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Sample records for enriched genomic libraries

  1. Gene enrichment in plant genomic shotgun libraries.

    PubMed

    Rabinowicz, Pablo D; McCombie, W Richard; Martienssen, Robert A

    2003-04-01

    The Arabidopsis genome (about 130 Mbp) has been completely sequenced; whereas a draft sequence of the rice genome (about 430 Mbp) is now available and the sequencing of this genome will be completed in the near future. The much larger genomes of several important crop species, such as wheat (about 16,000 Mbp) or maize (about 2500 Mbp), may not be fully sequenced with current technology. Instead, sequencing-analysis strategies are being developed to obtain sequencing and mapping information selectively for the genic fraction (gene space) of complex plant genomes.

  2. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    PubMed

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  3. Development of SSR markers derived from SSR-enriched genomic library of eggplant (Solanum melongena L.).

    PubMed

    Nunome, Tsukasa; Negoro, Satomi; Kono, Izumi; Kanamori, Hiroyuki; Miyatake, Koji; Yamaguchi, Hirotaka; Ohyama, Akio; Fukuoka, Hiroyuki

    2009-10-01

    Eggplant (Solanum melongena L.), also known as aubergine or brinjal, is an important vegetable in many countries. Few useful molecular markers have been reported for eggplant. We constructed simple sequence repeat (SSR)-enriched genomic libraries in order to develop SSR markers, and sequenced more than 14,000 clones. From these sequences, we designed 2,265 primer pairs to flank SSR motifs. We identified 1,054 SSR markers from amplification of 1,399 randomly selected primer pairs. The markers have an average polymorphic information content of 0.27 among eight lines of S. melongena. Of the 1,054 SSR markers, 214 segregated in an intraspecific mapping population. We constructed cDNA libraries from several eggplant tissues and obtained 6,144 expressed sequence tag (EST) sequences. From these sequences, we designed 209 primer pairs, 7 of which segregated in the mapping population. On the basis of the segregation data, we constructed a linkage map, and mapped the 236 segregating markers to 14 linkage groups. The linkage map spans a total length of 959.1 cM, with an average marker distance of 4.3 cM. The markers should be a useful resource for qualitative and quantitative trait mapping and for marker-assisted selection in eggplant breeding.

  4. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries.

    PubMed

    Carpenter, Meredith L; Buenrostro, Jason D; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M Thomas P; Willerslev, Eske; Greenleaf, William J; Bustamante, Carlos D

    2013-11-07

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.

  5. Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

    PubMed Central

    Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

    2013-01-01

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

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

    PubMed Central

    Nelson, William; Luo, Meizhong; Ma, Jianxin; Estep, Matt; Estill, James; He, Ruifeng; Talag, Jayson; Sisneros, Nicholas; Kudrna, David; Kim, HyeRan; Ammiraju, Jetty SS; Collura, Kristi; Bharti, Arvind K; Messing, Joachim; Wing, Rod A; SanMiguel, Phillip; Bennetzen, Jeffrey L; Soderlund, Carol

    2008-01-01

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

  7. Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

    PubMed

    Murata, Natsuko; Masuda, Kiyoshi; Nishiyama, Ryutaro; Nomura, Koji

    2005-06-01

    In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

  8. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    PubMed

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  9. Enriching screening libraries with bioactive fragment space.

    PubMed

    Zhang, Na; Zhao, Hongtao

    2016-08-01

    By deconvoluting 238,073 bioactive molecules in the ChEMBL library into extended Murcko ring systems, we identified a set of 2245 ring systems present in at least 10 molecules. These ring systems belong to 2221 clusters by ECFP4 fingerprints with a minimum intracluster similarity of 0.8. Their overlap with ring systems in commercial libraries was further quantified. Our findings suggest that success of a small fragment library is driven by the convergence of effective coverage of bioactive ring systems (e.g., 10% coverage by 1000 fragments vs. 40% by 2million HTS compounds), high enrichment of bioactive ring systems, and low molecular complexity enhancing the probability of a match with the protein targets. Reconciling with the previous studies, bioactive ring systems are underrepresented in screening libraries. As such, we propose a library of virtual fragments with key functionalities via fragmentation of bioactive molecules. Its utility is exemplified by a prospective application on protein kinase CK2, resulting in the discovery of a series of novel inhibitors with the most potent compound having an IC50 of 0.5μM and a ligand efficiency of 0.41kcal/mol per heavy atom.

  10. Microsatellite DNA capture from enriched libraries.

    PubMed

    Gonzalez, Elena G; Zardoya, Rafael

    2013-01-01

    Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

  11. Development of novel simple sequence repeat markers in bitter gourd (Momordica charantia L.) through enriched genomic libraries and their utilization in analysis of genetic diversity and cross-species transferability.

    PubMed

    Saxena, Swati; Singh, Archana; Archak, Sunil; Behera, Tushar K; John, Joseph K; Meshram, Sudhir U; Gaikwad, Ambika B

    2015-01-01

    Microsatellite or simple sequence repeat (SSR) markers are the preferred markers for genetic analyses of crop plants. The availability of a limited number of such markers in bitter gourd (Momordica charantia L.) necessitates the development and characterization of more SSR markers. These were developed from genomic libraries enriched for three dinucleotide, five trinucleotide, and two tetranucleotide core repeat motifs. Employing the strategy of polymerase chain reaction-based screening, the number of clones to be sequenced was reduced by 81 % and 93.7 % of the sequenced clones contained in microsatellite repeats. Unique primer-pairs were designed for 160 microsatellite loci, and amplicons of expected length were obtained for 151 loci (94.4 %). Evaluation of diversity in 54 bitter gourd accessions at 51 loci indicated that 20 % of the loci were polymorphic with the polymorphic information content values ranging from 0.13 to 0.77. Fifteen Indian varieties were clearly distinguished indicative of the usefulness of the developed markers. Markers at 40 loci (78.4 %) were transferable to six species, viz. Momordica cymbalaria, Momordica subangulata subsp. renigera, Momordica balsamina, Momordica dioca, Momordica cochinchinesis, and Momordica sahyadrica. The microsatellite markers reported will be useful in various genetic and molecular genetic studies in bitter gourd, a cucurbit of immense nutritive, medicinal, and economic importance.

  12. Whole-Genome Sequencing: Manual Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-01-03

    This protocol describes a manual approach for the preparation of genomic DNA libraries suitable for Illumina sequencing. Genomic DNA fragments produced by shearing by sonication are ligated to adaptors and amplified by polymerase chain reaction (PCR). The amplified DNA, separated by size and gel-purified, is suitable for use as template in whole-genome sequencing.

  13. Optimized Construction of SSR-enriched Libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a more efficient method to construct simple sequence repeat (SSR) libraries. We have designed and tested several new adapter. We changed the typical blunt-end ligation of adapters for the more effective sticky-end ligation. We optimized temperature, enzymes and length of each st...

  14. Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

    PubMed

    Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

    2017-01-01

    Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

  15. Selective enrichment of damaged DNA molecules for ancient genome sequencing

    PubMed Central

    2014-01-01

    Contamination by present-day human and microbial DNA is one of the major hindrances for large-scale genomic studies using ancient biological material. We describe a new molecular method, U selection, which exploits one of the most distinctive features of ancient DNA—the presence of deoxyuracils—for selective enrichment of endogenous DNA against a complex background of contamination during DNA library preparation. By applying the method to Neanderthal DNA extracts that are heavily contaminated with present-day human DNA, we show that the fraction of useful sequence information increases ∼10-fold and that the resulting sequences are more efficiently depleted of human contamination than when using purely computational approaches. Furthermore, we show that U selection can lead to a four- to fivefold increase in the proportion of endogenous DNA sequences relative to those of microbial contaminants in some samples. U selection may thus help to lower the costs for ancient genome sequencing of nonhuman samples also. PMID:25081630

  16. Ancient whole genome enrichment using baits built from modern DNA.

    PubMed

    Enk, Jacob M; Devault, Alison M; Kuch, Melanie; Murgha, Yusuf E; Rouillard, Jean-Marie; Poinar, Hendrik N

    2014-05-01

    We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of ∼80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.

  17. 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

  18. Large insert environmental genomic library production.

    PubMed

    Taupp, Marcus; Lee, Sangwon; Hawley, Alyse; Yang, Jinshu; Hallam, Steven J

    2009-09-23

    The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties

  19. A phloem-enriched cDNA library from Ricinus: insights into phloem function.

    PubMed

    Doering-Saad, C; Newbury, H J; Couldridge, C E; Bale, J S; Pritchard, J

    2006-01-01

    The aim of this study was to identify genes that are expressed in the phloem. Increased knowledge of phloem regulation will contribute to our understanding of its many roles, from transport of solutes to information about interactions with pathogens. A cDNA library constructed from phloem-enriched sap exuding from cut Ricinus communis (L.) hypocotyls was sequenced. To assess contamination from other tissues, two libraries were constructed: one using the first 15 min of exudation and the other from sap collected after 120 min of exudation had elapsed. Of 1012 clones sequenced, 158 unique transcripts were identified. The presence of marker molecules such as profilin, the low occurrence of chloroplast-related mRNAs, and the sieve element localization of constituent mRNA using in situ hybridization were consistent with a phloem origin of the sap. Functional analysis of the cDNAs revealed classifications including ribosomal function, interaction with the environment, transport, DNA/RNA binding, and protein turnover. An analysis of the closest Arabidopsis thaliana (L.) homologue for each clone indicated that genes involved in cell localization, protein synthesis, tissue localization, organ localization, organ differentiation, and cell fate were represented at twice the level occurring in the whole Arabidopsis genome. The transcripts found in this phloem-enriched library are discussed in the context of phloem function and the relationship between the companion cell and sieve element.

  20. Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening

    PubMed Central

    Lane, Andrew B.; Strzelecka, Magdalena; Ettinger, Andreas; Grenfell, Andrew W.; Wittmann, Torsten; Heald, Rebecca

    2015-01-01

    Summary CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency. PMID:26212133

  1. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents

    SciTech Connect

    Albertsen, H.M.; Abderrahim, H.; Cann, H.M.; Dausset, J.; Le Paslier, D.; Cohen, D. )

    1990-06-01

    Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, the authors performed a series of preliminary experiments aimed at developing a suitable protocol. They found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.

  2. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

    PubMed Central

    Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci. PMID:27327613

  3. Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria

    PubMed Central

    Russell, Joseph A.; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F.

    2016-01-01

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface. PMID:27242705

  4. Deep subsurface life from North Pond: Enrichment, isolation, characterization and genomes of heterotrophic bacteria

    DOE PAGES

    Russell, Joseph A.; Leon-Zayas, Rosa; Wrighton, Kelly; ...

    2016-05-10

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic watercolumn west of the Mid-Atlantic Ridge at 22° N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sedimentmore » column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. Furthermore, the cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.« less

  5. A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits.

    PubMed

    Karamitros, Timokratis; Magiorkinis, Gkikas

    2015-12-15

    The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.

  6. Enriching Critical Thinking and Language Learning with Educational Digital Libraries

    ERIC Educational Resources Information Center

    Lu, Hsin-lin

    2012-01-01

    As the amount of information available in online digital libraries increases exponentially, questions arise concerning the most productive way to use that information to advance learning. Applying the earlier information seeking theories advocated by Kelly (1963), Taylor (1968), and Belkin (1980) to the digital libraries experience, Carol Kuhlthau…

  7. Adaptation of a commercial robot for genome library replication

    SciTech Connect

    Uber, D.C.; Searles, W.L.

    1994-01-01

    This report describes tools and fixtures developed at the Human Genome Center at Lawrence Berkeley Laboratory for the Hewlett-Packard ORCA{trademark} (Optimized Robot for Chemical Analysis) to replicate large genome libraries. Photographs and engineering drawings of the various custom-designed components are included.

  8. 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

  9. Isolation and characterization of rice cesium transporter genes from a rice-transporter-enriched yeast expression library.

    PubMed

    Yamaki, Tomohiro; Otani, Masahiro; Ono, Kohei; Mimura, Takuro; Oda, Koshiro; Minamii, Takeshi; Matsumoto, Shingo; Matsuo, Yuzy; Kawamukai, Makoto; Akihiro, Takashi

    2017-03-28

    A considerable portion of agricultural land in central-east Japan has been contaminated by radioactive material, particularly radioactive Cs, due to the industrial accident at the Fukushima Daiichi nuclear power plant. Understanding the mechanism of absorption, translocation, and accumulation of Cs(+) in plants will greatly assist in developing approaches to help reduce the radioactive contamination of agricultural products. At present, however, little is known regarding the Cs(+) transporters in rice. A transporter-enriched yeast expression library was constructed and the library was screened for Cs(+) transporter genes. The 1452 full length cDNAs encoding transporter genes were obtained from the Rice Genome Resource Center and 1358 clones of these transporter genes were successively subcloned into yeast expression vectors; which were then transferred into yeast. Using this library, both positive and negative selection screens can be performed, which have not been previously possible. The constructed library is an excellent tool for the isolation of novel transporter genes. This library was screened for clones that were sensitive to Cs(+) using a SD-Gal medium containing either 30 or 70 mM CsCl; resulting in the isolation of thirteen Cs(+) sensitive clones. (137) Cs absorption experiments were conducted and confirmed that all of the identified clones were able to absorb (137) Cs. Three potassium transporters, two ABC transporters, and one NRAMP transporter were among the thirteen identified clones.

  10. Antigen discovery using whole-genome phage display libraries.

    PubMed

    Beghetto, Elisa; Gargano, Nicola

    2013-01-01

    In the last two decades phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda-display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.

  11. Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment.

    PubMed

    Kim, Jonghwan; Bhinge, Akshay A; Morgan, Xochitl C; Iyer, Vishwanath R

    2005-01-01

    Identifying the chromosomal targets of transcription factors is important for reconstructing the transcriptional regulatory networks underlying global gene expression programs. We have developed an unbiased genomic method called sequence tag analysis of genomic enrichment (STAGE) to identify the direct binding targets of transcription factors in vivo. STAGE is based on high-throughput sequencing of concatemerized tags derived from target DNA enriched by chromatin immunoprecipitation. We first used STAGE in yeast to confirm that RNA polymerase III genes are the most prominent targets of the TATA-box binding protein. We optimized the STAGE protocol and developed analysis methods to allow the identification of transcription factor targets in human cells. We used STAGE to identify several previously unknown binding targets of human transcription factor E2F4 that we independently validated by promoter-specific PCR and microarray hybridization. STAGE provides a means of identifying the chromosomal targets of DNA-associated proteins in any sequenced genome.

  12. Deep subsurface life from North Pond: Enrichment, isolation, characterization and genomes of heterotrophic bacteria

    SciTech Connect

    Russell, Joseph A.; Leon-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F.

    2016-05-10

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic watercolumn west of the Mid-Atlantic Ridge at 22° N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. Furthermore, the cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.

  13. Semantically Enriching the Search System of a Music Digital Library

    NASA Astrophysics Data System (ADS)

    de Juan, Paloma; Iglesias, Carlos

    Traditional search systems are usually based on keywords, a very simple and convenient mechanism to express a need for information. This is the most popular way of searching the Web, although it is not always an easy task to accurately summarize a natural language query in a few keywords. Working with keywords means losing the context, which is the only thing that can help us deal with ambiguity. This is the biggest problem of keyword-based systems. Semantic Web technologies seem a perfect solution to this problem, since they make it possible to represent the semantics of a given domain. In this chapter, we present three projects, Harmos, Semusici and Cantiga, whose aim is to provide access to a music digital library. We will describe two search systems, a traditional one and a semantic one, developed in the context of these projects and compare them in terms of usability and effectiveness.

  14. Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment

    PubMed Central

    Li, Tengguo; Unger, Elizabeth R.; Batra, Dhwani; Sheth, Mili; Steinau, Martin; Jasinski, Jean; Jones, Jennifer

    2016-01-01

    ABSTRACT We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology. PMID:27974548

  15. Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

    PubMed

    Naimuddin, Mohammed; Kitamura, Koichirou; Kinoshita, Yasunori; Honda-Takahashi, Yoko; Murakami, Marina; Ito, Masato; Yamamoto, Kenji; Hanada, Kazunori; Husimi, Yuzuru; Nishigaki, Koichi

    2007-01-01

    A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.

  16. Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1-butanol tolerance using a metagenomic DNA library.

    PubMed

    Freedman, Benjamin G; Zu, Theresah N K; Wallace, Robert S; Senger, Ryan S

    2016-07-01

    Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real-time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi-genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1-butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP-PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP-PCR enabled high efficiency library cloning (with no DNA shearing or end-polishing) and the inclusion un-culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent-tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering.

  17. Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

    PubMed

    Nishiyama, Milton Yutaka; Ferreira, Savio Siqueira; Tang, Pei-Zhong; Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100-130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

  18. Whole-Genome Sequencing: Automated, Nonindexed Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-03-01

    This protocol describes an automated procedure for constructing a nonindexed Illumina DNA library and relies on the use of a CyBi-SELMA automated pipetting machine, the Covaris E210 shearing instrument, and the epMotion 5075. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Here, double-stranded DNA is fragmented when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymerase chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing.

  19. Whole-Genome Sequencing: Automated, Indexed Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-03-01

    This protocol describes an automated procedure for constructing an indexed Illumina DNA library. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Double-stranded DNA (dsDNA) will fragment when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymer chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing.

  20. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis.

    PubMed

    Charlop-Powers, Zachary; Banik, Jacob J; Owen, Jeremy G; Craig, Jeffrey W; Brady, Sean F

    2013-01-18

    The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein, we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts.

  1. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis

    PubMed Central

    Charlop-Powers, Zachary; Banik, Jacob J.; Owen, Jeremy G.; Craig, Jeffrey W.; Brady, Sean F.

    2012-01-01

    The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts. PMID:23072412

  2. Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome

    PubMed Central

    Ostrovnaya, Irina; Rubnitz, Kaitlyn R.; Ali, Siraj M.; Miller, Vincent A.; Mossé, Yael P.; Maris, John M.

    2016-01-01

    Neuroblastoma is characterized by a relative paucity of recurrent somatic mutations at diagnosis. However, recent studies have shown that the mutational burden increases at relapse, likely as a result of clonal evolution of mutation-carrying cells during primary treatment. To inform the development of personalized therapies, we sought to further define the frequency of potentially actionable mutations in neuroblastoma, both at diagnosis and after chemotherapy. We performed a retrospective study to determine mutation frequency, the only inclusion criterion being availability of cancer gene panel sequencing data from Foundation Medicine. We analyzed 151 neuroblastoma tumor samples: 44 obtained at diagnosis, 42 at second look surgery or biopsy for stable disease after chemotherapy, and 59 at relapse (6 were obtained at unknown time points). Nine patients had multiple tumor biopsies. ALK was the most commonly mutated gene in this cohort, and we observed a higher frequency of suspected oncogenic ALK mutations in relapsed disease than at diagnosis. Patients with relapsed disease had, on average, a greater number of mutations reported to be recurrent in cancer, and a greater number of mutations in genes that are potentially targetable with available therapeutics. We also observed an enrichment of reported recurrent RAS/MAPK pathway mutations in tumors obtained after chemotherapy. Our data support recent evidence suggesting that neuroblastomas undergo substantial mutational evolution during therapy, and that relapsed disease is more likely to be driven by a targetable oncogenic pathway, highlighting that it is critical to base treatment decisions on the molecular profile of the tumor at the time of treatment. However, it will be necessary to conduct prospective clinical trials that match sequencing results to targeted therapeutic intervention to determine if cancer genomic profiling improves patient outcomes. PMID:27997549

  3. [Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

    PubMed

    Bai, Yichun; Xu, Kun; Wei, Zehui; Ma, Zheng; Zhang, Zhiying

    2016-01-01

    Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

  4. RecA-assisted rapid enrichment of specific clones from model DNA libraries.

    PubMed

    Teintze, M; Arzimanoglou, I I; Lovelace, C I; Xu, Z J; Rigas, B

    1995-06-26

    An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

  5. Enrichment of chemical libraries docked to protein conformational ensembles and application to aldehyde dehydrogenase 2.

