RNA sequencing library preparation

USDA-ARS?s Scientific Manuscript database

Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome as...

Protein-Protein Interaction Reagents | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu

Development of microsatellites from Fothergilla xintermedia (Hamamelidaceae) and cross transfer to four other genera within Hamamelidaceae

USDA-ARS?s Scientific Manuscript database

Premise of the study: Develop microsatellites from Fothergilla ×intermedia to establish loci capable of distinguishing species and cultivars, and assess genetic diversity for use by ornamental breeders, and for transfer within Hamamelidaceae. Methods and Results: A small insert genomic library enric...

Broad issues to consider for library involvement in bioinformatics*

PubMed Central

Geer, Renata C.

2006-01-01

Background: The information landscape in biological and medical research has grown far beyond literature to include a wide variety of databases generated by research fields such as molecular biology and genomics. The traditional role of libraries to collect, organize, and provide access to information can expand naturally to encompass these new data domains. Methods: This paper discusses the current and potential role of libraries in bioinformatics using empirical evidence and experience from eleven years of work in user services at the National Center for Biotechnology Information. Findings: Medical and science libraries over the last decade have begun to establish educational and support programs to address the challenges users face in the effective and efficient use of a plethora of molecular biology databases and retrieval and analysis tools. As more libraries begin to establish a role in this area, the issues they face include assessment of user needs and skills, identification of existing services, development of plans for new services, recruitment and training of specialized staff, and establishment of collaborations with bioinformatics centers at their institutions. Conclusions: Increasing library involvement in bioinformatics can help address information needs of a broad range of students, researchers, and clinicians and ultimately help realize the power of bioinformatics resources in making new biological discoveries. PMID:16888662

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

PubMed Central

Davidsson, Marcus; Diaz-Fernandez, Paula; Schwich, Oliver D.; Torroba, Marcos; Wang, Gang; Björklund, Tomas

2016-01-01

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode. PMID:27874090

Genomic attributes of extended-spectrum β-lactamase-producing Escherichia coli isolated from patients in Lebanon.

PubMed

Tokajian, Sima; Salloum, Tamara; Eisen, Jonathan A; Jospin, Guillaume; Farra, Anna; Mokhbat, Jacques E; Coil, David A

2017-03-01

Extended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs. Samples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced. The isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1 , bla TEM-1B , bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage. This is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

PubMed

Garst, Andrew D; Bassalo, Marcelo C; Pines, Gur; Lynch, Sean A; Halweg-Edwards, Andrea L; Liu, Rongming; Liang, Liya; Wang, Zhiwen; Zeitoun, Ramsey; Alexander, William G; Gill, Ryan T

2017-01-01

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

PubMed

Zeitoun, Ramsey I; Pines, Gur; Grau, Willliam C; Gill, Ryan T

2017-04-21

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform.

PubMed

Rutvisuttinunt, Wiriya; Chinnawirotpisan, Piyawan; Simasathien, Sriluck; Shrestha, Sanjaya K; Yoon, In-Kyu; Klungthong, Chonticha; Fernandez, Stefan

2013-11-01

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

PubMed Central

Ziegenhagen, Birgit; Liepelt, Sascha

2015-01-01

Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings. PMID:25924061

An Arrayed Genome-Scale Lentiviral-Enabled Short Hairpin RNA Screen Identifies Lethal and Rescuer Gene Candidates

PubMed Central

Bhinder, Bhavneet; Antczak, Christophe; Ramirez, Christina N.; Shum, David; Liu-Sullivan, Nancy; Radu, Constantin; Frattini, Mark G.

2013-01-01

Abstract RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder–Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general. PMID:23198867

Large-scale contamination of microbial isolate genomes by Illumina PhiX control.

PubMed

Mukherjee, Supratim; Huntemann, Marcel; Ivanova, Natalia; Kyrpides, Nikos C; Pati, Amrita

2015-01-01

With the rapid growth and development of sequencing technologies, genomes have become the new go-to for exploring solutions to some of the world's biggest challenges such as searching for alternative energy sources and exploration of genomic dark matter. However, progress in sequencing has been accompanied by its share of errors that can occur during template or library preparation, sequencing, imaging or data analysis. In this study we screened over 18,000 publicly available microbial isolate genome sequences in the Integrated Microbial Genomes database and identified more than 1000 genomes that are contaminated with PhiX, a control frequently used during Illumina sequencing runs. Approximately 10% of these genomes have been published in literature and 129 contaminated genomes were sequenced under the Human Microbiome Project. Raw sequence reads are prone to contamination from various sources and are usually eliminated during downstream quality control steps. Detection of PhiX contaminated genomes indicates a lapse in either the application or effectiveness of proper quality control measures. The presence of PhiX contamination in several publicly available isolate genomes can result in additional errors when such data are used in comparative genomics analyses. Such contamination of public databases have far-reaching consequences in the form of erroneous data interpretation and analyses, and necessitates better measures to proofread raw sequences before releasing them to the broader scientific community.