    PubMed

    Wang, Bo; Buchman, Cameron D; Li, Liwei; Hurley, Thomas D; Meroueh, Samy O

    2014-07-28

    Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein-compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5

  6. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1991-01-01

    We have made important progress since the beginning of the current grant year. We have further developed the microdissection and PCR- assisted microcloning techniques using the linker-adaptor method. We have critically evaluated the microdissection libraries constructed by this microtechnology and proved that they are of high quality. We further demonstrated that these microdissection clones are useful in identifying corresponding YAC clones for a thousand-fold expansion of the genomic coverage and for contig construction. We are also improving the technique of cloning the dissected fragments in test tube by the TDT method. We are applying both of these PCR cloning technique to human chromosomes 2 and 5 to construct region-specific libraries for physical mapping purposes of LLNL and LANL. Finally, we are exploring efficient procedures to use unique sequence microclones to isolate cDNA clones from defined chromosomal regions as valuable resources for identifying expressed gene sequences in the human genome. We believe that we are making important progress under the auspices of this DOE human genome program grant and we will continue to make significant contributions in the coming year. 4 refs., 4 figs.

  7. Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

    SciTech Connect

    McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

    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. A 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.

  8. The infectious BAC genomic DNA expression library: a high capacity vector system for functional genomics

    PubMed Central

    Lufino, Michele M. P.; Edser, Pauline A. H.; Quail, Michael A.; Rice, Stephen; Adams, David J.; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

  9. Draft Genome Sequence of Antarctic Methanogen Enriched from Dry Valley Permafrost

    PubMed Central

    Buongiorno, Joy; Bird, Jordan T.; Krivushin, Kirill; Oshurkova, Victoria; Shcherbakova, Victoria; Rivkina, Elizaveta M.

    2016-01-01

    A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics. PMID:27932654

  10. Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

    SciTech Connect

    Branscomb, E.; Slezak, T.; Pae, R.; Carrano, A.V. ); Galas, D.; Waterman, M. )

    1990-01-01

    The authors present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Their primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, they adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called contigs.' These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.

  11. A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes1

    PubMed Central

    Stull, Gregory W.; Moore, Michael J.; Mandala, Venkata S.; Douglas, Norman A.; Kates, Heather-Rose; Qi, Xinshuai; Brockington, Samuel F.; Soltis, Pamela S.; Soltis, Douglas E.; Gitzendanner, Matthew A.

    2013-01-01

    • Premise of the study: We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS) of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms. • Methods and Results: A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots), which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×), even for the two monocots. • Conclusions: Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving ∼50× mean coverage). However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96) available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms. PMID:25202518

  12. A library of TAL effector nucleases spanning the human genome.

    PubMed

    Kim, Yongsub; Kweon, Jiyeon; Kim, Annie; Chon, Jae Kyung; Yoo, Ji Yeon; Kim, Hye Joo; Kim, Sojung; Lee, Choongil; Jeong, Euihwan; Chung, Eugene; Kim, Doyoung; Lee, Mi Seon; Go, Eun Mi; Song, Hye Jung; Kim, Hwangbeom; Cho, Namjin; Bang, Duhee; Kim, Seokjoong; Kim, Jin-Soo

    2013-03-01

    Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

  13. Cost-effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies.

    PubMed

    Mariac, Cédric; Scarcelli, Nora; Pouzadou, Juliette; Barnaud, Adeline; Billot, Claire; Faye, Adama; Kougbeadjo, Ayite; Maillol, Vincent; Martin, Guillaume; Sabot, François; Santoni, Sylvain; Vigouroux, Yves; Couvreur, Thomas L P

    2014-11-01

    Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next-generation sequencing methods. In this study, we develop an easy and cost-effective protocol for in-solution enrichment hybridization capture of complete chloroplast genomes applicable at deep-multiplexed levels. The protocol uses cheap in-house species-specific probes developed via long-range PCR of the entire chloroplast. Barcoded libraries are constructed, and in-solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes.

  14. Gap Closing/Finishing by Targeted Genomic Region Enrichment and Sequencing

    SciTech Connect

    Singh, Kanwar; Froula, Jeff; Trice, Hope; Pennacchio, Len A.; Chen, Feng

    2010-05-27

    Gap Closing/Finishing of draft genome assemblies is a labor and cost intensive process where several rounds of repetitious amplification and sequencing are required. Here we demonstrate a high throughput procedure where custom primers flanking gaps in draft genomes are designed. Primer libraries containing up to 4,000 unique pairs in independent droplets are merged with a fragmented genomic template. From this millions of picoliter scale droplets are formed, each one being the functional equivalent of an individual PCR reaction. The PCR products are concatenated and sequenced by Illumina which is then assembled and used for gap closure. Here we present an overall experimental strategy, primer design algorithm and initial results.

  15. Construction of a llama bacterial artificial chromosome library with approximately 9-fold genome equivalent coverage.

    PubMed

    Airmet, K W; Hinckley, J D; Tree, L T; Moss, M; Blumell, S; Ulicny, K; Gustafson, A K; Weed, M; Theodosis, R; Lehnardt, M; Genho, J; Stevens, M R; Kooyman, D L

    2012-01-01

    The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4 × 10⁹ bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

  16. Halogen-enriched fragment libraries as chemical probes for harnessing halogen bonding in fragment-based lead discovery.

    PubMed

    Zimmermann, Markus O; Lange, Andreas; Wilcken, Rainer; Cieslik, Markus B; Exner, Thomas E; Joerger, Andreas C; Koch, Pierre; Boeckler, Frank M

    2014-04-01

    Halogen bonding has recently experienced a renaissance, gaining increased recognition as a useful molecular interaction in the life sciences. Halogen bonds are favorable, fairly directional interactions between an electropositive region on the halogen (the σ-hole) and a number of different nucleophilic interaction partners. Some aspects of halogen bonding are not yet understood well enough to take full advantage of its potential in drug discovery. We describe and present the concept of halogen-enriched fragment libraries. These libraries consist of unique chemical probes, facilitating the identification of favorable halogen bonds by sharing the advantages of classical fragment-based screening. Besides providing insights into the nature and applicability of halogen bonding, halogen-enriched fragment libraries provide smart starting points for hit-to-lead evolution.

  17. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    PubMed Central

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

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. 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 genome and transcriptome research of Bengal tigers. PMID:23708105

  18. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

    SciTech Connect

    Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John; Fitzsimmons, Kevin; Brown, Judith K

    2013-09-01

    With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

  19. Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections

    PubMed Central

    Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe

    2016-01-01

    Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3. PMID:27703859

  20. Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

    PubMed

    Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe; Avarre, Jean-Christophe

    2016-01-01

    Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×10(7). The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

  1. Enrichment of genomic DNA for polymorphism detection in a non-model highly polyploid crop plant.

    PubMed

    Bundock, Peter C; Casu, Rosanne E; Henry, Robert J

    2012-08-01

    Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8 megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270 000-280 000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13 000-16 000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.

  2. Robotic Enrichment Processing of Roche 454 Titanium Emlusion PCR at the DOE Joint Genome Institute

    SciTech Connect

    Hamilton, Matthew; Wilson, Steven; Bauer, Diane; Miller, Don; Duffy-Wei, Kecia; Hammon, Nancy; Lucas, Susan; Pollard, Martin; Cheng, Jan-Fang

    2010-05-28

    Enrichment of emulsion PCR product is the most laborious and pipette-intensive step in the 454 Titanium process, posing the biggest obstacle for production-oriented scale up. The Joint Genome Institute has developed a pair of custom-made robots based on the Microlab Star liquid handling deck manufactured by Hamilton to mediate the complexity and ergonomic demands of the 454 enrichment process. The robot includes a custom built centrifuge, magnetic deck positions, as well as heating and cooling elements. At present processing eight emulsion cup samples in a single 2.5 hour run, these robots are capable of processing up to 24 emulsion cup samples. Sample emulsions are broken using the standard 454 breaking process and transferred from a pair of 50ml conical tubes to a single 2ml tube and loaded on the robot. The robot performs the enrichment protocol and produces beads in 2ml tubes ready for counting. The robot follows the Roche 454 enrichment protocol with slight exceptions to the manner in which it resuspends beads via pipette mixing rather than vortexing and a set number of null bead removal washes. The robotic process is broken down in similar discrete steps: First Melt and Neutralization, Enrichment Primer Annealing, Enrichment Bead Incubation, Null Bead Removal, Second Melt and Neutralization and Sequencing Primer Annealing. Data indicating our improvements in enrichment efficiency and total number of bases per run will also be shown.

  3. Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

    PubMed

    Zhang, Hong-Bin; Scheuring, Chantel F; Zhang, Meiping; Zhang, Yang; Wu, Cheng-Cang; Dong, Jennifer J; Li, Yaning

    2012-02-16

    Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ∼7 d.

  4. Human genome libraries. Final progress report, February 1, 1994--August 31, 1997

    SciTech Connect

    Kao, Fa-Ten

    1998-01-01

    The goal of this program is to use a novel technology of chromosome microdissection and microcloning to construct chromosome region-specific libraries as resources for various human genome program studies. Region specific libraries have been constructed for the entire human chromosomes 2 and 18.

  5. Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli

    PubMed Central

    Reyes, Luis H.; Almario, Maria P.; Kao, Katy C.

    2011-01-01

    Background n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance. Methodology/Principal Findings Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%. Conclusions/Significance We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the

  6. Genomes of two new ammonia-oxidizing archaea enriched from deep marine sediments.

    PubMed

    Park, Soo-Je; Ghai, Rohit; Martín-Cuadrado, Ana-Belén; Rodríguez-Valera, Francisco; Chung, Won-Hyong; Kwon, KaeKyoung; Lee, Jung-Hyun; Madsen, Eugene L; Rhee, Sung-Keun

    2014-01-01

    Ammonia-oxidizing archaea (AOA) are ubiquitous and abundant and contribute significantly to the carbon and nitrogen cycles in the ocean. In this study, we assembled AOA draft genomes from two deep marine sediments from Donghae, South Korea, and Svalbard, Arctic region, by sequencing the enriched metagenomes. Three major microorganism clusters belonging to Thaumarchaeota, Epsilonproteobacteria, and Gammaproteobacteria were deduced from their 16S rRNA genes, GC contents, and oligonucleotide frequencies. Three archaeal genomes were identified, two of which were distinct and were designated Ca. "Nitrosopumilus koreensis" AR1 and "Nitrosopumilus sediminis" AR2. AR1 and AR2 exhibited average nucleotide identities of 85.2% and 79.5% to N. maritimus, respectively. The AR1 and AR2 genomes contained genes pertaining to energy metabolism and carbon fixation as conserved in other AOA, but, conversely, had fewer heme-containing proteins and more copper-containing proteins than other AOA. Most of the distinctive AR1 and AR2 genes were located in genomic islands (GIs) that were not present in other AOA genomes or in a reference water-column metagenome from the Sargasso Sea. A putative gene cluster involved in urea utilization was found in the AR2 genome, but not the AR1 genome, suggesting niche specialization in marine AOA. Co-cultured bacterial genome analysis suggested that bacterial sulfur and nitrogen metabolism could be involved in interactions with AOA. Our results provide fundamental information concerning the metabolic potential of deep marine sedimentary AOA.

  7. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

    PubMed

    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.

  8. Target enrichment of ultraconserved elements from arthropods provides a genomic perspective on relationships among Hymenoptera.

    PubMed

    Faircloth, Brant C; Branstetter, Michael G; White, Noor D; Brady, Seán G

    2015-05-01

    Gaining a genomic perspective on phylogeny requires the collection of data from many putatively independent loci across the genome. Among insects, an increasingly common approach to collecting this class of data involves transcriptome sequencing, because few insects have high-quality genome sequences available; assembling new genomes remains a limiting factor; the transcribed portion of the genome is a reasonable, reduced subset of the genome to target; and the data collected from transcribed portions of the genome are similar in composition to the types of data with which biologists have traditionally worked (e.g. exons). However, molecular techniques requiring RNA as a template, including transcriptome sequencing, are limited to using very high-quality source materials, which are often unavailable from a large proportion of biologically important insect samples. Recent research suggests that DNA-based target enrichment of conserved genomic elements offers another path to collecting phylogenomic data across insect taxa, provided that conserved elements are present in and can be collected from insect genomes. Here, we identify a large set (n = 1510) of ultraconserved elements (UCEs) shared among the insect order Hymenoptera. We used in silico analyses to show that these loci accurately reconstruct relationships among genome-enabled hymenoptera, and we designed a set of RNA baits (n = 2749) for enriching these loci that researchers can use with DNA templates extracted from a variety of sources. We used our UCE bait set to enrich an average of 721 UCE loci from 30 hymenopteran taxa, and we used these UCE loci to reconstruct phylogenetic relationships spanning very old (≥220 Ma) to very young (≤1 Ma) divergences among hymenopteran lineages. In contrast to a recent study addressing hymenopteran phylogeny using transcriptome data, we found ants to be sister to all remaining aculeate lineages with complete support, although this result could be explained by

  9. Rapid enrichment of leucocytes and genomic DNA from blood based on bifunctional core shell magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Nie, Xiaorong; Yu, Bingbin; Zhang, Xu

    2007-04-01

    A series of protocols are proposed to extract genomic DNA from whole blood at different scales using carboxyl-functionalized magnetic nanoparticles as solid-phase absorbents. The enrichment of leucocytes and the adsorption of genomic DNA can be achieved with the same carboxyl-functionalized magnetic nanoparticles. The DNA bound to the bead surfaces can be used directly as PCR templates. By coupling cell separation and DNA purification, the whole operation can be accomplished in a few minutes. Our simplified protocols proved to be rapid, low cost, and biologically and chemically non-hazardous, and are therefore promising for microfabrication of a DNA-preparation chip and routine laboratory use.

  10. Genomic features of uncultured methylotrophs in activated-sludge microbiomes grown under different enrichment procedures

    PubMed Central

    Fujinawa, Kazuki; Asai, Yusuke; Miyahara, Morio; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

    2016-01-01

    Methylotrophs are organisms that are able to grow on C1 compounds as carbon and energy sources. They play important roles in the global carbon cycle and contribute largely to industrial wastewater treatment. To identify and characterize methylotrophs that are involved in methanol degradation in wastewater-treatment plants, methanol-fed activated-sludge (MAS) microbiomes were subjected to phylogenetic and metagenomic analyses, and genomic features of dominant methylotrophs in MAS were compared with those preferentially grown in laboratory enrichment cultures (LECs). These analyses consistently indicate that Hyphomicrobium plays important roles in MAS, while Methylophilus occurred predominantly in LECs. Comparative analyses of bin genomes reconstructed for the Hyphomicrobium and Methylophilus methylotrophs suggest that they have different C1-assimilation pathways. In addition, function-module analyses suggest that their cell-surface structures are different. Comparison of the MAS bin genome with genomes of closely related Hyphomicrobium isolates suggests that genes unnecessary in MAS (for instance, genes for anaerobic respiration) have been lost from the genome of the dominant methylotroph. We suggest that genomic features and coded functions in the MAS bin genome provide us with insights into how this methylotroph adapts to activated-sludge ecosystems. PMID:27221669

  11. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    PubMed Central

    2011-01-01

    Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome

  12. Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics

    PubMed Central

    Delmont, Tom O.; Eren, A. Murat; Maccario, Lorrie; Prestat, Emmanuel; Esen, Özcan C.; Pelletier, Eric; Le Paslier, Denis; Simonet, Pascal; Vogel, Timothy M.

    2015-01-01

    Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard de novo assembly techniques (a total of 559,555 genes and 29,176 functions), from which we could recover novel bacterial genomes, plasmids and phages. The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2, novel nitrogen fixing taxon), Ktedonobacter (n = 1, second representative of the family Ktedonobacteraceae), Streptomyces (n = 3, novel polyketide synthase modules), and Burkholderia (n = 2, includes mega-plasmids conferring mercury resistance). Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment. PMID:25983722

  13. Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes.

    PubMed

    Weber, Evelyne; Seifert, Alexander; Antonovici, Mihaela; Geinitz, Christopher; Pleiss, Jürgen; Urlacher, Vlada B

    2011-01-21

    A minimal enriched P450 BM3 library was screened for the ability to oxidize inert cyclic and acyclic alkanes. The F87A/A328V mutant was found to effectively hydroxylate cyclooctane, cyclodecane and cyclododecane. F87V/A328F with high activity towards cyclooctane hydroxylated acyclic n-octane to 2-(R)-octanol (46% ee) with high regioselectivity (92%).

  14. Designing Focused Chemical Libraries Enriched in Protein-Protein Interaction Inhibitors using Machine-Learning Methods

    PubMed Central

    Reynès, Christelle; Host, Hélène; Camproux, Anne-Claude; Laconde, Guillaume; Leroux, Florence; Mazars, Anne; Deprez, Benoit; Fahraeus, Robin; Villoutreix, Bruno O.; Sperandio, Olivier

    2010-01-01

    Protein-protein interactions (PPIs) may represent one of the next major classes of therapeutic targets. So far, only a minute fraction of the estimated 650,000 PPIs that comprise the human interactome are known with a tiny number of complexes being drugged. Such intricate biological systems cannot be cost-efficiently tackled using conventional high-throughput screening methods. Rather, time has come for designing new strategies that will maximize the chance for hit identification through a rationalization of the PPI inhibitor chemical space and the design of PPI-focused compound libraries (global or target-specific). Here, we train machine-learning-based models, mainly decision trees, using a dataset of known PPI inhibitors and of regular drugs in order to determine a global physico-chemical profile for putative PPI inhibitors. This statistical analysis unravels two important molecular descriptors for PPI inhibitors characterizing specific molecular shapes and the presence of a privileged number of aromatic bonds. The best model has been transposed into a computer program, PPI-HitProfiler, that can output from any drug-like compound collection a focused chemical library enriched in putative PPI inhibitors. Our PPI inhibitor profiler is challenged on the experimental screening results of 11 different PPIs among which the p53/MDM2 interaction screened within our own CDithem platform, that in addition to the validation of our concept led to the identification of 4 novel p53/MDM2 inhibitors. Collectively, our tool shows a robust behavior on the 11 experimental datasets by correctly profiling 70% of the experimentally identified hits while removing 52% of the inactive compounds from the initial compound collections. We strongly believe that this new tool can be used as a global PPI inhibitor profiler prior to screening assays to reduce the size of the compound collections to be experimentally screened while keeping most of the true PPI inhibitors. PPI-HitProfiler is

  15. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.

    PubMed

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-04-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

  16. High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution

    PubMed Central

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-01-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the “natural” AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid–DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid–genome identity correlation. Overall, our observations demonstrate that production of “natural” AAVs results in low capsid mosaicism and high capsid–genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype–genotype correlation. PMID:25586687

  17. High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

    PubMed

    Malausa, Thibaut; Gilles, André; Meglécz, Emese; Blanquart, Hélène; Duthoy, Stéphanie; Costedoat, Caroline; Dubut, Vincent; Pech, Nicolas; Castagnone-Sereno, Philippe; Délye, Christophe; Feau, Nicolas; Frey, Pascal; Gauthier, Philippe; Guillemaud, Thomas; Hazard, Laurent; Le Corre, Valérie; Lung-Escarmant, Brigitte; Malé, Pierre-Jean G; Ferreira, Stéphanie; Martin, Jean-François

    2011-07-01

    Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

  18. 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.

  19. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    DOE PAGES

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

  20. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    SciTech Connect

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.

  1. Cell context dependent p53 genome-wide binding patterns and enrichment at repeats.

    PubMed

    Botcheva, Krassimira; McCorkle, Sean R

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.

  2. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    PubMed Central

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways. PMID:25415302

  3. Genome-wide BAC-end sequencing of Cucumis melo using two BAC libraries

    PubMed Central

    2010-01-01

    Background Although melon (Cucumis melo L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library. Results We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes. Conclusion The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes. PMID:21054843

  4. 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

  5. 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

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-10-12

    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.

  6. Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

    PubMed

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

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future.

  7. Local assemblies of paired-end reduced representation libraries sequenced with the illumina genome analyzer in maize.

    PubMed

    Deschamps, Stéphane; Nannapaneni, Kishore; Zhang, Yun; Hayes, Kevin

    2012-01-01

    The use of next-generation DNA sequencing technologies has greatly facilitated reference-guided variant detection in complex plant genomes. However, complications may arise when regions adjacent to a read of interest are used for marker assay development, or when reference sequences are incomplete, as short reads alone may not be long enough to ascertain their uniqueness. Here, the possibility of generating longer sequences in discrete regions of the large and complex genome of maize is demonstrated, using a modified version of a paired-end RAD library construction strategy. Reads are generated from DNA fragments first digested with a methylation-sensitive restriction endonuclease, sheared, enriched with biotin and a selective PCR amplification step, and then sequenced at both ends. Sequences are locally assembled into contigs by subgrouping pairs based on the identity of the read anchored by the restriction site. This strategy applied to two maize inbred lines (B14 and B73) generated 183,609 and 129,018 contigs, respectively, out of which at least 76% were >200 bps in length. A subset of putative single nucleotide polymorphisms from contigs aligning to the B73 reference genome with at least one mismatch was resequenced, and 90% of those in B14 were confirmed, indicating that this method is a potent approach for variant detection and marker development in species with complex genomes or lacking extensive reference sequences.

  8. Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

    PubMed Central

    2010-01-01

    Background Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads. Results A modified rapid chloroplast isolation protocol was used to obtain plant DNA that was enriched for chloroplast DNA, but nevertheless contained nuclear and mitochondrial DNA. Multiply-primed rolling circle amplification of this mixed template produced sufficient quantities of chloroplast DNA, even when the amount of starting material was small, and improved the template quality for Illumina Genome Analyzer II (hereafter Illumina GAII) sequencing. We demonstrate, using independent samples of karaka (Corynocarpus laevigatus), that there is high fidelity in the sequence obtained from this template. Although less than 20% of our sequenced reads could be mapped to chloroplast genome, it was relatively easy to assemble complete chloroplast genome sequences from the mixture of nuclear, mitochondrial and chloroplast reads. Conclusions We report successful whole genome sequencing of chloroplast DNA from karaka, obtained efficiently and with high fidelity. PMID:20920211

  9. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    PubMed Central

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Michael S.; Yang, Zamin K.; Klingeman, Dawn M.; Land, Miriam L.; Allman, Steve L.; Lu, Tse-Yuan S.; Brown, Steven D.; Schadt, Christopher W.; Podar, Mircea; Doktycz, Mitchel J.

    2016-01-01

    ABSTRACT Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses

  10. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

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

    PubMed Central

    Tian, Wenlan; Paudel, Dev

    2017-01-01

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

  12. Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

    PubMed

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

    2014-03-07

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

  13. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    SciTech Connect

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Jr., Michael S.; Yang, Zamin Koo; Klingeman, Dawn Marie; Land, Miriam L.; Allman, Steve L.; Lu, Tse-Yuan S.; Brown, Steven D.; Schadt, Christopher Warren; Podar, Mircea; Doktycz, Mitchel J.; Pelletier, Dale A.

    2016-07-15

    Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.

  14. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    DOE PAGES

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Jr., Michael S.; ...

    2016-07-15

    Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from themore » plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.« less

  15. Focused chemical libraries--design and enrichment: an example of protein-protein interaction chemical space.

    PubMed

    Zhang, Xu; Betzi, Stéphane; Morelli, Xavier; Roche, Philippe

    2014-07-01

    One of the many obstacles in the development of new drugs lies in the limited number of therapeutic targets and in the quality of screening collections of compounds. In this review, we present general strategies for building target-focused chemical libraries with a particular emphasis on protein-protein interactions (PPIs). We describe the chemical spaces spanned by nine commercially available PPI-focused libraries and compare them to our 2P2I3D academic library, dedicated to orthosteric PPI modulators. We show that although PPI-focused libraries have been designed using different strategies, they share common subspaces. PPI inhibitors are larger and more hydrophobic than standard drugs; however, an effort has been made to improve the drug-likeness of focused chemical libraries dedicated to this challenging class of targets.

  16. Scalable whole-genome single-cell library preparation without preamplification.

    PubMed

    Zahn, Hans; Steif, Adi; Laks, Emma; Eirew, Peter; VanInsberghe, Michael; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

    2017-02-01

    Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

  17. 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…

  18. A novel ammonia-oxidizing archaeon from wastewater treatment plant: Its enrichment, physiological and genomic characteristics

    NASA Astrophysics Data System (ADS)

    Li, Yuyang; Ding, Kun; Wen, Xianghua; Zhang, Bing; Shen, Bo; Yang, Yunfeng

    2016-03-01

    Ammonia-oxidizing archaea (AOA) are recently found to participate in the ammonia removal processes in wastewater treatment plants (WWTPs), similar to their bacterial counterparts. However, due to lack of cultivated AOA strains from WWTPs, their functions and contributions in these systems remain unclear. Here we report a novel AOA strain SAT1 enriched from activated sludge, with its physiological and genomic characteristics investigated. The maximal 16S rRNA gene similarity between SAT1 and other reported AOA strain is 96% (with “Ca. Nitrosotenuis chungbukensis”), and it is affiliated with Wastewater Cluster B (WWC-B) based on amoA gene phylogeny, a cluster within group I.1a and specific for activated sludge. Our strain is autotrophic, mesophilic (25 °C–33 °C) and neutrophilic (pH 5.0–7.0). Its genome size is 1.62 Mb, with a large fragment inversion (accounted for 68% genomic size) inside. The strain could not utilize urea due to truncation of the urea transporter gene. The lack of the pathways to synthesize usual compatible solutes makes it intolerant to high salinity (>0.03%), but could adapt to low salinity (0.005%) environments. This adaptation, together with possibly enhanced cell-biofilm attachment ability, makes it suitable for WWTPs environment. We propose the name “Candidatus Nitrosotenuis cloacae” for the strain SAT1.

  19. A novel ammonia-oxidizing archaeon from wastewater treatment plant: Its enrichment, physiological and genomic characteristics

    PubMed Central

    Li, Yuyang; Ding, Kun; Wen, Xianghua; Zhang, Bing; Shen, Bo; Yang, Yunfeng

    2016-01-01

    Ammonia-oxidizing archaea (AOA) are recently found to participate in the ammonia removal processes in wastewater treatment plants (WWTPs), similar to their bacterial counterparts. However, due to lack of cultivated AOA strains from WWTPs, their functions and contributions in these systems remain unclear. Here we report a novel AOA strain SAT1 enriched from activated sludge, with its physiological and genomic characteristics investigated. The maximal 16S rRNA gene similarity between SAT1 and other reported AOA strain is 96% (with “Ca. Nitrosotenuis chungbukensis”), and it is affiliated with Wastewater Cluster B (WWC-B) based on amoA gene phylogeny, a cluster within group I.1a and specific for activated sludge. Our strain is autotrophic, mesophilic (25 °C–33 °C) and neutrophilic (pH 5.0–7.0). Its genome size is 1.62 Mb, with a large fragment inversion (accounted for 68% genomic size) inside. The strain could not utilize urea due to truncation of the urea transporter gene. The lack of the pathways to synthesize usual compatible solutes makes it intolerant to high salinity (>0.03%), but could adapt to low salinity (0.005%) environments. This adaptation, together with possibly enhanced cell-biofilm attachment ability, makes it suitable for WWTPs environment. We propose the name “Candidatus Nitrosotenuis cloacae” for the strain SAT1. PMID:27030530

  20. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

    PubMed Central

    Futamura, Norihiro; Totoki, Yasushi; Toyoda, Atsushi; Igasaki, Tomohiro; Nanjo, Tokihiko; Seki, Motoaki; Sakaki, Yoshiyuki; Mari, Adriano; Shinozaki, Kazuo; Shinohara, Kenji

    2008-01-01

    Background Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. Results We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. Conclusion Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species. PMID:18691438

  1. The 19 Genomes of Drosophila: A BAC Library Resource for Genus-Wide and Genome-Scale Comparative Evolutionary Research

    PubMed Central

    Song, Xiang; Goicoechea, Jose Luis; Ammiraju, Jetty S. S.; Luo, Meizhong; He, Ruifeng; Lin, Jinke; Lee, So-Jeong; Sisneros, Nicholas; Watts, Tom; Kudrna, David A.; Golser, Wolfgang; Ashley, Elizabeth; Collura, Kristi; Braidotti, Michele; Yu, Yeisoo; Matzkin, Luciano M.; McAllister, Bryant F.; Markow, Therese Ann; Wing, Rod A.

    2011-01-01

    The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125–168 kb), low nonrecombinant clone content (0.3–5.3%), and deep coverage (9.1–42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny. PMID:21321134

  2. The 19 genomes of Drosophila: a BAC library resource for genus-wide and genome-scale comparative evolutionary research.

    PubMed

    Song, Xiang; Goicoechea, Jose Luis; Ammiraju, Jetty S S; Luo, Meizhong; He, Ruifeng; Lin, Jinke; Lee, So-Jeong; Sisneros, Nicholas; Watts, Tom; Kudrna, David A; Golser, Wolfgang; Ashley, Elizabeth; Collura, Kristi; Braidotti, Michele; Yu, Yeisoo; Matzkin, Luciano M; McAllister, Bryant F; Markow, Therese Ann; Wing, Rod A

    2011-04-01

    The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125-168 kb), low nonrecombinant clone content (0.3-5.3%), and deep coverage (9.1-42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny.

  3. BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

    PubMed Central

    Guimarães, Patricia M; Garsmeur, Olivier; Proite, Karina; Leal-Bertioli, Soraya CM; Seijo, Guilhermo; Chaine, Christian; Bertioli, David J; D'Hont, Angelique

    2008-01-01

    Background Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively. Results We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues. Conclusion These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map. PMID:18230166

  4. Synthesis of an arrayed sgRNA library targeting the human genome

    PubMed Central

    Schmidt, Tobias; Schmid-Burgk, Jonathan L.; Hornung, Veit

    2015-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested. PMID:26446710

  5. Genome-wide enrichment analysis between endometriosis and obesity-related traits reveals novel susceptibility loci

    PubMed Central

    Rahmioglu, Nilufer; Macgregor, Stuart; Drong, Alexander W.; Hedman, Åsa K.; Harris, Holly R.; Randall, Joshua C.; Prokopenko, Inga; Nyholt, Dale R.; Morris, Andrew P.; Montgomery, Grant W.; Missmer, Stacey A.; Lindgren, Cecilia M.; Zondervan, Krina T.

    2015-01-01

    Endometriosis is a chronic inflammatory condition in women that results in pelvic pain and subfertility, and has been associated with decreased body mass index (BMI). Genetic variants contributing to the heritable component have started to emerge from genome-wide association studies (GWAS), although the majority remain unknown. Unexpectedly, we observed an intergenic locus on 7p15.2 that was genome-wide significantly associated with both endometriosis and fat distribution (waist-to-hip ratio adjusted for BMI; WHRadjBMI) in an independent meta-GWAS of European ancestry individuals. This led us to investigate the potential overlap in genetic variants underlying the aetiology of endometriosis, WHRadjBMI and BMI using GWAS data. Our analyses demonstrated significant enrichment of common variants between fat distribution and endometriosis (P = 3.7 × 10−3), which was stronger when we restricted the investigation to more severe (Stage B) cases (P = 4.5 × 10−4). However, no genetic enrichment was observed between endometriosis and BMI (P = 0.79). In addition to 7p15.2, we identify four more variants with statistically significant evidence of involvement in both endometriosis and WHRadjBMI (in/near KIFAP3, CAB39L, WNT4, GRB14); two of these, KIFAP3 and CAB39L, are novel associations for both traits. KIFAP3, WNT4 and 7p15.2 are associated with the WNT signalling pathway; formal pathway analysis confirmed a statistically significant (P = 6.41 × 10−4) overrepresentation of shared associations in developmental processes/WNT signalling between the two traits. Our results demonstrate an example of potential biological pleiotropy that was hitherto unknown, and represent an opportunity for functional follow-up of loci and further cross-phenotype comparisons to assess how fat distribution and endometriosis pathogenesis research fields can inform each other. PMID:25296917

  6. Core and region-enriched networks of behaviorally regulated genes and the singing genome

    PubMed Central

    Whitney, Osceola; Pfenning, Andreas R.; Howard, Jason T.; Blatti, Charles A; Liu, Fang; Ward, James M.; Wang, Rui; Audet, Jean-Nicolas; Kellis, Manolis; Mukherjee, Sayan; Sinha, Saurabh; Hartemink, Alexander J.; West, Anne E.; Jarvis, Erich D.

    2015-01-01

    Songbirds represent an important model organism for elucidating molecular mechanisms that link genes with complex behaviors, in part because they have discrete vocal learning circuits that have parallels with those that mediate human speech. We found that ~10% of the genes in the avian genome were regulated by singing, and we found a striking regional diversity of both basal and singing-induced programs in the four key song nuclei of the zebra finch, a vocal learning songbird. The region-enriched patterns were a result of distinct combinations of region-enriched transcription factors (TFs), their binding motifs, and presinging acetylation of histone 3 at lysine 27 (H3K27ac) enhancer activity in the regulatory regions of the associated genes. RNA interference manipulations validated the role of the calcium-response transcription factor (CaRF) in regulating genes preferentially expressed in specific song nuclei in response to singing. Thus, differential combinatorial binding of a small group of activity-regulated TFs and predefined epigenetic enhancer activity influences the anatomical diversity of behaviorally regulated gene networks. PMID:25504732

  7. Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library.

    PubMed

    Yoshino, Seiko; Hara, Toshiro; Weng, Jane S; Takahashi, Yuka; Seiki, Motoharu; Sakamoto, Takeharu

    2012-01-01

    Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.

  8. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    PubMed Central

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-01-01

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved. Images PMID:6296782

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

    PubMed Central

    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. PMID:26442115

  10. 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.

  11. Near-Complete Genome Sequence of Thalassospira sp. Strain KO164 Isolated from a Lignin-Enriched Marine Sediment Microcosm

    PubMed Central

    Woo, Hannah L.; O’Dell, Kaela B.; Utturkar, Sagar; McBride, Kathryn R.; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Stamatis, Dimitrios; Reddy, T. B. K.; Ngan, Chew Yee; Daum, Chris; Shapiro, Nicole; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Woyke, Tanja; Brown, Steven D.

    2016-01-01

    Thalassospira sp. strain KO164 was isolated from eastern Mediterranean seawater and sediment laboratory microcosms enriched on insoluble organosolv lignin under oxic conditions. The near-complete genome sequence presented here will facilitate analyses into this deep-ocean bacterium’s ability to degrade recalcitrant organics such as lignin. PMID:27881538

  12. Near-Complete Genome Sequence of Thalassospira sp. Strain KO164 Isolated from a Lignin-Enriched Marine Sediment Microcosm.

    PubMed

    Woo, Hannah L; O'Dell, Kaela B; Utturkar, Sagar; McBride, Kathryn R; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Stamatis, Dimitrios; Reddy, T B K; Ngan, Chew Yee; Daum, Chris; Shapiro, Nicole; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Woyke, Tanja; Brown, Steven D; Hazen, Terry C

    2016-11-23

    Thalassospira sp. strain KO164 was isolated from eastern Mediterranean seawater and sediment laboratory microcosms enriched on insoluble organosolv lignin under oxic conditions. The near-complete genome sequence presented here will facilitate analyses into this deep-ocean bacterium's ability to degrade recalcitrant organics such as lignin.

  13. Fidelity by design: Yoctoreactor and binder trap enrichment for small-molecule DNA-encoded libraries and drug discovery.

    PubMed

    Blakskjaer, Peter; Heitner, Tara; Hansen, Nils Jakob Vest

    2015-06-01

    DNA-encoded small-molecule library (DEL) technology allows vast drug-like small molecule libraries to be efficiently synthesized in a combinatorial fashion and screened in a single tube method for binding, with an assay readout empowered by advances in next generation sequencing technology. This approach has increasingly been applied as a viable technology for the identification of small-molecule modulators to protein targets and as precursors to drugs in the past decade. Several strategies for producing and for screening DELs have been devised by both academic and industrial institutions. This review highlights some of the most significant and recent strategies along with important results. A special focus on the production of high fidelity DEL technologies with the ability to eliminate screening noise and false positives is included: using a DNA junction called the Yoctoreactor, building blocks (BBs) are spatially confined at the center of the junction facilitating both the chemical reaction between BBs and encoding of the synthetic route. A screening method, known as binder trap enrichment, permits DELs to be screened robustly in a homogeneous manner delivering clean data sets and potent hits for even the most challenging targets.

  14. 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 (AatII, PstI, MspI) and methylation-insensitive (SphI, MseI) 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.

  15. Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization

    PubMed Central

    Bai, Jianing; McAteer, Sean P.; Paxton, Edith; Mahajan, Arvind; Gally, David L.; Tree, Jai J.

    2011-01-01

    Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to ruminant feces containing the bacterium. The main colonization site of EHEC O157:H7 in cattle is the terminal rectum where the bacteria intimately attach to the epithelium and multiply in the intestinal mucus. This study aimed to identify genomic regions of EHEC O157:H7 that contribute to colonization and multiplication at this site. A bacterial artificial chromosome (BAC) library was generated from a derivative of the sequenced E. coli O157:H7 Sakai strain. The library contains 1152 clones averaging 150 kbp. To verify the library, clones containing a complete locus of enterocyte effacement (LEE) were identified by DNA hybridization. In line with a previous report, these did not confer a type III secretion (T3S) capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored T3S. Three hundred eighty-four clones from the library were subjected to two different selective screens; one involved three rounds of adherence assays to bovine primary rectal epithelial cells while the other competed the clones over three rounds of growth in bovine rectal mucus. The input strain DNA was then compared with the selected strains using comparative genomic hybridization (CGH) on an E. coli microarray. The adherence assay enriched for pO157 DNA indicating the importance of this plasmid for colonization of rectal epithelial cells. The mucus assay enriched for multiple regions involved in carbohydrate utilization, including hexuronate uptake, indicating that these regions provide a competitive growth advantage in bovine mucus. This BAC-CGH approach provides a positive selection screen that complements negative selection transposon-based screens. As demonstrated, this may be of particular use for identifying genes with redundant functions such as adhesion and carbon

  16. Characterization of a BAC Library from Channel Catfish Ictalurus punctatus: Indications of High Rates of Evolution Among Teleost Genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The CHORI-212 bacterial artificial chromosome (BAC) library was constructed by cloning EcoRI/EcoRI partially digested DNA into the pTARBAC2.1 vector. The library has an average insert size of 161 kb, and provides 10.6-fold coverage of the channel catfish haploid genome. Screening of 32 genes using o...

  17. Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

    PubMed

    Yang, Tae-Jin; Yu, Yeisoo; Nah, Gyoungju; Atkins, Michael; Lee, Seunghee; Frisch, David A; Wing, Rod A

    2003-08-01

    Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

  18. 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.

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

    PubMed Central

    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. PMID:26909353

  20. Chronic periodontitis genome-wide association studies: gene-centric and gene set enrichment analyses.

    PubMed

    Rhodin, K; Divaris, K; North, K E; Barros, S P; Moss, K; Beck, J D; Offenbacher, S

    2014-09-01

    Recent genome-wide association studies (GWAS) of chronic periodontitis (CP) offer rich data sources for the investigation of candidate genes, functional elements, and pathways. We used GWAS data of CP (n = 4,504) and periodontal pathogen colonization (n = 1,020) from a cohort of adult Americans of European descent participating in the Atherosclerosis Risk in Communities study and employed a MAGENTA approach (i.e., meta-analysis gene set enrichment of variant associations) to obtain gene-centric and gene set association results corrected for gene size, number of single-nucleotide polymorphisms, and local linkage disequilibrium characteristics based on the human genome build 18 (National Center for Biotechnology Information build 36). We used the Gene Ontology, Ingenuity, KEGG, Panther, Reactome, and Biocarta databases for gene set enrichment analyses. Six genes showed evidence of statistically significant association: 4 with severe CP (NIN, p = 1.6 × 10(-7); ABHD12B, p = 3.6 × 10(-7); WHAMM, p = 1.7 × 10(-6); AP3B2, p = 2.2 × 10(-6)) and 2 with high periodontal pathogen colonization (red complex-KCNK1, p = 3.4 × 10(-7); Porphyromonas gingivalis-DAB2IP, p = 1.0 × 10(-6)). Top-ranked genes for moderate CP were HGD (p = 1.4 × 10(-5)), ZNF675 (p = 1.5 × 10(-5)), TNFRSF10C (p = 2.0 × 10(-5)), and EMR1 (p = 2.0 × 10(-5)). Loci containing NIN, EMR1, KCNK1, and DAB2IP had showed suggestive evidence of association in the earlier single-nucleotide polymorphism-based analyses, whereas WHAMM and AP2B2 emerged as novel candidates. The top gene sets included severe CP ("endoplasmic reticulum membrane," "cytochrome P450," "microsome," and "oxidation reduction") and moderate CP ("regulation of gene expression," "zinc ion binding," "BMP signaling pathway," and "ruffle"). Gene-centric analyses offer a promising avenue for efficient interrogation of large-scale GWAS data. These results highlight genes in previously identified loci and new candidate genes and pathways

  1. Pre-capture multiplexing improves efficiency and cost-effectiveness of targeted genomic enrichment

    PubMed Central

    2012-01-01

    Background Targeted genomic enrichment (TGE) is a widely used method for isolating and enriching specific genomic regions prior to massively parallel sequencing. To make effective use of sequencer output, barcoding and sample pooling (multiplexing) after TGE and prior to sequencing (post-capture multiplexing) has become routine. While previous reports have indicated that multiplexing prior to capture (pre-capture multiplexing) is feasible, no thorough examination of the effect of this method has been completed on a large number of samples. Here we compare standard post-capture TGE to two levels of pre-capture multiplexing: 12 or 16 samples per pool. We evaluated these methods using standard TGE metrics and determined the ability to identify several classes of genetic mutations in three sets of 96 samples, including 48 controls. Our overall goal was to maximize cost reduction and minimize experimental time while maintaining a high percentage of reads on target and a high depth of coverage at thresholds required for variant detection. Results We adapted the standard post-capture TGE method for pre-capture TGE with several protocol modifications, including redesign of blocking oligonucleotides and optimization of enzymatic and amplification steps. Pre-capture multiplexing reduced costs for TGE by at least 38% and significantly reduced hands-on time during the TGE protocol. We found that pre-capture multiplexing reduced capture efficiency by 23 or 31% for pre-capture pools of 12 and 16, respectively. However efficiency losses at this step can be compensated by reducing the number of simultaneously sequenced samples. Pre-capture multiplexing and post-capture TGE performed similarly with respect to variant detection of positive control mutations. In addition, we detected no instances of sample switching due to aberrant barcode identification. Conclusions Pre-capture multiplexing improves efficiency of TGE experiments with respect to hands-on time and reagent use compared

  2. Validation of SCALE 4. 0 -- CSAS25 module and the 27-group ENDF/B-IV cross-section library for low-enriched uranium systems

    SciTech Connect

    Jordan, W.C.