Inverse PCR-based method for isolating novel SINEs from genome.

PubMed

Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

2014-04-01

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

Northern Spotted Owl (Strix occidentalis caurina) Genome: Divergence with the Barred Owl (Strix varia) and Characterization of Light-Associated Genes

PubMed Central

Henderson, James B.; Wall, Jeffrey D.; Emerling, Christopher A.; Fuchs, Jérôme; Runckel, Charles; Mindell, David P.; Bowie, Rauri C. K.; DeRisi, Joseph L.; Dumbacher, John P.

2017-01-01

Abstract We report here the assembly of a northern spotted owl (Strix occidentalis caurina) genome. We generated Illumina paired-end sequence data at 90× coverage using nine libraries with insert lengths ranging from ∼250 to 9,600 nt and read lengths from 100 to 375 nt. The genome assembly is comprised of 8,108 scaffolds totaling 1.26 × 109 nt in length with an N50 length of 3.98 × 106 nt. We calculated the genome-wide fixation index (FST) of S. o. caurina with the closely related barred owl (Strix varia) as 0.819. We examined 19 genes that encode proteins with light-dependent functions in our genome assembly as well as in that of the barn owl (Tyto alba). We present genomic evidence for loss of three of these in S. o. caurina and four in T. alba. We suggest that most light-associated gene functions have been maintained in owls and their loss has not proceeded to the same extent as in other dim-light-adapted vertebrates. PMID:28992302

Population-based structural variation discovery with Hydra-Multi.

PubMed

Lindberg, Michael R; Hall, Ira M; Quinlan, Aaron R

2015-04-15

Current strategies for SNP and INDEL discovery incorporate sequence alignments from multiple individuals to maximize sensitivity and specificity. It is widely accepted that this approach also improves structural variant (SV) detection. However, multisample SV analysis has been stymied by the fundamental difficulties of SV calling, e.g. library insert size variability, SV alignment signal integration and detecting long-range genomic rearrangements involving disjoint loci. Extant tools suffer from poor scalability, which limits the number of genomes that can be co-analyzed and complicates analysis workflows. We have developed an approach that enables multisample SV analysis in hundreds to thousands of human genomes using commodity hardware. Here, we describe Hydra-Multi and measure its accuracy, speed and scalability using publicly available datasets provided by The 1000 Genomes Project and by The Cancer Genome Atlas (TCGA). Hydra-Multi is written in C++ and is freely available at https://github.com/arq5x/Hydra. aaronquinlan@gmail.com or ihall@genome.wustl.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

SOBA: sequence ontology bioinformatics analysis.

PubMed

Moore, Barry; Fan, Guozhen; Eilbeck, Karen

2010-07-01

The advent of cheaper, faster sequencing technologies has pushed the task of sequence annotation from the exclusive domain of large-scale multi-national sequencing projects to that of research laboratories and small consortia. The bioinformatics burden placed on these laboratories, some with very little programming experience can be daunting. Fortunately, there exist software libraries and pipelines designed with these groups in mind, to ease the transition from an assembled genome to an annotated and accessible genome resource. We have developed the Sequence Ontology Bioinformatics Analysis (SOBA) tool to provide a simple statistical and graphical summary of an annotated genome. We envisage its use during annotation jamborees, genome comparison and for use by developers for rapid feedback during annotation software development and testing. SOBA also provides annotation consistency feedback to ensure correct use of terminology within annotations, and guides users to add new terms to the Sequence Ontology when required. SOBA is available at http://www.sequenceontology.org/cgi-bin/soba.cgi.

Ocean biogeochemistry modeled with emergent trait-based genomics.