    1993-02-01

    A version of KENO V.a and the 27-group library in SCALE-4.0 were validated for use in evaluating the nuclear criticality safety of low-enriched uranium systems. A total of 59 critical systems were analyzed. A statistical analysis of the results was performed, and subcritical acceptanced criteria are established.

  3. Validation of SCALE 4.0 -- CSAS25 module and the 27-group ENDF/B-IV cross-section library for low-enriched uranium systems

    SciTech Connect

    Jordan, W.C.

    1993-02-01

    A version of KENO V.a and the 27-group library in SCALE-4.0 were validated for use in evaluating the nuclear criticality safety of low-enriched uranium systems. A total of 59 critical systems were analyzed. A statistical analysis of the results was performed, and subcritical acceptanced criteria are established.

  4. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    PubMed

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  5. Genome-wide association analysis and pathways enrichment for lactation persistency in Canadian Holstein cattle.

    PubMed

    Do, D N; Bissonnette, N; Lacasse, P; Miglior, F; Sargolzaei, M; Zhao, X; Ibeagha-Awemu, E M

    2017-03-01

    Lactation persistency (LP), defined as the rate of declining milk yield after milk peak, is an economically important trait for dairy cattle. Improving LP is considered a good alternative method for increasing overall milk production because it does not cause the negative energy balance and other health issues that cows experience during peak milk production. However, little is known about the biology of LP. A genome-wide association study (GWAS) and pathway enrichment were used to explore the genetic mechanisms underlying LP. The GWAS was performed using a univariate regression mixed linear model on LP data of 3,796 cows and 44,100 single nucleotide polymorphisms (SNP). Eight and 47 SNP were significantly and suggestively associated with LP, respectively. The 2 most important quantitative trait loci regions for LP were (1) a region from 106 to 108 Mb on Bos taurus autosome (BTA) 5, where the most significant SNP (ARS-BFGL-NGS-2399) was located and also formed a linkage disequilibrium block with 3 other SNP; and (2) a region from 29.3 to 31.3 Mb on BTA 20, which contained 3 significant SNP. Based on physical positions, MAN1C1, MAP3K5, HCN1, TSPAN9, MRPS30, TEX14, and CCL28 are potential candidate genes for LP because the significant SNP were located in their intronic regions. Enrichment analyses of a list of 536 genes in 0.5-Mb flanking regions of significant and suggestive SNP indicates that synthesis of milk components, regulation of cell apoptosis processes and insulin, and prolactin signaling pathways are important for LP. Upstream regulators relevant for LP positional candidate genes were prolactin (PRL), peroxisome proliferator-activated receptor gamma (PPARG), and Erb-B2 receptor tyrosine kinase 2 (ERBB2). Several networks related to cellular development, proliferation and death were significantly enriched for LP positional candidate genes. In conclusion, this study detected several SNP, genes, and interesting regions for fine mapping and validation of

  6. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

  7. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.

    PubMed

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine E; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten

    2017-03-01

    This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

  8. A genome-wide library of CB4856/N2 introgression lines of Caenorhabditis elegans

    PubMed Central

    Doroszuk, Agnieszka; Snoek, L. Basten; Fradin, Emilie; Riksen, Joost; Kammenga, Jan

    2009-01-01

    Recombinant inbred lines (RILs) derived from Caenorhabditis elegans wild-type N2 and CB4856 are increasingly being used for mapping genes underlying complex traits. To speed up mapping and gene discovery, introgression lines (ILs) offer a powerful tool for more efficient QTL identification. We constructed a library of 90 ILs, each carrying a single homozygous CB4856 genomic segment introgressed into the genetic background of N2. The ILs were genotyped by 123 single-nucleotide polymorphism (SNP) markers. The proportion of the CB4856 segments in most lines does not exceed 3%, and together the introgressions cover 96% of the CB4856 genome. The value of the IL library was demonstrated by identifying novel loci underlying natural variation in two ageing-related traits, i.e. lifespan and pharyngeal pumping rate. Bin mapping of lifespan resulted in six QTLs, which all have a lifespan-shortening effect on the CB4856 allele. We found five QTLs for the decrease in pumping rate, of which four colocated with QTLs found for average lifespan. This suggests pleiotropic or closely linked QTL associated with lifespan and pumping rate. Overall, the presented IL library provides a versatile resource toward easier and efficient fine mapping and functional analyses of loci and genes underlying complex traits in C. elegans. PMID:19542186

  9. A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

    PubMed Central

    Liles, Mark R.; Manske, Brian F.; Bintrim, Scott B.; Handelsman, Jo; Goodman, Robert M.

    2003-01-01

    We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division. PMID:12732537

  10. 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.

  11. Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

    PubMed Central

    Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

    2016-01-01

    The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

  12. 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

  13. Genome sequence of Candidatus Nitrososphaera evergladensis from group I.1b enriched from Everglades soil reveals novel genomic features of the ammonia-oxidizing archaea.

    PubMed

    Zhalnina, Kateryna V; Dias, Raquel; Leonard, Michael T; Dorr de Quadros, Patricia; Camargo, Flavio A O; Drew, Jennifer C; Farmerie, William G; Daroub, Samira H; Triplett, Eric W

    2014-01-01

    The activity of ammonia-oxidizing archaea (AOA) leads to the loss of nitrogen from soil, pollution of water sources and elevated emissions of greenhouse gas. To date, eight AOA genomes are available in the public databases, seven are from the group I.1a of the Thaumarchaeota and only one is from the group I.1b, isolated from hot springs. Many soils are dominated by AOA from the group I.1b, but the genomes of soil representatives of this group have not been sequenced and functionally characterized. The lack of knowledge of metabolic pathways of soil AOA presents a critical gap in understanding their role in biogeochemical cycles. Here, we describe the first complete genome of soil archaeon Candidatus Nitrososphaera evergladensis, which has been reconstructed from metagenomic sequencing of a highly enriched culture obtained from an agricultural soil. The AOA enrichment was sequenced with the high throughput next generation sequencing platforms from Pacific Biosciences and Ion Torrent. The de novo assembly of sequences resulted in one 2.95 Mb contig. Annotation of the reconstructed genome revealed many similarities of the basic metabolism with the rest of sequenced AOA. Ca. N. evergladensis belongs to the group I.1b and shares only 40% of whole-genome homology with the closest sequenced relative Ca. N. gargensis. Detailed analysis of the genome revealed coding sequences that were completely absent from the group I.1a. These unique sequences code for proteins involved in control of DNA integrity, transporters, two-component systems and versatile CRISPR defense system. Notably, genomes from the group I.1b have more gene duplications compared to the genomes from the group I.1a. We suggest that the presence of these unique genes and gene duplications may be associated with the environmental versatility of this group.

  14. Genome Sequence of Candidatus Nitrososphaera evergladensis from Group I.1b Enriched from Everglades Soil Reveals Novel Genomic Features of the Ammonia-Oxidizing Archaea

    PubMed Central

    Zhalnina, Kateryna V.; Dias, Raquel; Leonard, Michael T.; Dorr de Quadros, Patricia; Camargo, Flavio A. O.; Drew, Jennifer C.; Farmerie, William G.; Daroub, Samira H.; Triplett, Eric W.

    2014-01-01

    The activity of ammonia-oxidizing archaea (AOA) leads to the loss of nitrogen from soil, pollution of water sources and elevated emissions of greenhouse gas. To date, eight AOA genomes are available in the public databases, seven are from the group I.1a of the Thaumarchaeota and only one is from the group I.1b, isolated from hot springs. Many soils are dominated by AOA from the group I.1b, but the genomes of soil representatives of this group have not been sequenced and functionally characterized. The lack of knowledge of metabolic pathways of soil AOA presents a critical gap in understanding their role in biogeochemical cycles. Here, we describe the first complete genome of soil archaeon Candidatus Nitrososphaera evergladensis, which has been reconstructed from metagenomic sequencing of a highly enriched culture obtained from an agricultural soil. The AOA enrichment was sequenced with the high throughput next generation sequencing platforms from Pacific Biosciences and Ion Torrent. The de novo assembly of sequences resulted in one 2.95 Mb contig. Annotation of the reconstructed genome revealed many similarities of the basic metabolism with the rest of sequenced AOA. Ca. N. evergladensis belongs to the group I.1b and shares only 40% of whole-genome homology with the closest sequenced relative Ca. N. gargensis. Detailed analysis of the genome revealed coding sequences that were completely absent from the group I.1a. These unique sequences code for proteins involved in control of DNA integrity, transporters, two-component systems and versatile CRISPR defense system. Notably, genomes from the group I.1b have more gene duplications compared to the genomes from the group I.1a. We suggest that the presence of these unique genes and gene duplications may be associated with the environmental versatility of this group. PMID:24999826

  15. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

    PubMed Central

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

    2015-01-01

    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. PMID:26165496

  16. 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.

  17. Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

    PubMed Central

    Weichhart, Thomas; Horky, Markus; Söllner, Johannes; Gangl, Susanne; Henics, Tamàs; Nagy, Eszter; Meinke, Andreas; von Gabain, Alexander; Fraser, Claire M.; Gill, Steve R.; Hafner, Martin; von Ahsen, Uwe

    2003-01-01

    An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens. PMID:12874343

  18. Construction and analysis of Pst I DNA library for RFLP mapping of the rye genome

    SciTech Connect

    Korzun, V.N.; Kartel, N.A.; Boerner, A.

    1995-06-01

    Pst I, a methylation-sensitive restriction enzyme, was used for producing a library of rye genome DNA rich in low-copy sequences, and intended as probes for genetic mapping. Dot-hybridization and Southern blot analysis showed that 43.6% of the library is represented by low-copy DNA sequences. To locate these sequences on chromosomes and determine the degree of their repetitiveness, 11 clones were hybridized with DNA of nulli-tetrasomic lines of Chinese Spring wheat, wheat-rye addition lines, and barley cleaved by Hind III, EcoR I, EcoR V, Dra I, and BamH I restriction enzymes. Each of the rye DNA clones studied hybridized with wheat and barley DNA, suggesting that low-copy Pst I clones of rye correspond to the evolutionary conservative DNA fraction in cereals. 21 refs., 3 figs., 2 tabs.

  19. Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

    PubMed Central

    Ozga, Andrew T.; Nieves‐Colón, Maria A.; Honap, Tanvi P.; Sankaranarayanan, Krithivasan; Hofman, Courtney A.; Milner, George R.; Lewis, Cecil M.; Stone, Anne C.

    2016-01-01

    ABSTRACT Objectives Archaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Materials and Methods Extracted DNA from six individuals at the 700‐year‐old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in‐solution capture techniques, followed by Illumina high‐throughput sequencing. Results Full mitogenomes (7–34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Discussion Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220–228, 2016. © 2016 The Authors American Journal of Physical Anthropology

  20. Microsatellite markers isolated from Cabomba aquatica s.l. (Cabombaceae) from an enriched genomic library1

    PubMed Central

    Barbosa, Tiago D. M.; Trad, Rafaela J.; Bajay, Miklos M.; Amaral, Maria C. E.

    2015-01-01

    Premise of the study: Microsatellite primers were designed for the submersed aquatic plant Cabomba aquatica s.l. (Cabombaceae) and characterized to estimate genetic diversity parameters. Methods and Results: Using a selective hybridization method, we designed and tested 30 simple sequence repeat loci using two natural populations of C. aquatica s.l., resulting in 13 amplifiable loci. Twelve loci were polymorphic, and alleles per locus ranged from two to four across the 49 C. aquatica s.l. individuals. Observed heterozygosity, expected heterozygosity, and fixation index varied from 0.0 to 1.0, 0.0 to 0.5, and −1.0 to −0.0667, respectively, for the Manaus population and from 0.0 to 1.0, 0.0 to 0.6, and −1.0 to 0.4643 for the Viruá population. Conclusions: The developed markers will be used in further taxonomic and population studies within Cabomba. This set of microsatellite primers represents the first report on rapid molecular markers in the genus. PMID:26649271

  1. Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences.

    PubMed

    Vardi, Oriya; Shamir, Inbal; Javasky, Elisheva; Goren, Alon; Simon, Itamar

    2017-01-01

    Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5' end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.

  2. Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences

    PubMed Central

    Shamir, Inbal; Javasky, Elisheva; Goren, Alon; Simon, Itamar

    2017-01-01

    Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5′ end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation. PMID:28235101

  3. Alzheimer's Disease Variants with the Genome-Wide Significance are Significantly Enriched in Immune Pathways and Active in Immune Cells.

    PubMed

    Jiang, Qinghua; Jin, Shuilin; Jiang, Yongshuai; Liao, Mingzhi; Feng, Rennan; Zhang, Liangcai; Liu, Guiyou; Hao, Junwei

    2017-01-01

    The existing large-scale genome-wide association studies (GWAS) datasets provide strong support for investigating the mechanisms of Alzheimer's disease (AD) by applying multiple methods of pathway analysis. Previous studies using selected single nucleotide polymorphisms (SNPs) with several thresholds of nominal significance for pathway analysis determined that the threshold chosen for SNPs can reflect the disease model. Presumably, then, pathway analysis with a stringent threshold to define "associated" SNPs would test the hypothesis that highly associated SNPs are enriched in one or more particular pathways. Here, we selected 599 AD variants (P < 5.00E-08) to investigate the pathways in which these variants are enriched and the cell types in which these variants are active. Our results showed that AD variants are significantly enriched in pathways of the immune system. Further analysis indicated that AD variants are significantly enriched for enhancers in a number of cell types, in particular the B-lymphocyte, which is the most substantially enriched cell type. This cell type maintains its dominance among the strongest enhancers. AD SNPs also display significant enrichment for DNase in 12 cell types, among which the top 6 significant signals are from immune cell types, including 4 B cells (top 4 significant signals) and CD14+ and CD34+ cells. In summary, our results show that these AD variants with P < 5.00E-08 are significantly enriched in pathways of the immune system and active in immune cells. To a certain degree, the genetic predisposition for development of AD is rooted in the immune system, rather than in neuronal cells.

  4. Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA.

    PubMed

    Gallagher, Ryan R; Li, Zhe; Lewis, Aaron O; Isaacs, Farren J

    2014-10-01

    Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. Alternatively, MAGE can introduce precise mutant alleles at many loci for genome-wide editing or for recoding projects that are not possible with other methods. In recent technological advances, MAGE has been improved by strain modifications and selection techniques that enhance allelic replacement. This protocol describes the manual execution of MAGE wherein each cycle takes ≈ 2.5 h, which, if carried out by two people, allows ≈ 10 continuous cycles of MAGE-based mutagenesis per day.

  5. Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.

    PubMed

    Goodman, Andrew L; Wu, Meng; Gordon, Jeffrey I

    2011-11-17

    Insertion sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16-17 bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18 h), easy to scale up, amenable to automation and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in a multiwell format is provided.

  6. Draft Genome Sequence of Paenibacillus sp. Strain DMB5, Acclimatized and Enriched for Catabolizing Anthropogenic Compounds

    PubMed Central

    Johnson, Jenny; Shah, Binal; Jain, Kunal; Parmar, Nidhi; Hinsu, Ankit; Patel, Namrata

    2016-01-01

    Here, we present the draft genome sequence of Paenibacillus sp. strain DMB5, isolated from polluted sediments of the Kharicut Canal, Vatva, India, having a genome size of 7.5 Mbp and 7,077 coding sequences. The genome of this dye-degrading bacterium provides valuable information on the microbe-mediated biodegradation of anthropogenic compounds. PMID:27034501

  7. 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

  8. Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

    PubMed Central

    James, P.; Halladay, J.; Craig, E. A.

    1996-01-01

    The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections. PMID:8978031

  9. High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.

    PubMed

    Reimer, Lorenz C; Spura, Jana; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known

  10. A kingdom-specific protein domain HMM library for improved annotation of fungal genomes

    PubMed Central

    Alam, Intikhab; Hubbard, Simon J; Oliver, Stephen G; Rattray, Magnus

    2007-01-01

    Background Pfam is a general-purpose database of protein domain alignments and profile Hidden Markov Models (HMMs), which is very popular for the annotation of sequence data produced by genome sequencing projects. Pfam provides models that are often very general in terms of the taxa that they cover and it has previously been suggested that such general models may lack some of the specificity or selectivity that would be provided by kingdom-specific models. Results Here we present a general approach to create domain libraries of HMMs for sub-taxa of a kingdom. Taking fungal species as an example, we construct a domain library of HMMs (called Fungal Pfam or FPfam) using sequences from 30 genomes, consisting of 24 species from the ascomycetes group and two basidiomycetes, Ustilago maydis, a fungal pathogen of maize, and the white rot fungus Phanerochaete chrysosporium. In addition, we include the Microsporidion Encephalitozoon cuniculi, an obligate intracellular parasite, and two non-fungal species, the oomycetes Phytophthora sojae and Phytophthora ramorum, both plant pathogens. We evaluate the performance in terms of coverage against the original 30 genomes used in training FPfam and against five more recently sequenced fungal genomes that can be considered as an independent test set. We show that kingdom-specific models such as FPfam can find instances of both novel and well characterized domains, increases overall coverage and detects more domains per sequence with typically higher bitscores than Pfam for the same domain families. An evaluation of the effect of changing E-values on the coverage shows that the performance of FPfam is consistent over the range of E-values applied. Conclusion Kingdom-specific models are shown to provide improved coverage. However, as the models become more specific, some sequences found by Pfam may be missed by the models in FPfam and some of the families represented in the test set are not present in FPfam. Therefore, we recommend

  11. Exon-Enriched Libraries Reveal Large Genic Differences Between Aedes aegypti from Senegal, West Africa, and Populations Outside Africa.

    PubMed

    Dickson, Laura B; Campbell, Corey L; Juneja, Punita; Jiggins, Francis M; Sylla, Massamba; Black, William C

    2017-02-09

    Aedes aegypti is one of the most studied mosquito species, and the principal vector of several arboviruses pathogenic to humans. Recently failure to oviposit, low fecundity, and poor egg-to-adult survival were observed when Ae. aegypti from Senegal (SenAae) West Africa were crossed with Ae. aegypti (Aaa) from outside of Africa, and in SenAae intercrosses. Fluorescent in situ hybridization analyses indicated rearrangements on chromosome 1, and pericentric inversions on chromosomes 2 and 3. Herein, high throughput sequencing (HTS) of exon-enriched libraries was used to compare chromosome-wide genetic diversity among Aaa collections from rural Thailand and Mexico, a sylvatic collection from southeastern Senegal (PK10), and an urban collection from western Senegal (Kaolack). Sex-specific polymorphisms were analyzed in Thailand and PK10 to assess genetic differences between sexes. Expected heterozygosity was greatest in SenAae FST distributions of 15,735 genes among all six pairwise comparisons of the four collections indicated that Mexican and Thailand collections are genetically similar, while FST distributions between PK10 and Kaolack were distinct. All four comparisons of SenAae with Aaa indicated extreme differentiation. FST was uniform between sexes across all chromosomes in Thailand, but were different, especially on the sex autosome 1, in PK10. These patterns correlate with the reproductive isolation noted earlier. We hypothesize that cryptic Ae. aegypti taxa may exist in West Africa, and the large genic differences between Aaa and SenAae detected in the present study have accumulated over a long period following the evolution of chromosome rearrangements in allopatric populations that subsequently cause reproductive isolation when these populations became sympatric.

  12. Exon-Enriched Libraries Reveal Large Genic Differences Between Aedes aegypti from Senegal, West Africa, and Populations Outside Africa

    PubMed Central

    Dickson, Laura B.; Campbell, Corey L.; Juneja, Punita; Jiggins, Francis M.; Sylla, Massamba; Black, William C.

    2016-01-01

    Aedes aegypti is one of the most studied mosquito species, and the principal vector of several arboviruses pathogenic to humans. Recently failure to oviposit, low fecundity, and poor egg-to-adult survival were observed when Ae. aegypti from Senegal (SenAae) West Africa were crossed with Ae. aegypti (Aaa) from outside of Africa, and in SenAae intercrosses. Fluorescent in situ hybridization analyses indicated rearrangements on chromosome 1, and pericentric inversions on chromosomes 2 and 3. Herein, high throughput sequencing (HTS) of exon-enriched libraries was used to compare chromosome-wide genetic diversity among Aaa collections from rural Thailand and Mexico, a sylvatic collection from southeastern Senegal (PK10), and an urban collection from western Senegal (Kaolack). Sex-specific polymorphisms were analyzed in Thailand and PK10 to assess genetic differences between sexes. Expected heterozygosity was greatest in SenAae. FST distributions of 15,735 genes among all six pairwise comparisons of the four collections indicated that Mexican and Thailand collections are genetically similar, while FST distributions between PK10 and Kaolack were distinct. All four comparisons of SenAae with Aaa indicated extreme differentiation. FST was uniform between sexes across all chromosomes in Thailand, but were different, especially on the sex autosome 1, in PK10. These patterns correlate with the reproductive isolation noted earlier. We hypothesize that cryptic Ae. aegypti taxa may exist in West Africa, and the large genic differences between Aaa and SenAae detected in the present study have accumulated over a long period following the evolution of chromosome rearrangements in allopatric populations that subsequently cause reproductive isolation when these populations became sympatric. PMID:28007834

  13. A support vector machines approach for virtual screening of active compounds of single and multiple mechanisms from large libraries at an improved hit-rate and enrichment factor.

    PubMed

    Han, L Y; Ma, X H; Lin, H H; Jia, J; Zhu, F; Xue, Y; Li, Z R; Cao, Z W; Ji, Z L; Chen, Y Z

    2008-06-01

    Support vector machines (SVM) and other machine-learning (ML) methods have been explored as ligand-based virtual screening (VS) tools for facilitating lead discovery. While exhibiting good hit selection performance, in screening large compound libraries, these methods tend to produce lower hit-rate than those of the best performing VS tools, partly because their training-sets contain limited spectrum of inactive compounds. We tested whether the performance of SVM can be improved by using training-sets of diverse inactive compounds. In retrospective database screening of active compounds of single mechanism (HIV protease inhibitors, DHFR inhibitors, dopamine antagonists) and multiple mechanisms (CNS active agents) from large libraries of 2.986 million compounds, the yields, hit-rates, and enrichment factors of our SVM models are 52.4-78.0%, 4.7-73.8%, and 214-10,543, respectively, compared to those of 62-95%, 0.65-35%, and 20-1200 by structure-based VS and 55-81%, 0.2-0.7%, and 110-795 by other ligand-based VS tools in screening libraries of >or=1 million compounds. The hit-rates are comparable and the enrichment factors are substantially better than the best results of other VS tools. 24.3-87.6% of the predicted hits are outside the known hit families. SVM appears to be potentially useful for facilitating lead discovery in VS of large compound libraries.

  14. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    PubMed

    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.

  15. 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

  16. Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis

    SciTech Connect

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

    2002-02-26

    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 and 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.

  17. Chromosome region-specific libraries for human genome analysis. Final progress report, 1 March 1991--28 February 1994

    SciTech Connect

    Kao, F.T.

    1994-04-01

    The objectives of this grant proposal include (1) development of a chromosome microdissection and PCR-mediated microcloning technology, (2) application of this microtechnology to the construction of region-specific libraries for human genome analysis. During this grant period, the authors have successfully developed this microtechnology and have applied it to the construction of microdissection libraries for the following chromosome regions: a whole chromosome 21 (21E), 2 region-specific libraries for the long arm of chromosome 2, 2q35-q37 (2Q1) and 2q33-q35 (2Q2), and 4 region-specific libraries for the entire short arm of chromosome 2, 2p23-p25 (2P1), 2p21-p23 (2P2), 2p14-p16 (wP3) and 2p11-p13 (2P4). In addition, 20--40 unique sequence microclones have been isolated and characterized for genomic studies. These region-specific libraries and the single-copy microclones from the library have been used as valuable resources for (1) isolating microsatellite probes in linkage analysis to further refine the disease locus; (2) isolating corresponding clones with large inserts, e.g. YAC, BAC, P1, cosmid and phage, to facilitate construction of contigs for high resolution physical mapping; and (3) isolating region-specific cDNA clones for use as candidate genes. These libraries are being deposited in the American Type Culture Collection (ATCC) for general distribution.

  18. Characterization of Uncultured Genome Fragment from Soil Metagenomic Library Exposed Rare Mismatch of Internal Tetranucleotide Frequency

    PubMed Central

    Liu, Yunpeng; Yang, Dongqing; Zhang, Nan; Chen, Lin; Cui, Zhongli; Shen, Qirong; Zhang, Ruifu

    2016-01-01

    Exploring the genomic information of a specific uncultured soil bacterium is vital to understand its function in the ecosystem but is still a challenge due to the lack of culture techniques. To examine the genomes of uncultured bacteria, a metagenomic bacterial artificial chromosome library derived from a soil sample was screened for 16S rDNA-containing clones. Five clones (4C6, 5E7, 5G4, 5G12, and 5H7) containing uncultured soil bacteria genome fragment (with low 16S rDNA similarity to isolated bacteria) were selected for sequencing. Clone 5E7 and 5G4 showed only 82 and 83% of 16S rDNA identity to known sequences. Phylogenetic analysis of 16S rDNA indicated that 5E7 and 5G4 were potentially from new class of Chloroflexi. Only one-third of the 5G4 open reading frames have significant hits against HMMER. Internal tetranucleotide frequency analysis indicated that the unknown region of 5G4 was poorly correlated with other parts of the clone, indicating that this section might be obtained through lateral transfer. It was suggested that this region rich for unknown genes is under fast evolution. PMID:28066395

  19. Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

    PubMed

    Miyata, Haruhiko; Castaneda, Julio M; Fujihara, Yoshitaka; Yu, Zhifeng; Archambeault, Denise R; Isotani, Ayako; Kiyozumi, Daiji; Kriseman, Maya L; Mashiko, Daisuke; Matsumura, Takafumi; Matzuk, Ryan M; Mori, Masashi; Noda, Taichi; Oji, Asami; Okabe, Masaru; Prunskaite-Hyyrylainen, Renata; Ramirez-Solis, Ramiro; Satouh, Yuhkoh; Zhang, Qian; Ikawa, Masahito; Matzuk, Martin M

    2016-07-12

    Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

  20. Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

    PubMed

    Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

    2017-02-03

    This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene

  1. Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

    PubMed Central

    Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W.; Gallardo-Escarate, Cristian; Planas, Josep V.; Mackenzie, Simon

    2017-01-01

    This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene

  2. SiRNA sequence model: redesign algorithm based on available genome-wide libraries.

    PubMed

    Kozak, Karol

    2013-12-01

    The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

  3. USE OF COMPETITIVE GENOMIC HYBRIDIZATION TO ENRICH FOR GENOME-SPECIFIC DIFFERENCES BETWEEN TWO CLOSELY RELATED HUMAN FECAL INDICATOR BACTERIA

    EPA Science Inventory

    Enterococci are frequently used as indicators of fecal pollution in surface waters. To accelerate the identification of Enterococcus faecalis-specific DNA sequences, we employed a comparative genomic strategy utilizing a positive selection process to compare E. faec...

  4. IDENTIFICATION OF AVIAN-SPECIFIC FECAL METAGENOMIC SEQUENCES USING GENOME FRAGMENT ENRICHMENTS

    EPA Science Inventory

    Sequence analysis of microbial genomes has provided biologists the opportunity to compare genetic differences between closely related microorganisms. While random sequencing has also been used to study natural microbial communities, metagenomic comparisons via sequencing analysis...

  5. Enriching public descriptions of marine phages using the Genomic Standards Consortium MIGS standard.

    PubMed

    Duhaime, Melissa Beth; Kottmann, Renzo; Field, Dawn; Glöckner, Frank Oliver

    2011-04-29

    In any sequencing project, the possible depth of comparative analysis is determined largely by the amount and quality of the accompanying contextual data. The structure, content, and storage of this contextual data should be standardized to ensure consistent coverage of all sequenced entities and facilitate comparisons. The Genomic Standards Consortium (GSC) has developed the "Minimum Information about Genome/Metagenome Sequences (MIGS/MIMS)" checklist for the description of genomes and here we annotate all 30 publicly available marine bacteriophage sequences to the MIGS standard. These annotations build on existing International Nucleotide Sequence Database Collaboration (INSDC) records, and confirm, as expected that current submissions lack most MIGS fields. MIGS fields were manually curated from the literature and placed in XML format as specified by the Genomic Contextual Data Markup Language (GCDML). These "machine-readable" reports were then analyzed to highlight patterns describing this collection of genomes. Completed reports are provided in GCDML. This work represents one step towards the annotation of our complete collection of genome sequences and shows the utility of capturing richer metadata along with raw sequences.

  6. Enriching public descriptions of marine phages using the Genomic Standards Consortium MIGS standard

    PubMed Central

    Duhaime, Melissa Beth; Kottmann, Renzo; Field, Dawn; Glöckner, Frank Oliver

    2011-01-01

    In any sequencing project, the possible depth of comparative analysis is determined largely by the amount and quality of the accompanying contextual data. The structure, content, and storage of this contextual data should be standardized to ensure consistent coverage of all sequenced entities and facilitate comparisons. The Genomic Standards Consortium (GSC) has developed the “Minimum Information about Genome/Metagenome Sequences (MIGS/MIMS)” checklist for the description of genomes and here we annotate all 30 publicly available marine bacteriophage sequences to the MIGS standard. These annotations build on existing International Nucleotide Sequence Database Collaboration (INSDC) records, and confirm, as expected that current submissions lack most MIGS fields. MIGS fields were manually curated from the literature and placed in XML format as specified by the Genomic Contextual Data Markup Language (GCDML). These “machine-readable” reports were then analyzed to highlight patterns describing this collection of genomes. Completed reports are provided in GCDML. This work represents one step towards the annotation of our complete collection of genome sequences and shows the utility of capturing richer metadata along with raw sequences. PMID:21677864

  7. Integrating molecular QTL data into genome-wide genetic association analysis: Probabilistic assessment of enrichment and colocalization

    PubMed Central

    2017-01-01

    We propose a novel statistical framework for integrating the result from molecular quantitative trait loci (QTL) mapping into genome-wide genetic association analysis of complex traits, with the primary objectives of quantitatively assessing the enrichment of the molecular QTLs in complex trait-associated genetic variants and the colocalizations of the two types of association signals. We introduce a natural Bayesian hierarchical model that treats the latent association status of molecular QTLs as SNP-level annotations for candidate SNPs of complex traits. We detail a computational procedure to seamlessly perform enrichment, fine-mapping and colocalization analyses, which is a distinct feature compared to the existing colocalization analysis procedures in the literature. The proposed approach is computationally efficient and requires only summary-level statistics. We evaluate and demonstrate the proposed computational approach through extensive simulation studies and analyses of blood lipid data and the whole blood eQTL data from the GTEx project. In addition, a useful utility from our proposed method enables the computation of expected colocalization signals using simple characteristics of the association data. Using this utility, we further illustrate the importance of enrichment analysis on the ability to discover colocalized signals and the potential limitations of currently available molecular QTL data. The software pipeline that implements the proposed computation procedures, enloc, is freely available at https://github.com/xqwen/integrative. PMID:28278150

  8. Genomic Contributors to Rhythm Outcome of Atrial Fibrillation Catheter Ablation – Pathway Enrichment Analysis of GWAS Data

    PubMed Central

    Ueberham, Laura; Dinov, Borislav; Sommer, Philipp; Arya, Arash; Hindricks, Gerhard; Bollmann, Andreas

    2016-01-01

    Background Left atrial enlargement and persistent atrial fibrillation (AF) are well-known predictors for arrhythmia recurrence after AF catheter ablation (LRAF). In this study, by using pathway enrichment analysis of GWAS data, we tested the hypothesis that genetic pathways associated with these phenotypes are also associated with LRAF. Methods Samples from 660 patients with paroxysmal (n = 370) or persistent AF (n = 290) undergoing de-novo AF catheter ablation were genotyped for ~1,000,000 SNPs. SNPs found to be significantly associated with left atrial diameter (LAD) or AF type were used for gene-based association tests in a systematic biological Knowledge-based mining system for Genome-wide Genetic studies (KGG). Associated genes were tested for pathway enrichment using WEB-based Gene SeT AnaLysis Toolkit (WebGestalt), the Gene Annotation Tool to Help Explain Relationships (GATHER) and the databases provided by Kyoto Encyclopedia of Genes and Genomes (KEGG). In a second step, the association of consistently enriched pathways and LRAF was tested. Results By using sequential 7-day Holter ECGs, LRAF between 3 and 12 months was observed in 48% and was associated with LAD (B = 1.801, 95% CI 0.760–2.841, p = 1.0E-3) and persistent AF (OR = 2.1; 95% CI 1.567–2.931, p = 2.0E-6). WebGestalt (adj. p = 2.7E-22) and GATHER (adj. p = 5.2E-3) identified the calcium signaling pathway (hsa04020) as the only consistently enriched pathway for LAD, while the extracellular matrix (ECM) -receptor interaction pathway (hsa04512) was the only consistently enriched pathway for AF type (adj. p = 2.1E-15 in WebGestalt; adj. p = 9.3E-4 in GATHER). Both calcium signaling (adj. p = 2.2E-17 in WebGestalt; adj. p = 2.9E-2 in GATHER) and ECM-receptor interaction (adj. p = 1.2E-10 in WebGestalt; adj. p = 2.9E-2 in GATHER) were significantly associated with LRAF. Conclusions Calcium signaling and ECM-receptor interaction pathways are associated with LAD and AF type and, in turn, with LRAF

  9. The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza.

    PubMed

    Ammiraju, Jetty S S; Luo, Meizhong; Goicoechea, José L; Wang, Wenming; Kudrna, Dave; Mueller, Christopher; Talag, Jayson; Kim, HyeRan; Sisneros, Nicholas B; Blackmon, Barbara; Fang, Eric; Tomkins, Jeffery B; Brar, Darshan; MacKill, David; McCouch, Susan; Kurata, Nori; Lambert, Georgina; Galbraith, David W; Arumuganathan, K; Rao, Kiran; Walling, Jason G; Gill, Navdeep; Yu, Yeisoo; SanMiguel, Phillip; Soderlund, Carol; Jackson, Scott; Wing, Rod A

    2006-01-01

    Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

  10. Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

    PubMed

    Krueger, R W; Holland, M A; Chisholm, D; Polacco, J C

    1987-01-01

    We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

  11. Genome-wide analyses in bacteria show small-RNA enrichment for long and conserved intergenic regions.

    PubMed

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael; Contreras, Lydia M

    2015-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5' and 3' untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms.

  12. Genome-Wide Analyses in Bacteria Show Small-RNA Enrichment for Long and Conserved Intergenic Regions

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael

    2014-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5′ and 3′ untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms. PMID:25313390

  13. Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.

    PubMed

    Waterfield, Nicholas R; Sanchez-Contreras, Maria; Eleftherianos, Ioannis; Dowling, Andrea; Yang, Guowei; Wilkinson, Paul; Parkhill, Julian; Thomson, Nicholas; Reynolds, Stuart E; Bode, Helge B; Dorus, Steven; Ffrench-Constant, Richard H

    2008-10-14

    Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

  14. Genome wide analysis of Silurana (Xenopus) tropicalis development reveals dynamic expression using network enrichment analysis.

    PubMed

    Langlois, Valérie S; Martyniuk, Christopher J

    2013-01-01

    Development involves precise timing of gene expression and coordinated pathways for organogenesis and morphogenesis. Functional and sub-network enrichment analysis provides an integrated approach for identifying networks underlying development. The objectives of this study were to characterize early gene regulatory networks over Silurana tropicalis development from NF stage 2 to 46 using a custom Agilent 4×44K microarray. There were >8000 unique gene probes that were differentially expressed between Nieuwkoop-Faber (NF) stage 2 and stage 16, and >2000 gene probes differentially expressed between NF 34 and 46. Gene ontology revealed that genes involved in nucleosome assembly, cell division, pattern specification, neurotransmission, and general metabolism were increasingly regulated throughout development, consistent with active development. Sub-network enrichment analysis revealed that processes such as membrane hyperpolarisation, retinoic acid, cholesterol, and dopamine metabolic gene networks were activated/inhibited over time. This study identifies RNA transcripts that are potentially maternally inherited in an anuran species, provides evidence that the expression of genes involved in retinoic acid receptor signaling may increase prior to those involved in thyroid receptor signaling, and characterizes novel gene expression networks preceding organogenesis which increases understanding of the spatiotemporal embryonic development in frogs.

  15. Identification of Susceptible Loci and Enriched Pathways for Bipolar II Disorder Using Genome-Wide Association Studies

    PubMed Central

    Kao, Chung-Feng; Chen, Hui-Wen; Chen, Hsi-Chung; Yang, Jenn-Hwai; Huang, Ming-Chyi; Chiu, Yi-Hang; Lin, Shih-Ku; Lee, Ya-Chin; Liu, Chih-Min; Chuang, Li-Chung; Chen, Chien-Hsiun; Wu, Jer-Yuarn

    2016-01-01

    Background: This study aimed to identify susceptible loci and enriched pathways for bipolar disorder subtype II. Methods: We conducted a genome-wide association scan in discovery samples with 189 bipolar disorder subtype II patients and 1773 controls, and replication samples with 283 bipolar disorder subtype II patients and 500 controls in a Taiwanese Han population using Affymetrix Axiom Genome-Wide CHB1 Array. We performed single-marker and gene-based association analyses, as well as calculated polygeneic risk scores for bipolar disorder subtype II. Pathway enrichment analyses were employed to reveal significant biological pathways. Results: Seven markers were found to be associated with bipolar disorder subtype II in meta-analysis combining both discovery and replication samples (P<5.0×10–6), including markers in or close to MYO16, HSP90AB3P, noncoding gene LOC100507632, and markers in chromosomes 4 and 10. A novel locus, ETF1, was associated with bipolar disorder subtype II (P<6.0×10–3) in gene-based association tests. Results of risk evaluation demonstrated that higher genetic risk scores were able to distinguish bipolar disorder subtype II patients from healthy controls in both discovery (P=3.9×10–4~1.0×10–3) and replication samples (2.8×10–4~1.7×10–3). Genetic variance explained by chip markers for bipolar disorder subtype II was substantial in the discovery (55.1%) and replication (60.5%) samples. Moreover, pathways related to neurodevelopmental function, signal transduction, neuronal system, and cell adhesion molecules were significantly associated with bipolar disorder subtype II. Conclusion: We reported novel susceptible loci for pure bipolar subtype II disorder that is less addressed in the literature. Future studies are needed to confirm the roles of these loci for bipolar disorder subtype II. PMID:27450446

  16. A Genomic and Protein-Protein Interaction Analyses of Nonsyndromic Hearing Impairment in Cameroon Using Targeted Genomic Enrichment and Massively Parallel Sequencing.