PubMed

Coles, V J; Stukel, M R; Brooks, M T; Burd, A; Crump, B C; Moran, M A; Paul, J H; Satinsky, B M; Yager, P L; Zielinski, B L; Hood, R R

2017-12-01

Marine ecosystem models have advanced to incorporate metabolic pathways discovered with genomic sequencing, but direct comparisons between models and "omics" data are lacking. We developed a model that directly simulates metagenomes and metatranscriptomes for comparison with observations. Model microbes were randomly assigned genes for specialized functions, and communities of 68 species were simulated in the Atlantic Ocean. Unfit organisms were replaced, and the model self-organized to develop community genomes and transcriptomes. Emergent communities from simulations that were initialized with different cohorts of randomly generated microbes all produced realistic vertical and horizontal ocean nutrient, genome, and transcriptome gradients. Thus, the library of gene functions available to the community, rather than the distribution of functions among specific organisms, drove community assembly and biogeochemical gradients in the model ocean. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Conserved microstructure of the Brassica B Genome of Brassica nigra in relation to homologous regions of Arabidopsis thaliana, B. rapa and B. oleracea

PubMed Central

2013-01-01

Background The Brassica B genome is known to carry several important traits, yet there has been limited analyses of its underlying genome structure, especially in comparison to the closely related A and C genomes. A bacterial artificial chromosome (BAC) library of Brassica nigra was developed and screened with 17 genes from a 222 kb region of A. thaliana that had been well characterised in both the Brassica A and C genomes. Results Fingerprinting of 483 apparently non-redundant clones defined physical contigs for the corresponding regions in B. nigra. The target region is duplicated in A. thaliana and six homologous contigs were found in B. nigra resulting from the whole genome triplication event shared by the Brassiceae tribe. BACs representative of each region were sequenced to elucidate the level of microscale rearrangements across the Brassica species divide. Conclusions Although the B genome species separated from the A/C lineage some 6 Mya, comparisons between the three paleopolyploid Brassica genomes revealed extensive conservation of gene content and sequence identity. The level of fractionation or gene loss varied across genomes and genomic regions; however, the greatest loss of genes was observed to be common to all three genomes. One large-scale chromosomal rearrangement differentiated the B genome suggesting such events could contribute to the lack of recombination observed between B genome species and those of the closely related A/C lineage. PMID:23586706

GenomeRNAi: a database for cell-based RNAi phenotypes.

PubMed

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

GenomeRNAi: a database for cell-based RNAi phenotypes

PubMed Central

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at PMID:17135194

Impact of HIPAA’s Minimum Necessary Standard on Genomic Data Sharing

PubMed Central

Evans, Barbara J.; Jarvik, Gail P.

2017-01-01

Purpose This article provides a brief introduction to the HIPAA Privacy Rule’s minimum necessary standard, which applies to sharing of genomic data, particularly clinical data, following 2013 Privacy Rule revisions. Methods This research used the Thomson Reuters Westlaw™ database and law library resources in its legal analysis of the HIPAA privacy tiers and the impact of the minimum necessary standard on genomic data-sharing. We considered relevant example cases of genomic data-sharing needs. Results In a climate of stepped-up HIPAA enforcement, this standard is of concern to laboratories that generate, use, and share genomic information. How data-sharing activities are characterized—whether for research, public health, or clinical interpretation and medical practice support—affects how the minimum necessary standard applies and its overall impact on data access and use. Conclusion There is no clear regulatory guidance on how to apply HIPAA’s minimum necessary standard when considering the sharing of information in the data-rich environment of genomic testing. Laboratories that perform genomic testing should engage with policy-makers to foster sound, well-informed policies and appropriate characterization of data-sharing activities to minimize adverse impacts on day-to-day workflows. PMID:28914268

Impact of HIPAA's minimum necessary standard on genomic data sharing.

PubMed

Evans, Barbara J; Jarvik, Gail P

2018-04-01

This article provides a brief introduction to the Health Insurance Portability and Accountability Act of 1996 (HIPAA) Privacy Rule's minimum necessary standard, which applies to sharing of genomic data, particularly clinical data, following 2013 Privacy Rule revisions. This research used the Thomson Reuters Westlaw database and law library resources in its legal analysis of the HIPAA privacy tiers and the impact of the minimum necessary standard on genomic data sharing. We considered relevant example cases of genomic data-sharing needs. In a climate of stepped-up HIPAA enforcement, this standard is of concern to laboratories that generate, use, and share genomic information. How data-sharing activities are characterized-whether for research, public health, or clinical interpretation and medical practice support-affects how the minimum necessary standard applies and its overall impact on data access and use. There is no clear regulatory guidance on how to apply HIPAA's minimum necessary standard when considering the sharing of information in the data-rich environment of genomic testing. Laboratories that perform genomic testing should engage with policy makers to foster sound, well-informed policies and appropriate characterization of data-sharing activities to minimize adverse impacts on day-to-day workflows.