    PubMed

    Lebeko, Kamogelo; Manyisa, Noluthando; Chimusa, Emile R; Mulder, Nicola; Dandara, Collet; Wonkam, Ambroise

    2017-02-01

    Hearing impairment (HI) is one of the leading causes of disability in the world, impacting the social, economic, and psychological well-being of the affected individual. This is particularly true in sub-Saharan Africa, which carries one of the highest burdens of this condition. Despite this, there are limited data on the most prevalent genes or mutations that cause HI among sub-Saharan Africans. Next-generation technologies, such as targeted genomic enrichment and massively parallel sequencing, offer new promise in this context. This study reports, for the first time to the best of our knowledge, on the prevalence of novel mutations identified through a platform of 116 HI genes (OtoSCOPE(®)), among 82 African probands with HI. Only variants OTOF NM_194248.2:c.766-2A>G and MYO7A NM_000260.3:c.1996C>T, p.Arg666Stop were found in 3 (3.7%) and 5 (6.1%) patients, respectively. In addition and uniquely, the analysis of protein-protein interactions (PPI), through interrogation of gene subnetworks, using a custom script and two databases (Enrichr and PANTHER), and an algorithm in the igraph package of R, identified the enrichment of sensory perception and mechanical stimulus biological processes, and the most significant molecular functions of these variants pertained to binding or structural activity. Furthermore, 10 genes (MYO7A, MYO6, KCTD3, NUMA1, MYH9, KCNQ1, UBC, DIAPH1, PSMC2, and RDX) were identified as significant hubs within the subnetworks. Results reveal that the novel variants identified among familial cases of HI in Cameroon are not common, and PPI analysis has highlighted the role of 10 genes, potentially important in understanding HI genomics among Africans.

  17. Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome.

    PubMed

    Gu, Zhuoya; Jin, Ke; Crabbe, M James C; Zhang, Yang; Liu, Xiaolin; Huang, Yanyan; Hua, Mengyi; Nan, Peng; Zhang, Zhaolei; Zhong, Yang

    2016-04-01

    Transposable elements (TEs) have no longer been totally considered as "junk DNA" for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r = 0.9, P < 2.2 × 10(16); IMR90 fibroblasts: r = 0.94, P < 2.2 × 10(16)) and also have a significant positive correlation with some remote functional DNA elements like enhancers and promoters (Enhancer: hESC: r = 0.997, P = 2.3 × 10(-4); IMR90: r = 0.934, P = 2 × 10(-2); Promoter: hESC: r = 0.995, P = 3.8 × 10(-4); IMR90: r = 0.996, P = 3.2 × 10(-4)). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes.

  18. Genome-wide identification of splicing QTLs in the human brain and their enrichment among schizophrenia-associated loci

    PubMed Central

    Takata, Atsushi; Matsumoto, Naomichi; Kato, Tadafumi

    2017-01-01

    Detailed analyses of transcriptome have revealed complexity in regulation of alternative splicing (AS). These AS events often undergo modulation by genetic variants. Here we analyse RNA-sequencing data of prefrontal cortex from 206 individuals in combination with their genotypes and identify cis-acting splicing quantitative trait loci (sQTLs) throughout the genome. These sQTLs are enriched among exonic and H3K4me3-marked regions. Moreover, we observe significant enrichment of sQTLs among disease-associated loci identified by GWAS, especially in schizophrenia risk loci. Closer examination of each schizophrenia-associated loci revealed four regions (each encompasses NEK4, FXR1, SNAP91 or APOPT1), where the index SNP in GWAS is in strong linkage disequilibrium with sQTL SNP(s), suggesting dysregulation of AS as the underlying mechanism of the association signal. Our study provides an informative resource of sQTL SNPs in the human brain, which can facilitate understanding of the genetic architecture of complex brain disorders such as schizophrenia. PMID:28240266

  19. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome.

    PubMed

    Cui, Pin; Löber, Ulrike; Alquezar-Planas, David E; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N; Lenz, Dorina; Helgen, Kristofer M; Roca, Alfred L; Hartman, Stefanie; Greenwood, Alex D

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small.

  20. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    PubMed Central

    Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  1. Towards a Library of Standard Operating Procedures (SOPs) for (meta)genomic annotation

    SciTech Connect

    Kyrpides, Nikos; Angiuoli, Samuel V.; Cochrane, Guy; Field, Dawn; Garrity, George; Gussman, Aaron; Kodira, Chinnappa D.; Klimke, William; Kyrpides, Nikos; Madupu, Ramana; Markowitz, Victor; Tatusova, Tatiana; Thomson, Nick; White, Owen

    2008-04-01

    Genome annotations describe the features of genomes and accompany sequences in genome databases. The methodologies used to generate genome annotation are diverse and typically vary amongst groups. Descriptions of the annotation procedure are helpful in interpreting genome annotation data. Standard Operating Procedures (SOPs) for genome annotation describe the processes that generate genome annotations. Some groups are currently documenting procedures but standards are lacking for structure and content of annotation SOPs. In addition, there is no central repository to store and disseminate procedures and protocols for genome annotation. We highlight the importance of SOPs for genome annotation and endorse a central online repository of SOPs.

  2. Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis M-135

    NASA Astrophysics Data System (ADS)

    Yoshikazu, Kawata; Shin-Ichi, Yano; Hiroyuki, Kojima

    1998-03-01

    An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends with Taq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase gene crt B from Spirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

  3. Genome-Wide Association Studies Suggest Limited Immune Gene Enrichment in Schizophrenia Compared to 5 Autoimmune Diseases.

    PubMed

    Pouget, Jennie G; Gonçalves, Vanessa F; Spain, Sarah L; Finucane, Hilary K; Raychaudhuri, Soumya; Kennedy, James L; Knight, Jo

    2016-09-01

    There has been intense debate over the immunological basis of schizophrenia, and the potential utility of adjunct immunotherapies. The major histocompatibility complex is consistently the most powerful region of association in genome-wide association studies (GWASs) of schizophrenia and has been interpreted as strong genetic evidence supporting the immune hypothesis. However, global pathway analyses provide inconsistent evidence of immune involvement in schizophrenia, and it remains unclear whether genetic data support an immune etiology per se. Here we empirically test the hypothesis that variation in immune genes contributes to schizophrenia. We show that there is no enrichment of immune loci outside of the MHC region in the largest genetic study of schizophrenia conducted to date, in contrast to 5 diseases of known immune origin. Among 108 regions of the genome previously associated with schizophrenia, we identify 6 immune candidates (DPP4, HSPD1, EGR1, CLU, ESAM, NFATC3) encoding proteins with alternative, nonimmune roles in the brain. While our findings do not refute evidence that has accumulated in support of the immune hypothesis, they suggest that genetically mediated alterations in immune function may not play a major role in schizophrenia susceptibility. Instead, there may be a role for pleiotropic effects of a small number of immune genes that also regulate brain development and plasticity. Whether immune alterations drive schizophrenia progression is an important question to be addressed by future research, especially in light of the growing interest in applying immunotherapies in schizophrenia.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  5. Genome Sequence of Halomonas sp. Strain KO116, an Ionic Liquid- Tolerant Marine Bacterium Isolated from a Lignin-Enriched Seawater Microcosm

    DOE PAGES

    O'Dell, Kaela; Woo, Hannah L.; Utturkar, Sagar M.; ...

    2015-05-07

    Halomonas sp. strain KO116 was isolated from Nile Delta Mediterranean Sea surface water enriched with insoluble organosolv lignin. It was further screened for growth on alkali lignin minimal salts medium agar. The strain tolerates the ionic liquid 1-ethyl-3-methylimidazolium acetate. Its complete genome sequence is presented in this report.

  6. Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms.

    PubMed

    Coorey, Namal V C; Matthews, James H; Bellows, David S; Atkinson, Paul H

    2015-11-01

    Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA). However, the biological activities of many xenobiotics are masked by the pleiotropic drug resistance (PDR) network which effluxes xenobiotics that are PDR substrates. The PDR network in S. cerevisiae is almost entirely under the control of two functionally homologous transcription factors Pdr1p and Pdr3p. Herein we report the construction of a PDR-attenuated haploid non-essential DMA (PA-DMA), lacking PDR1 and PDR3, which permits the MoA elucidation of xenobiotics that are PDR substrates at low concentrations. The functionality of four key cellular processes commonly activated in response to xenobiotic stress: oxidative stress response, general stress response, unfolded stress response and calcium signalling pathways were assessed in the absence of PDR1 and PDR3 genes and were found to unaltered, therefore, these key chemogenomic signatures are not lost when using the PA-DMA. Efficacy of the PA-DMA was demonstrated using cycloheximide and latrunculin A at low nanomolar concentrations to attain chemical genetic profiles that were more specific to their known main mechanisms. We also found a two-fold increase in the number of compounds that are bioactive in the pdr1Δpdr3Δ compared to the wild type strain in screening the commercially available LOPAC(1280) library. The PA-DMA should be particularly applicable to mechanism determination of xenobiotics that have limited availability, such as natural products.

  7. Adventures in the Enormous: A 1.8 Million Clone BAC Library for the 21.7 Gb Genome of Loblolly Pine

    PubMed Central

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

    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 constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu). PMID:21283709

  8. Availability of birth defects and genetic disease information in public libraries -- implications for the Human Genome Project

    SciTech Connect

    Sell, S.; Gettig, E.; Mulvihill, J.J.

    1994-09-01

    In order to better educate the public about birth defects and genetic diseases/testing, access to information is critical. The public library system of the United States is extensive and serves as an invaluable resource to citizens. We surveyed reference librarians at each of 87 public libraries in Allegheny and Westmoreland Counties, Pennsylvania. The study design included a questionnaire to ascertain the genetic knowledge of reference librarians and cataloged current resources in print and via telecommunications available to the public. A high compliance rate was achieved due to the incentive of providing copies of the Alliance of Genetic Support Group Directory to those who responded to the survey along with complete sets of the forty-three March of Dimes Information Sheets currently available. Analysis of demographic data related to the age, gender, and educational background, in addition to the occurrence of personal experiences with genetic disease was ascertained. Reference librarians were chosen as the study group due to the common experience of families seeking further information from the public library after or prior to a genetic consultation. As the Human Genome Project identifies new genes for conditions, people will seek public information more frequently. The study shows that public libraries are an appropriate point of education to and for the public.

  9. 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.

  10. All SNPs are not created equal: genome-wide association studies reveal a consistent pattern of enrichment among functionally annotated SNPs.

    PubMed

    Schork, Andrew J; Thompson, Wesley K; Pham, Phillip; Torkamani, Ali; Roddey, J Cooper; Sullivan, Patrick F; Kelsoe, John R; O'Donovan, Michael C; Furberg, Helena; Schork, Nicholas J; Andreassen, Ole A; Dale, Anders M

    2013-04-01

    Recent results indicate that genome-wide association studies (GWAS) have the potential to explain much of the heritability of common complex phenotypes, but methods are lacking to reliably identify the remaining associated single nucleotide polymorphisms (SNPs). We applied stratified False Discovery Rate (sFDR) methods to leverage genic enrichment in GWAS summary statistics data to uncover new loci likely to replicate in independent samples. Specifically, we use linkage disequilibrium-weighted annotations for each SNP in combination with nominal p-values to estimate the True Discovery Rate (TDR = 1-FDR) for strata determined by different genic categories. We show a consistent pattern of enrichment of polygenic effects in specific annotation categories across diverse phenotypes, with the greatest enrichment for SNPs tagging regulatory and coding genic elements, little enrichment in introns, and negative enrichment for intergenic SNPs. Stratified enrichment directly leads to increased TDR for a given p-value, mirrored by increased replication rates in independent samples. We show this in independent Crohn's disease GWAS, where we find a hundredfold variation in replication rate across genic categories. Applying a well-established sFDR methodology we demonstrate the utility of stratification for improving power of GWAS in complex phenotypes, with increased rejection rates from 20% in height to 300% in schizophrenia with traditional FDR and sFDR both fixed at 0.05. Our analyses demonstrate an inherent stratification among GWAS SNPs with important conceptual implications that can be leveraged by statistical methods to improve the discovery of loci.

  11. Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum

    PubMed Central

    Huang, Cui-Qin; Gasser, Robin B.; Cantacessi, Cinzia; Nisbet, Alasdair J.; Zhong, Weiwei; Sternberg, Paul W.; Loukas, Alex; Mulvenna, Jason; Lin, Rui-Qing; Chen, Ning; Zhu, Xing-Quan

    2008-01-01

    Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality

  12. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library.

    PubMed

    Kumar, Anand; Drozd, Mary; Pina-Mimbela, Ruby; Xu, Xiulan; Helmy, Yosra A; Antwi, Janet; Fuchs, James R; Nislow, Corey; Templeton, Jillian; Blackall, Patrick J; Rajashekara, Gireesh

    2016-01-01

    Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a "bioactive" library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

  13. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

    PubMed Central

    Kumar, Anand; Drozd, Mary; Pina-Mimbela, Ruby; Xu, Xiulan; Helmy, Yosra A.; Antwi, Janet; Fuchs, James R.; Nislow, Corey; Templeton, Jillian; Blackall, Patrick J.; Rajashekara, Gireesh

    2016-01-01

    Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine. PMID:27092106

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    • 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 ...

  15. 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

  16. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    PubMed

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  17. Functional Classification, Genomic Organization, Putatively cis-Acting Regulatory Elements, and Relationship to Quantitative Trait Loci, of Sorghum Genes with Rhizome-Enriched Expression1[W

    PubMed Central

    Jang, Cheol Seong; Kamps, Terry L.; Skinner, D. Neil; Schulze, Stefan R.; Vencill, William K.; Paterson, Andrew H.

    2006-01-01

    Rhizomes are organs of fundamental importance to plant competitiveness and invasiveness. We have identified genes expressed at substantially higher levels in rhizomes than other plant parts, and explored their functional categorization, genomic organization, regulatory motifs, and association with quantitative trait loci (QTLs) conferring rhizomatousness. The finding that genes with rhizome-enriched expression are distributed across a wide range of functional categories suggests some degree of specialization of individual members of many gene families in rhizomatous plants. A disproportionate share of genes with rhizome-enriched expression was implicated in secondary and hormone metabolism, and abiotic stimuli and development. A high frequency of unknown-function genes reflects our still limited knowledge of this plant organ. A putative oligosaccharyl transferase showed the highest degree of rhizome-specific expression, with several transcriptional or regulatory protein complex factors also showing high (but lesser) degrees of specificity. Inferred by the upstream sequences of their putative rice (Oryza sativa) homologs, sorghum (Sorghum bicolor) genes that were relatively highly expressed in rhizome tip tissues were enriched for cis-element motifs, including the pyrimidine box, TATCCA box, and CAREs box, implicating the gibberellins in regulation of many rhizome-specific genes. From cDNA clones showing rhizome-enriched expression, expressed sequence tags forming 455 contigs were plotted on the rice genome and aligned to QTL likelihood intervals for ratooning and rhizomatous traits in rice and sorghum. Highly expressed rhizome genes were somewhat enriched in QTL likelihood intervals for rhizomatousness or ratooning, with specific candidates including some of the most rhizome-specific genes. Some rhizomatousness and ratooning QTLs were shown to be potentially related to one another as a result of ancient duplication, suggesting long-term functional conservation of

  18. An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts

    PubMed Central

    2012-01-01

    Background The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. Results A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. Conclusions The new gene-enriched high-resolution turbot map represents a

  19. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML

    PubMed Central

    Kurata, Morito; Rathe, Susan K.; Bailey, Natashay J.; Aumann, Natalie K.; Jones, Justine M.; Veldhuijzen, G. Willemijn; Moriarity, Branden S.; Largaespada, David A.

    2016-01-01

    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. PMID:27808171

  20. 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.

  1. KENeV: A web-application for the automated reconstruction and visualization of the enriched metabolic and signaling super-pathways deriving from genomic experiments

    PubMed Central

    Pilalis, Eleftherios; Koutsandreas, Theodoros; Valavanis, Ioannis; Athanasiadis, Emmanouil; Spyrou, George; Chatziioannou, Aristotelis

    2015-01-01

    Gene expression analysis, using high throughput genomic technologies,has become an indispensable step for the meaningful interpretation of the underlying molecular complexity, which shapes the phenotypic manifestation of the investigated biological mechanism. The modularity of the cellular response to different experimental conditions can be comprehended through the exploitation of molecular pathway databases, which offer a controlled, curated background for statistical enrichment analysis. Existing tools enable pathway analysis, visualization, or pathway merging but none integrates a fully automated workflow, combining all above-mentioned modules and destined to non-programmer users. We introduce an online web application, named KEGG Enriched Network Visualizer (KENeV), which enables a fully automated workflow starting from a list of differentially expressed genes and deriving the enriched KEGG metabolic and signaling pathways, merged into two respective, non-redundant super-networks. The final networks can be downloaded as SBML files, for further analysis, or instantly visualized through an interactive visualization module. In conclusion, KENeV (available online at http://www.grissom.gr/kenev) provides an integrative tool, suitable for users with no programming experience, for the functional interpretation, at both the metabolic and signaling level, of differentially expressed gene subsets deriving from genomic experiments. PMID:26925206

  2. All SNPs Are Not Created Equal: Genome-Wide Association Studies Reveal a Consistent Pattern of Enrichment among Functionally Annotated SNPs

    PubMed Central

    Schork, Andrew J.; Thompson, Wesley K.; Pham, Phillip; Torkamani, Ali; Roddey, J. Cooper; Sullivan, Patrick F.; Kelsoe, John R.; O'Donovan, Michael C.; Furberg, Helena; Schork, Nicholas J.; Andreassen, Ole A.; Dale, Anders M.

    2013-01-01

    Recent results indicate that genome-wide association studies (GWAS) have the potential to explain much of the heritability of common complex phenotypes, but methods are lacking to reliably identify the remaining associated single nucleotide polymorphisms (SNPs). We applied stratified False Discovery Rate (sFDR) methods to leverage genic enrichment in GWAS summary statistics data to uncover new loci likely to replicate in independent samples. Specifically, we use linkage disequilibrium-weighted annotations for each SNP in combination with nominal p-values to estimate the True Discovery Rate (TDR = 1−FDR) for strata determined by different genic categories. We show a consistent pattern of enrichment of polygenic effects in specific annotation categories across diverse phenotypes, with the greatest enrichment for SNPs tagging regulatory and coding genic elements, little enrichment in introns, and negative enrichment for intergenic SNPs. Stratified enrichment directly leads to increased TDR for a given p-value, mirrored by increased replication rates in independent samples. We show this in independent Crohn's disease GWAS, where we find a hundredfold variation in replication rate across genic categories. Applying a well-established sFDR methodology we demonstrate the utility of stratification for improving power of GWAS in complex phenotypes, with increased rejection rates from 20% in height to 300% in schizophrenia with traditional FDR and sFDR both fixed at 0.05. Our analyses demonstrate an inherent stratification among GWAS SNPs with important conceptual implications that can be leveraged by statistical methods to improve the discovery of loci. PMID:23637621

  3. Mitogenome assembly from genomic multiplex libraries: comparison of strategies and novel mitogenomes for five species of frogs.

    PubMed

    Machado, D J; Lyra, M L; Grant, T

    2016-05-01

    Next-generation sequencing continues to revolutionize biodiversity studies by generating unprecedented amounts of DNA sequence data for comparative genomic analysis. However, these data are produced as millions or billions of short reads of variable quality that cannot be directly applied in comparative analyses, creating a demand for methods to facilitate assembly. We optimized an in silico strategy to efficiently reconstruct high-quality mitochondrial genomes directly from genomic reads. We tested this strategy using sequences from five species of frogs: Hylodes meridionalis (Hylodidae), Hyloxalus yasuni (Dendrobatidae), Pristimantis fenestratus (Craugastoridae), and Melanophryniscus simplex and Rhinella sp. (Bufonidae). These are the first mitogenomes published for these species, the genera Hylodes, Hyloxalus, Pristimantis, Melanophryniscus and Rhinella, and the families Craugastoridae and Hylodidae. Sequences were generated using only half of one lane of a standard Illumina HiqSeq 2000 flow cell, resulting in fewer than eight million reads. We analysed the reads of Hylodes meridionalis using three different assembly strategies: (1) reference-based (using bowtie2); (2) de novo (using abyss, soapdenovo2 and velvet); and (3) baiting and iterative mapping (using mira and mitobim). Mitogenomes were assembled exclusively with strategy 3, which we employed to assemble the remaining mitogenomes. Annotations were performed with mitos and confirmed by comparison with published amphibian mitochondria. In most cases, we recovered all 13 coding genes, 22 tRNAs, and two ribosomal subunit genes, with minor gene rearrangements. Our results show that few raw reads can be sufficient to generate high-quality scaffolds, making any Illumina machine run using genomic multiplex libraries a potential source of data for organelle assemblies as by-catch.

  4. Final report. Human artificial episomal chromosome (HAEC) for building large genomic libraries

    SciTech Connect

    Jean-Michael H. Vos

    1999-12-09

    Collections of human DNA fragments are maintained for research purposes as clones in bacterial host cells. However for unknown reasons, some regions of the human genome appear to be unclonable or unstable in bacteria. Their team has developed a system using episomes (extrachromosomal, autonomously replication DNA) that maintains large DNA fragments in human cells. This human artificial episomal chromosomal (HAEC) system may prove useful for coverage of these especially difficult regions. In the broader biomedical community, the HAEC system also shows promise for use in functional genomics and gene therapy. Recent improvements to the HAEC system and its application to mapping, sequencing, and functionally studying human and mouse DNA are summarized. Mapping and sequencing the human genome and model organisms are only the first steps in determining the function of various genetic units critical for gene regulation, DNA replication, chromatin packaging, chromosomal stability, and chromatid segregation. Such studies will require the ability to transfer and manipulate entire functional units into mammalian cells.

  5. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    SciTech Connect

    Mitchell, S.C.; Bocskai, D.; Cao, Y.

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  6. Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

    PubMed Central

    2012-01-01

    Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

  7. Improving the genome annotation of the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5'-ends of primary transcripts.

    PubMed

    Schwientek, Patrick; Neshat, Armin; Kalinowski, Jörn; Klein, Andreas; Rückert, Christian; Schneiker-Bekel, Susanne; Wendler, Sergej; Stoye, Jens; Pühler, Alfred

    2014-11-20

    Actinoplanes sp. SE50/110 is the producer of the alpha-glucosidase inhibitor acarbose, which is an economically relevant and potent drug in the treatment of type-2 diabetes mellitus. In this study, we present the detection of transcription start sites on this genome by sequencing enriched 5'-ends of primary transcripts. Altogether, 1427 putative transcription start sites were initially identified. With help of the annotated genome sequence, 661 transcription start sites were found to belong to the leader region of protein-coding genes with the surprising result that roughly 20% of these genes rank among the class of leaderless transcripts. Next, conserved promoter motifs were identified for protein-coding genes with and without leader sequences. The mapped transcription start sites were finally used to improve the annotation of the Actinoplanes sp. SE50/110 genome sequence. Concerning protein-coding genes, 41 translation start sites were corrected and 9 novel protein-coding genes could be identified. In addition to this, 122 previously undetermined non-coding RNA (ncRNA) genes of Actinoplanes sp. SE50/110 were defined. Focusing on antisense transcription start sites located within coding genes or their leader sequences, it was discovered that 96 of those ncRNA genes belong to the class of antisense RNA (asRNA) genes. The remaining 26 ncRNA genes were found outside of known protein-coding genes. Four chosen examples of prominent ncRNA genes, namely the transfer messenger RNA gene ssrA, the ribonuclease P class A RNA gene rnpB, the cobalamin riboswitch RNA gene cobRS, and the selenocysteine-specific tRNA gene selC, are presented in more detail. This study demonstrates that sequencing of enriched 5'-ends of primary transcripts and the identification of transcription start sites are valuable tools for advanced genome annotation of Actinoplanes sp. SE50/110 and most probably also for other bacteria.

  8. Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

    PubMed

    Schmickl, Roswitha; Liston, Aaron; Zeisek, Vojtěch; Oberlander, Kenneth; Weitemier, Kevin; Straub, Shannon C K; Cronn, Richard C; Dreyer, Léanne L; Suda, Jan

    2016-09-01

    Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

  9. Genome Clone Libraries and Data from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium

    DOE Data Explorer

    The I.M.A.G.E. Consortium was initiated in 1993 by four academic groups on a collaborative basis after informal discussions led to a common vision of how to achieve an important goal in the study of the human genome: the Integrated Molecular Analysis of Genomes and their Expression Consortium's primary goal is to create arrayed cDNA libraries and associated bioinformatics tools, and make them publicly available to the research community. The primary organisms of interest include intensively studied mammalian species, including human, mouse, rat and non-human primate species. The Consortium has also focused on several commonly studied model organisms; as part of this effort it has arrayed cDNAs from zebrafish, and Fugu (pufferfish) as well as Xenopus laevis and X. tropicalis (frog). Utilizing high speed robotics, over nine million individual cDNA clones have been arrayed into 384-well microtiter plates, and sufficient replicas have been created to distribute copies both to sequencing centers and to a network of five distributors located worldwide. The I.M.A.G.E. Consortium represents the world's largest public cDNA collection, and works closely with the National Institutes of Health's Mammalian Gene Collection(MGC) to help it achieve its goal of creating a full-length cDNA clone for every human and mouse gene. I.M.A.G.E. is also a member of the ORFeome Collaboration, working to generate a complete set of expression-ready open reading frame clones representing each human gene. Custom informatics tools have been developed in support of these projects to better allow the research community to select clones of interest and track and collect all data deposited into public databases about those clones and their related sequences. I.M.A.G.E. clones are publicly available, free of any royalties, and may be used by anyone agreeing with the Consortium's guidelines.

  10. A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): an important genetic resource for comparative genomics and phylogenetic analysis.

    PubMed

    Li, Songryong; Ma, Yibao; Jang, Shenghun; Wu, Yingliang; Liu, Hui; Cao, Zhijian; Li, Wenxin

    2009-12-01

    Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.

  11. Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource.

    PubMed

    Romanov, Michael N; Koriabine, Maxim; Nefedov, Mikhail; de Jong, Pieter J; Ryder, Oliver A

    2006-12-01

    To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.

  12. Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

    PubMed Central

    Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, Neil; Peng, Donghai; Ruan, Lifang

    2012-01-01

    We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes. PMID:22544259

  13. Genome-Wide Anaplasma phagocytophilum AnkA-DNA Interactions Are Enriched in Intergenic Regions and Gene Promoters and Correlate with Infection-Induced Differential Gene Expression

    PubMed Central

    Dumler, J. Stephen; Sinclair, Sara H.; Pappas-Brown, Valeria; Shetty, Amol C.

    2016-01-01

    Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils, and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR)-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: (i) intergenic regions predicted to be MARs; (ii) within predicted lamina-associated domains; and (iii) at promoters ≤ 3000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome “re-organizer.” AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity. PMID:27703927

  14. Epigenetic Patterns in Blood Associated With Lipid Traits Predict Incident Coronary Heart Disease Events and Are Enriched for Results From Genome-Wide Association Studies

    PubMed Central

    Hedman, Åsa K.; Mendelson, Michael M.; Marioni, Riccardo E.; Gustafsson, Stefan; Joehanes, Roby; Irvin, Marguerite R.; Zhi, Degui; Sandling, Johanna K.; Yao, Chen; Liu, Chunyu; Liang, Liming; Huan, Tianxiao; McRae, Allan F.; Demissie, Serkalem; Shah, Sonia; Starr, John M.; Cupples, L. Adrienne; Deloukas, Panos; Spector, Timothy D.; Sundström, Johan; Krauss, Ronald M.; Arnett, Donna K.; Deary, Ian J.; Lind, Lars; Levy, Daniel

    2017-01-01

    Background— Genome-wide association studies have identified loci influencing circulating lipid concentrations in humans; further information on novel contributing genes, pathways, and biology may be gained through studies of epigenetic modifications. Methods and Results— To identify epigenetic changes associated with lipid concentrations, we assayed genome-wide DNA methylation at cytosine–guanine dinucleotides (CpGs) in whole blood from 2306 individuals from 2 population-based cohorts, with replication of findings in 2025 additional individuals. We identified 193 CpGs associated with lipid levels in the discovery stage (P<1.08E-07) and replicated 33 (at Bonferroni-corrected P<0.05), including 25 novel CpGs not previously associated with lipids. Genes at lipid-associated CpGs were enriched in lipid and amino acid metabolism processes. A differentially methylated locus associated with triglycerides and high-density lipoprotein cholesterol (HDL-C; cg27243685; P=8.1E-26 and 9.3E-19) was associated with cis-expression of a reverse cholesterol transporter (ABCG1; P=7.2E-28) and incident cardiovascular disease events (hazard ratio per SD increment, 1.38; 95% confidence interval, 1.15–1.66; P=0.0007). We found significant cis-methylation quantitative trait loci at 64% of the 193 CpGs with an enrichment of signals from genome-wide association studies of lipid levels (PTC=0.004, PHDL-C=0.008 and Ptriglycerides=0.00003) and coronary heart disease (P=0.0007). For example, genome-wide significant variants associated with low-density lipoprotein cholesterol and coronary heart disease at APOB were cis-methylation quantitative trait loci for a low-density lipoprotein cholesterol–related differentially methylated locus. Conclusions— We report novel associations of DNA methylation with lipid levels, describe epigenetic mechanisms related to previous genome-wide association studies discoveries, and provide evidence implicating epigenetic regulation of reverse cholesterol

  15. Whole Genome Duplication and Enrichment of Metal Cation Transporters Revealed by De Novo Genome Sequencing of Extremely Halotolerant Black Yeast Hortaea werneckii

    PubMed Central

    Jackman, Shaun; Turk, Martina; Sadowski, Ivan; Nislow, Corey; Jones, Steven; Birol, Inanc; Cimerman, Nina Gunde; Plemenitaš, Ana

    2013-01-01

    Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H+ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H+ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also

  16. Quick genome sequencing of “Candidatus Liberibacter” strains by use of Enrichment-Enlargement-Next generation sequencing (EEN)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Members of “Candidatus Liberibacter” are associated with several important plant diseases such as citrus Huanglongbing (HLB) and potato zebra chip (ZC) disease. Inability to culture and low titers in infected hosts have been major obstacles for research on these bacteria. The use of whole genome seq...

  17. Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco.

    PubMed

    Shi, B-J; Gustafson, J P; Button, J; Miyazaki, J; Pallotta, M; Gustafson, N; Zhou, H; Langridge, P; Collins, N C

    2009-08-01

    Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.

  18. Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2).

    PubMed

    Rodrigues, F; Zeeman, A M; Alves, C; Sousa, M J; Steensma, H Y; Côrte-Real, M; Leão, C

    2001-04-01

    A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.

  19. Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries, promoter sequence and gene expression analysis.

    PubMed

    Kanjanawattanawong, Supanath; Tangphatsornruang, Sithichoke; Triwitayakorn, Kanokporn; Ruang-areerate, Panthita; Sangsrakru, Duangjai; Poopear, Supannee; Somyong, Suthasinee; Narangajavana, Jarunya

    2014-10-01

    The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.

  20. Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353*

    PubMed Central

    Cho, Sung Mi; Jeoung, Sae Chae; Song, Ji-Young; Kupriyanova, Elena V.; Pronina, Natalia A.; Lee, Bong-Woo; Jo, Seong-Whan; Park, Beom-Seok; Choi, Sang-Bong; Song, Ji-Joon; Park, Youn-Il

    2015-01-01

    Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, Microcoleus IPPAS B353 (Microcoleus B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant Escherichia coli indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: 10 GAFs contained PCB chromophores, whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of Microcoleus B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found. PMID:26405033

  1. Enriching the Catalog

    ERIC Educational Resources Information Center

    Tennant, Roy

    2004-01-01

    After decades of costly and time-consuming effort, nearly all libraries have completed the retrospective conversion of their card catalogs to electronic form. However, bibliographic systems still are really not much more than card catalogs on wheels. Enriched content that Amazon.com takes for granted--such as digitized tables of contents, cover…

  2. Maize genome sequencing by methylation filtration.

    PubMed

    Palmer, Lance E; Rabinowicz, Pablo D; O'Shaughnessy, Andrew L; Balija, Vivekanand S; Nascimento, Lidia U; Dike, Sujit; de la Bastide, Melissa; Martienssen, Robert A; McCombie, W Richard

    2003-12-19

    Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.

  3. ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens

    PubMed Central

    Trebes, Amy; Brown, Anthony; Klenerman, Paul; Buck, David; Piazza, Paolo; Barnes, Eleanor; Bowden, Rory

    2015-01-01

    The routine availability of high-depth virus sequence data would allow the sensitive detection of resistance-associated variants that can jeopardize HIV or hepatitis C virus (HCV) treatment. We introduce ve-SEQ, a high-throughput method for sequence-specific enrichment and characterization of whole-virus genomes at up to 20% divergence from a reference sequence and 1,000-fold greater sensitivity than direct sequencing. The extreme genetic diversity of HCV led us to implement an algorithm for the efficient design of panels of oligonucleotide probes to capture any sequence among a defined set of targets without detectable bias. ve-SEQ enables efficient detection and sequencing of any HCV genome, including mixtures and intra-host variants, in a single experiment, with greater tolerance of sequence diversity than standard amplification methods and greater sensitivity than metagenomic sequencing, features that are directly applicable to other pathogens or arbitrary groups of target organisms, allowing the combination of sensitive detection with sequencing in many settings. PMID:27092241

  4. Generation of a Genome Scale Lentiviral Vector Library for EF1α Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer

    PubMed Central

    Škalamera, Dubravka; Dahmer, Mareike; Purdon, Amy S.; Wilson, Benjamin M.; Ranall, Max V.; Blumenthal, Antje; Gabrielli, Brian; Gonda, Thomas J.

    2012-01-01

    The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors. PMID:23251614

  5. Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA.

    PubMed

    Parkinson, Nicholas J; Maslau, Siarhei; Ferneyhough, Ben; Zhang, Gang; Gregory, Lorna; Buck, David; Ragoussis, Jiannis; Ponting, Chris P; Fischer, Michael D

    2012-01-01

    New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.

  6. Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA

    PubMed Central

    Parkinson, Nicholas J.; Maslau, Siarhei; Ferneyhough, Ben; Zhang, Gang; Gregory, Lorna; Buck, David; Ragoussis, Jiannis; Ponting, Chris P.; Fischer, Michael D.

    2012-01-01

    New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures. PMID:22090378

  7. Selective Enrichment and Sequencing of Whole Mitochondrial Genomes in the Presence of Nuclear Encoded Mitochondrial Pseudogenes (Numts)

    PubMed Central

    Wolff, Jonci N.; Shearman, Deborah C. A.; Brooks, Rob C.; Ballard, John W. O.

    2012-01-01

    Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error. PMID:22606342

  8. Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

    PubMed

    Al-Jarbou, Ahmed Nasser

    2012-01-01

    Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

  9. Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

    PubMed

    Pardo, Belén G; Álvarez-Dios, José Antonio; Cao, Asunción; Ramilo, Andrea; Gómez-Tato, Antonio; Planas, Josep V; Villalba, Antonio; Martínez, Paulino

    2016-12-01

    The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in

  10. Recurrent Rare Genomic Copy Number Variants and Bicuspid Aortic Valve Are Enriched in Early Onset Thoracic Aortic Aneurysms and Dissections

    PubMed Central

    Prakash, Siddharth; Kuang, Shao-Qing; Regalado, Ellen; Guo, Dongchuan; Milewicz, Dianna

    2016-01-01

    Thoracic Aortic Aneurysms and Dissections (TAAD) are a major cause of death in the United States. The spectrum of TAAD ranges from genetic disorders, such as Marfan syndrome, to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to early onset TAAD (ETAAD). We conducted a genome-wide SNP array analysis of ETAAD patients of European descent who were enrolled in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). Genotyping was performed on the Illumina Omni-Express platform, using PennCNV, Nexus and CNVPartition for CNV detection. ETAAD patients (n = 108, 100% European American, 28% female, average age 20 years, 55% with bicuspid aortic valves) were compared to 7013 dbGAP controls without a history of vascular disease using downsampled Omni 2.5 data. For comparison, 805 sporadic TAAD patients with late onset aortic disease (STAAD cohort) and 192 affected probands from families with at least two affected relatives (FTAAD cohort) from our institution were screened for additional CNVs at these loci with SNP arrays. We identified 47 recurrent CNV regions in the ETAAD, FTAAD and STAAD groups that were absent or extremely rare in controls. Nine rare CNVs that were either very large (>1 Mb) or shared by ETAAD and STAAD or FTAAD patients were also identified. Four rare CNVs involved genes that cause arterial aneurysms when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (two duplications and two deletions) and 17q25.1 (three duplications). The percentage of individuals harboring rare CNVs was significantly greater in the ETAAD cohort (32%) than in the FTAAD (23%) or STAAD (17%) cohorts. We identified multiple loci affected by rare CNVs in one-third of ETAAD patients, confirming the genetic heterogeneity of TAAD. Alterations of candidate genes at these loci may contribute to the pathogenesis of TAAD. PMID:27092555

  11. Recurrent Rare Genomic Copy Number Variants and Bicuspid Aortic Valve Are Enriched in Early Onset Thoracic Aortic Aneurysms and Dissections.

    PubMed

    Prakash, Siddharth; Kuang, Shao-Qing; Regalado, Ellen; Guo, Dongchuan; Milewicz, Dianna

    2016-01-01

    Thoracic Aortic Aneurysms and Dissections (TAAD) are a major cause of death in the United States. The spectrum of TAAD ranges from genetic disorders, such as Marfan syndrome, to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to early onset TAAD (ETAAD). We conducted a genome-wide SNP array analysis of ETAAD patients of European descent who were enrolled in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). Genotyping was performed on the Illumina Omni-Express platform, using PennCNV, Nexus and CNVPartition for CNV detection. ETAAD patients (n = 108, 100% European American, 28% female, average age 20 years, 55% with bicuspid aortic valves) were compared to 7013 dbGAP controls without a history of vascular disease using downsampled Omni 2.5 data. For comparison, 805 sporadic TAAD patients with late onset aortic disease (STAAD cohort) and 192 affected probands from families with at least two affected relatives (FTAAD cohort) from our institution were screened for additional CNVs at these loci with SNP arrays. We identified 47 recurrent CNV regions in the ETAAD, FTAAD and STAAD groups that were absent or extremely rare in controls. Nine rare CNVs that were either very large (>1 Mb) or shared by ETAAD and STAAD or FTAAD patients were also identified. Four rare CNVs involved genes that cause arterial aneurysms when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (two duplications and two deletions) and 17q25.1 (three duplications). The percentage of individuals harboring rare CNVs was significantly greater in the ETAAD cohort (32%) than in the FTAAD (23%) or STAAD (17%) cohorts. We identified multiple loci affected by rare CNVs in one-third of ETAAD patients, confirming the genetic heterogeneity of TAAD. Alterations of candidate genes at these loci may contribute to the pathogenesis of TAAD.

  12. Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9

    PubMed Central

    Park, Jeongbin; Kim, Jin-Soo; Bae, Sangsu

    2016-01-01

    Motivation: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. Results: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. Availability and implementation: Free access at http://www.rgenome.net/cas-database/. Contact: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153724

  13. Genome-Centric Analysis of Microbial Populations Enriched by Hydraulic Fracture Fluid Additives in a Coal Bed Methane Production Well.

    PubMed

    Robbins, Steven J; Evans, Paul N; Parks, Donovan H; Golding, Suzanne D; Tyson, Gene W

    2016-01-01

    Coal bed methane (CBM) is generated primarily through the microbial degradation of coal. Despite a limited understanding of the microorganisms responsible for this process, there is significant interest in developing methods to stimulate additional methane production from CBM wells. Physical techniques including hydraulic fracture stimulation are commonly applied to CBM wells, however the effects of specific additives contained in hydraulic fracture fluids on native CBM microbial communities are poorly understood. Here, metagenomic sequencing was applied to the formation waters of a hydraulically fractured and several non-fractured CBM production wells to determine the effect of this stimulation technique on the in-situ microbial community. The hydraulically fractured well was dominated by two microbial populations belonging to the class Phycisphaerae (within phylum Planctomycetes) and candidate phylum Aminicenantes. Populations from these phyla were absent or present at extremely low abundance in non-fractured CBM wells. Detailed metabolic reconstruction of near-complete genomes from these populations showed that their high relative abundance in the hydraulically fractured CBM well could be explained by the introduction of additional carbon sources, electron acceptors, and biocides contained in the hydraulic fracture fluid.

  14. Genome-Centric Analysis of Microbial Populations Enriched by Hydraulic Fracture Fluid Additives in a Coal Bed Methane Production Well

    PubMed Central

    Robbins, Steven J.; Evans, Paul N.; Parks, Donovan H.; Golding, Suzanne D.; Tyson, Gene W.

    2016-01-01

    Coal bed methane (CBM) is generated primarily through the microbial degradation of coal. Despite a limited understanding of the microorganisms responsible for this process, there is significant interest in developing methods to stimulate additional methane production from CBM wells. Physical techniques including hydraulic fracture stimulation are commonly applied to CBM wells, however the effects of specific additives contained in hydraulic fracture fluids on native CBM microbial communities are poorly understood. Here, metagenomic sequencing was applied to the formation waters of a hydraulically fractured and several non-fractured CBM production wells to determine the effect of this stimulation technique on the in-situ microbial community. The hydraulically fractured well was dominated by two microbial populations belonging to the class Phycisphaerae (within phylum Planctomycetes) and candidate phylum Aminicenantes. Populations from these phyla were absent or present at extremely low abundance in non-fractured CBM wells. Detailed metabolic reconstruction of near-complete genomes from these populations showed that their high relative abundance in the hydraulically fractured CBM well could be explained by the introduction of additional carbon sources, electron acceptors, and biocides contained in the hydraulic fracture fluid. PMID:27375557

  15. Genomic Signatures of North American Soybean Improvement Inform Diversity Enrichment Strategies and Clarify the Impact of Hybridization.