A phasing and imputation method for pedigreed populations that results in a single-stage genomic evaluation

PubMed Central

2012-01-01

Background Efficient, robust, and accurate genotype imputation algorithms make large-scale application of genomic selection cost effective. An algorithm that imputes alleles or allele probabilities for all animals in the pedigree and for all genotyped single nucleotide polymorphisms (SNP) provides a framework to combine all pedigree, genomic, and phenotypic information into a single-stage genomic evaluation. Methods An algorithm was developed for imputation of genotypes in pedigreed populations that allows imputation for completely ungenotyped animals and for low-density genotyped animals, accommodates a wide variety of pedigree structures for genotyped animals, imputes unmapped SNP, and works for large datasets. The method involves simple phasing rules, long-range phasing and haplotype library imputation and segregation analysis. Results Imputation accuracy was high and computational cost was feasible for datasets with pedigrees of up to 25 000 animals. The resulting single-stage genomic evaluation increased the accuracy of estimated genomic breeding values compared to a scenario in which phenotypes on relatives that were not genotyped were ignored. Conclusions The developed imputation algorithm and software and the resulting single-stage genomic evaluation method provide powerful new ways to exploit imputation and to obtain more accurate genetic evaluations. PMID:22462519

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Library of molecular associations: curating the complex molecular basis of liver diseases.

PubMed

Buchkremer, Stefan; Hendel, Jasmin; Krupp, Markus; Weinmann, Arndt; Schlamp, Kai; Maass, Thorsten; Staib, Frank; Galle, Peter R; Teufel, Andreas

2010-03-20

Systems biology approaches offer novel insights into the development of chronic liver diseases. Current genomic databases supporting systems biology analyses are mostly based on microarray data. Although these data often cover genome wide expression, the validity of single microarray experiments remains questionable. However, for systems biology approaches addressing the interactions of molecular networks comprehensive but also highly validated data are necessary. We have therefore generated the first comprehensive database for published molecular associations in human liver diseases. It is based on PubMed published abstracts and aimed to close the gap between genome wide coverage of low validity from microarray data and individual highly validated data from PubMed. After an initial text mining process, the extracted abstracts were all manually validated to confirm content and potential genetic associations and may therefore be highly trusted. All data were stored in a publicly available database, Library of Molecular Associations http://www.medicalgenomics.org/databases/loma/news, currently holding approximately 1260 confirmed molecular associations for chronic liver diseases such as HCC, CCC, liver fibrosis, NASH/fatty liver disease, AIH, PBC, and PSC. We furthermore transformed these data into a powerful resource for molecular liver research by connecting them to multiple biomedical information resources. Together, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.

BioCircos.js: an interactive Circos JavaScript library for biological data visualization on web applications.

PubMed

Cui, Ya; Chen, Xiaowei; Luo, Huaxia; Fan, Zhen; Luo, Jianjun; He, Shunmin; Yue, Haiyan; Zhang, Peng; Chen, Runsheng

2016-06-01

We here present BioCircos.js, an interactive and lightweight JavaScript library especially for biological data interactive visualization. BioCircos.js facilitates the development of web-based applications for circular visualization of various biological data, such as genomic features, genetic variations, gene expression and biomolecular interactions. BioCircos.js and its manual are freely available online at http://bioinfo.ibp.ac.cn/biocircos/ rschen@ibp.ac.cn Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

University of Texas Southwestern Medical Center: High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

University of Texas Southwestern Medical Center (UTSW): High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

SCARF: maximizing next-generation EST assemblies for evolutionary and population genomic analyses.

PubMed

Barker, Michael S; Dlugosch, Katrina M; Reddy, A Chaitanya C; Amyotte, Sarah N; Rieseberg, Loren H

2009-02-15

Scaffolded and Corrected Assembly of Roche 454 (SCARF) is a next-generation sequence assembly tool for evolutionary genomics that is designed especially for assembling 454 EST sequences against high-quality reference sequences from related species. The program was created to knit together 454 contigs that do not assemble during traditional de novo assembly, using a reference sequence library to orient the 454 sequences. SCARF is freely available at http://msbarker.com/software.htm, and is released under the open source GPLv3 license (http://www.opensource.org/licenses/gpl-3.0.html.

Screening a yeast promoter library leads to the isolation of the RP29/L32 and SNR17B/RPL37A divergent promoters and the discovery of a gene encoding ribosomal protein L37.

PubMed

Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

1991-08-30

Two promoters (A7 and A23), isolated at random from the Saccharomyces cerevisiae genome by virtue of their capacity to activate transcription, are identical to known intergenic bidirectional promoters. Sequence analysis of the genomic DNA adjacent to the A7 promoter identified a split gene encoding ribosomal (r) protein L37, which is homologous to the tRNA-binding r-proteins, L35a (from human and rat) and L32 (from frogs).