    PubMed

    Vaughn, Justin N; Li, Zenglu

    2016-09-08

    Crop improvement represents a long-running experiment in artificial selection on a complex trait, namely yield. How such selection relates to natural populations is unclear, but the analysis of domesticated populations could offer insights into the relative role of selection, drift, and recombination in all species facing major shifts in selective regimes. Because of the extreme autogamy exhibited by soybean (Glycine max), many "immortalized" genotypes of elite varieties spanning the last century have been preserved and characterized using ∼50,000 single nucleotide polymorphic (SNP) markers. Also due to autogamy, the history of North American soybean breeding can be roughly divided into pre- and posthybridization eras, allowing for direct interrogation of the role of recombination in improvement and selection. Here, we report on genome-wide characterization of the structure and history of North American soybean populations and the signature of selection in these populations. Supporting previous work, we find that maturity defines population structure. Though the diversity of North American ancestors is comparable to available landraces, prehybridization line selections resulted in a clonal structure that dominated early breeding and explains many of the reductions in diversity found in the initial generations of soybean hybridization. The rate of allele frequency change does not deviate sharply from neutral expectation, yet some regions bare hallmarks of strong selection, suggesting a highly variable range of selection strengths biased toward weak effects. We also discuss the importance of haplotypes as units of analysis when complex traits fall under novel selection regimes.

  16. Genome-wide analysis of loss of heterozygosity in breast infiltrating ductal carcinoma distant normal tissue highlights arm specific enrichment and expansion across tumor stages.

    PubMed

    Ruan, Xiaoyang; Liu, Hongfang; Boardman, Lisa; Kocher, Jean-Pierre A

    2014-01-01

    Studies have shown concurrent loss of heterozygosity (LOH) in breast infiltrating ductal carcinoma (IDC) and adjacent or distant normal tissue. However, the overall extent of LOH in normal tissue and their significance to tumorigenesis remain unknown, as existing studies are largely based on selected microsatellite markers. Here we present the first autosome-wide study of LOH in IDC and distant normal tissue using informative loci deduced from SNP array-based and sequencing-based techniques. We show a consistently high LOH concurrence rate in IDC (mean = 24%) and distant normal tissue (m = 54%), suggesting for most patients (31/33) histologically normal tissue contains genomic instability that can be a potential marker of increased IDC risk. Concurrent LOH is more frequent in fragile site related genes like WWOX (9/31), NTRK2 (10/31), and FHIT (7/31) than traditional genetic markers like BRCA1 (0/23), BRCA2 (2/29) and TP53 (1/13). Analysis at arm level shows distant normal tissue has low level but non-random enrichment of LOH (topped by 8p and 16q) significantly correlated with matched IDC (Pearson r = 0.66, p = 3.5E-6) (topped by 8p, 11q, 13q, 16q, 17p, and 17q). The arm-specific LOH enrichment was independently observed in tumor samples from 548 IDC patients when stratified by tumor size based T stages. Fine LOH structure from sequencing data indicates LOH in low order tissues non-randomly overlap (∼67%) with LOH that usually has longer tract length (the length of genomic region affected by LOH) in high order tissues. The consistent observations from multiple datasets suggest progressive LOH in the development of IDC potentially through arm-specific pile up effect with discernible signature in normal tissue. Our finding also suggests that LOH detected in IDC by comparing to paired adjacent or distant normal tissue are more likely underestimated.

  17. Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures.

    PubMed

    Takishita, Kiyotaka; Yubuki, Naoji; Kakizoe, Natsuki; Inagaki, Yuji; Maruyama, Tadashi

    2007-07-01

    Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the "hidden" microeukaryotic diversity in the environment examined.

  18. Sequencing the maize genome.

    PubMed

    Martienssen, Robert A; Rabinowicz, Pablo D; O'Shaughnessy, Andrew; McCombie, W Richard

    2004-04-01

    Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

  19. Chromosome region-specific libraries for human genome analysis. Progress report, September 1, 1991--August 31, 1992

    SciTech Connect

    Kao, Fa-Ten

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  20. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

    PubMed

    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-03-01

    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

  1. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    PubMed

    Saski, Christopher A; Bhattacharjee, Ranjana; Scheffler, Brian E; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  2. The highest-copy repeats are methylated in the small genome of the early divergent vascular plant Selaginella moellendorffii

    PubMed Central

    Chan, Agnes P; Melake-Berhan, Admasu; O'Brien, Kimberly; Buckley, Stephanie; Quan, Hui; Chen, Dan; Lewis, Matthew; Banks, Jo Ann; Rabinowicz, Pablo D

    2008-01-01

    Background The lycophyte Selaginella moellendorffii is a vascular plant that diverged from the fern/seed plant lineage at least 400 million years ago. Although genomic information for S. moellendorffii is starting to be produced, little is known about basic aspects of its molecular biology. In order to provide the first glimpse to the epigenetic landscape of this early divergent vascular plant, we used the methylation filtration technique. Methylation filtration genomic libraries select unmethylated DNA clones due to the presence of the methylation-dependent restriction endonuclease McrBC in the bacterial host. Results We conducted a characterization of the DNA methylation patterns of the S. moellendorffii genome by sequencing a set of S. moellendorffii shotgun genomic clones, along with a set of methylation filtered clones. Chloroplast DNA, which is typically unmethylated, was enriched in the filtered library relative to the shotgun library, showing that there is DNA methylation in the extremely small S. moellendorffii genome. The filtered library also showed enrichment in expressed and gene-like sequences, while the highest-copy repeats were largely under-represented in this library. These results show that genes and repeats are differentially methylated in the S. moellendorffii genome, as occurs in other plants studied. Conclusion Our results shed light on the genome methylation pattern in a member of a relatively unexplored plant lineage. The DNA methylation data reported here will help understanding the involvement of this epigenetic mark in fundamental biological processes, as well as the evolutionary aspects of epigenetics in land plants. PMID:18549478

  3. In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

    PubMed Central

    Jacobs, W R; Barrett, J F; Clark-Curtiss, J E; Curtiss, R

    1986-01-01

    Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts. Images PMID:2937735

  4. A Prospective Virtual Screening Study: Enriching Hit Rates and Designing Focus Libraries To Find Inhibitors of PI3Kδ and PI3Kγ.

    PubMed

    Damm-Ganamet, Kelly L; Bembenek, Scott D; Venable, Jennifer W; Castro, Glenda G; Mangelschots, Lieve; Peeters, Daniëlle C G; Mcallister, Heather M; Edwards, James P; Disepio, Daniel; Mirzadegan, Taraneh

    2016-05-12

    Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.

  5. 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.

  6. Reexamining Content-Enriched Access: Its Effect on Usage and Discovery

    ERIC Educational Resources Information Center

    Tosaka, Yuji; Weng, Cathy

    2011-01-01

    Content-enriched metadata in bibliographic records is considered helpful to library users in identifying and selecting library materials for their needs. The paper presents a study, using circulation data from a medium-sized academic library, of the effect of content-enriched records on library materials usage. The study also examines OPAC search…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name,…

  9. Construction and Characterization of a Repetitive DNA Library in Parodontidae (Actinopterygii: Characiformes): A Genomic and Evolutionary Approach to the Degeneration of the W Sex Chromosome

    PubMed Central

    Oliveira, Jordana Inácio Nascimento; Nogaroto, Viviane; Almeida, Mara Cristina; Artoni, Roberto Ferreira; Cestari, Marta Margarete; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

    2014-01-01

    Abstract Repetitive DNA sequences, including tandem and dispersed repeats, comprise a large portion of eukaryotic genomes and are important for gene regulation, sex chromosome differentiation, and karyotype evolution. In Parodontidae, only the repetitive DNAs WAp and pPh2004 and rDNAs were previously studied using fluorescence in situ hybridization. This study aimed to build a library of repetitive DNA in Parodontidae. We isolated 40 clones using Cot-1; 17 of these clones exhibited similarity to repetitive DNA sequences, including satellites, minisatellites, microsatellites, and class I and class II transposable elements (TEs), from Danio rerio and other organisms. The physical mapping of the clones to chromosomes revealed the presence of a satellite DNA, a Helitron element, and degenerate short interspersed element (SINE), long interspersed element (LINE), and tc1-mariner elements on the sex chromosomes. Some clones exhibited dispersed signals; other sequences were not detected. The 5S rDNA was detected on an autosomal pair. These elements likely function in the molecular degeneration of the W chromosome in Parodontidae. Thus, the location of these elements on the chromosomes is important for understanding the function of these repetitive DNAs and for integrative studies with genome sequencing. The presented data demonstrate that an intensive invasion of TEs occurred during W sex chromosome differentiation in the Parodontidae. PMID:25122415

  10. Strategies for complete plastid genome sequencing.

    PubMed

    Twyford, Alex D; Ness, Rob W

    2016-10-28

    Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.

  11. Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations.

    PubMed

    Jupe, Florian; Witek, Kamil; Verweij, Walter; Sliwka, Jadwiga; Pritchard, Leighton; Etherington, Graham J; Maclean, Dan; Cock, Peter J; Leggett, Richard M; Bryan, Glenn J; Cardle, Linda; Hein, Ingo; Jones, Jonathan D G

    2013-11-01

    RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum 'Heinz 1706' extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines.

  12. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  13. The genome of the of the generalist plant pathogenic fungus Fusarium avenaceum is enriched with genes involved in redox, signaling and secondary metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium avenaceum is a fungus commonly isolated from soil and with a wide range of host plants. We present here three genome sequences of F. avenaceum, one isolated from barley in Finland and two from spring and winter wheat in Canada. The physical sizes of the three genomes range from 41.6-43.2 MB...

  14. The draft genome sequence of the ascomycete fungus Penicillium subrubescens reveals a highly enriched content of plant biomass related CAZymes compared to related fungi.

    PubMed

    Peng, Mao; Dilokpimol, Adiphol; Mäkelä, Miia R; Hildén, Kristiina; Bervoets, Sander; Riley, Robert; Grigoriev, Igor V; Hainaut, Matthieu; Henrissat, Bernard; de Vries, Ronald P; Granchi, Zoraide

    2017-03-20

    Here we report the genome sequence of the ascomycete saprobic fungus Penicillium subrubescens FBCC1632/CBS132785 isolated from a Jerusalem artichoke field in Finland. The 39.75Mb genome containing 14,188 gene models is highly similar for that reported for other Penicillium species, but contains a significantly higher number of putative carbohydrate active enzyme (CAZyme) encoding genes.

  15. G-quadruplex (G4) motifs in the maize (Zea mays L.) genome are enriched at specific locations in thousands of genes coupled to energy status, hypoxia, low sugar, and nutrient deprivation.

    PubMed

    Andorf, Carson M; Kopylov, Mykhailo; Dobbs, Drena; Koch, Karen E; Stroupe, M Elizabeth; Lawrence, Carolyn J; Bass, Hank W

    2014-12-20

    The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5' UTR and the other at the 5' end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

  16. Progress in the characterization of a human genomic YAC library selected on the basis of homology to T{sub 2}AG{sub 3}

    SciTech Connect

    Vocero-Akbani, A.; Sanjurjo, H.; Fair, K.

    1994-09-01

    Using a combination of physical and genetic mapping methods we have characterized more than 190 YAC clones originally isolated on the basis of hybridization to the human telomere regions by FISH (using Alu-PCR products or YAC subclones individually or pooled as probes). Thirty-seven of the YACs mapped to single telomeres while 16 mapped to more than one telomere, or to interstitial regions, including centromeres. Subclone libraries were constructed for a subset of YACs, genetic markers developed, and the loci incorporated into genetic maps for chromosomes 2, 6, 7, 8, 10, 12, 13, 14 and 20. Altogether 28 different telomeres are now defined by chromosomally mapped STSs which were derived from YACs that were FISH mapped to the termini of 1p, 2p{sup *}, 2q{sup +}, 3p, 3q, 4q, 5q, 6q{sup *}, 7p, 7q{sup *+}, 8p{sup +}, 9q, 10p{sup *}, 10q, 11q, 12p{sup *}, 13q{sup *+}, 14q{sup *+}, 16p, 16q, 17p, 17q, 18p, 18q, 20p, 21q, and 22q ({sup *} microsatellite marker, {sup +}RFLP). Development of microsatellite genetic markers for the five additional telomeres is currently in progress [7p (50 b), 10q (275 kb). 17p (100 kb), 17q (175 kb), and 18p (225 kb)]. For YACs that have been localized to telomeres by FISH and to chromosomes by STS mapping to a rodent/human somatic cell hybrid chromosome panel, five genome equivalent bacteriophage lamda subclone libraries have been constructed and screened for the presence of human DNA and CA{sub n} dinucleotide repeats by plaque filter hybridization. A number of CA positive clones have been sequenced revealing simple repeats of 12 or more CAs per clone. STS development and testing for polymorphism using the CEPH pedigree resource is in progress.

  17. Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures.

    PubMed

    Kim, Wonyong; Park, Hee Kuk; Thanh, Hien Dang; Lee, Bo-Young; Shin, Jong Wook; Shin, Hyoung-Shik

    2011-12-01

    Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.

  18. Project ENRICH.

    ERIC Educational Resources Information Center

    Gwaley, Elizabeth; And Others

    Project ENRICH was conceived in Beaver County, Pennsylvania, to: (1) identify preschool children with learning disabilities, and (2) to develop a program geared to the remediation of the learning disabilities within a school year, while allowing the child to be enrolled in a regular class situation for the following school year. Through…

  19. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  20. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

    This paper suggests that by 2004, the traditional public libraries will have ceased to exist and new, attractive future libraries will have taken their place. The Libraries 2040 project of the Netherlands is initiating seven different libraries of the future. The Brabant library is the "ultimate library of the future" for the Dutch…

  1. Motif enrichment tool.

    PubMed

    Blatti, Charles; Sinha, Saurabh

    2014-07-01

    The Motif Enrichment Tool (MET) provides an online interface that enables users to find major transcriptional regulators of their gene sets of interest. MET searches the appropriate regulatory region around each gene and identifies which transcription factor DNA-binding specificities (motifs) are statistically overrepresented. Motif enrichment analysis is currently available for many metazoan species including human, mouse, fruit fly, planaria and flowering plants. MET also leverages high-throughput experimental data such as ChIP-seq and DNase-seq from ENCODE and ModENCODE to identify the regulatory targets of a transcription factor with greater precision. The results from MET are produced in real time and are linked to a genome browser for easy follow-up analysis. Use of the web tool is free and open to all, and there is no login requirement. ADDRESS: http://veda.cs.uiuc.edu/MET/.

  2. Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum.

    PubMed

    Grativol, Clícia; Regulski, Michael; Bertalan, Marcelo; McCombie, W Richard; da Silva, Felipe Rodrigues; Zerlotini Neto, Adhemar; Vicentini, Renato; Farinelli, Laurent; Hemerly, Adriana Silva; Martienssen, Robert A; Ferreira, Paulo Cavalcanti Gomes

    2014-07-01

    Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene-rich regions. Gene-enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene-enrichment strategy, we have compared assemblies using methyl-filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA-seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single-nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.

  3. Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

    PubMed Central

    Grativol, Clícia; Regulski, Michael; Bertalan, Marcelo; McCombie, W. Richard; da Silva, Felipe Rodrigues; Neto, Adhemar Zerlotini; Vicentini, Renato; Farinelli, Laurent; Hemerly, Adriana Silva; Martienssen, Robert A.; Ferreira, Paulo Cavalcanti Gomes

    2015-01-01

    SUMMARY Many economically important crops have large and complex genomes, which hampers sequencing of their genome by standard methods such as WGS. Large tracts of methylated repeats occur at plant genomes interspersed by hypomethylated gene-rich regions. Gene enrichment strategies based on methylation profile offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration (MF) with McrBC digestion to enrich for euchromatic regions of sugarcane genome. To verify the efficiency of MF and the assembly quality of sequences submitted to gene-enrichment strategy, we have compared assemblies using MF and unfiltered (UF) libraries. The MF allowed the achievement of a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5 times more scaffolds and 1.7 times more assembled Mb compared to unfiltered scaffolds. The coverage of sorghum CDS by MF scaffolds was at least 36% higher than by UF scaffolds. Using MF technology, we increased by 134X the coverage of genic regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds covering all genes at sugarcane BACs, 97.2% of sugarcane ESTs, 92.7% of sugarcane RNA-seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds encoding enzymes of the sucrose/starch pathway discovered 291 SNPs in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes were also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and improvement of sugarcane as a biofuel crop. PMID:24773339

  4. Pilot Sequencing of Onion Genomic DNA Reveals Fragments of Transposable Elements, Low Gene Densities, and Significant Gene Enrichment After Methyl Filtration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Onion (Allium cepa) is a diploid (2n=2x=16) monocot with one of the largest nuclear genomes among cultivated plants, over 6 and 16 times that of maize and rice, respectively. In this study, we sequenced onion BACs to estimate gene densities and investigate the nature and distribution of repetitive ...

  5. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    PubMed

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

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

  6. Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA.

    PubMed

    Gould, Matthew K; Bachmaier, Sabine; Ali, Juma A M; Alsford, Sam; Tagoe, Daniel N A; Munday, Jane C; Schnaufer, Achim C; Horn, David; Boshart, Michael; de Koning, Harry P

    2013-10-01

    One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

  7. pico-PLAZA, a genome database of microbial photosynthetic eukaryotes.

    PubMed

    Vandepoele, Klaas; Van Bel, Michiel; Richard, Guilhem; Van Landeghem, Sofie; Verhelst, Bram; Moreau, Hervé; Van de Peer, Yves; Grimsley, Nigel; Piganeau, Gwenael

    2013-08-01

    With the advent of next generation genome sequencing, the number of sequenced algal genomes and transcriptomes is rapidly growing. Although a few genome portals exist to browse individual genome sequences, exploring complete genome information from multiple species for the analysis of user-defined sequences or gene lists remains a major challenge. pico-PLAZA is a web-based resource (http://bioinformatics.psb.ugent.be/pico-plaza/) for algal genomics that combines different data types with intuitive tools to explore genomic diversity, perform integrative evolutionary sequence analysis and study gene functions. Apart from homologous gene families, multiple sequence alignments, phylogenetic trees, Gene Ontology, InterPro and text-mining functional annotations, different interactive viewers are available to study genome organization using gene collinearity and synteny information. Different search functions, documentation pages, export functions and an extensive glossary are available to guide non-expert scientists. To illustrate the versatility of the platform, different case studies are presented demonstrating how pico-PLAZA can be used to functionally characterize large-scale EST/RNA-Seq data sets and to perform environmental genomics. Functional enrichments analysis of 16 Phaeodactylum tricornutum transcriptome libraries offers a molecular view on diatom adaptation to different environments of ecological relevance. Furthermore, we show how complementary genomic data sources can easily be combined to identify marker genes to study the diversity and distribution of algal species, for example in metagenomes, or to quantify intraspecific diversity from environmental strains.

  8. Helitrons shaping the genomic architecture of Drosophila: enrichment of DINE-TR1 in α- and β-heterochromatin, satellite DNA emergence, and piRNA expression.

    PubMed

    Dias, Guilherme B; Heringer, Pedro; Svartman, Marta; Kuhn, Gustavo C S

    2015-09-01

    Drosophila INterspersed Elements (DINEs) constitute an abundant but poorly understood group of Helitrons present in several Drosophila species. The general structure of DINEs includes two conserved blocks that may or not contain a region with tandem repeats in between. These central tandem repeats (CTRs) are similar within species but highly divergent between species. It has been assumed that CTRs have independent origins. Herein, we identify a subset of DINEs, termed DINE-TR1, which contain homologous CTRs of approximately 150 bp. We found DINE-TR1 in the sequenced genomes of several Drosophila species and in Bactrocera tryoni (Acalyptratae, Diptera). However, interspecific high sequence identity (∼ 88 %) is limited to the first ∼ 30 bp of each tandem repeat, implying that evolutionary constraints operate differently over the monomer length. DINE-TR1 is unevenly distributed across the Drosophila phylogeny. Nevertheless, sequence analysis suggests vertical transmission. We found that CTRs within DINE-TR1 have independently expanded into satellite DNA-like arrays at least twice within Drosophila. By analyzing the genome of Drosophila virilis and Drosophila americana, we show that DINE-TR1 is highly abundant in pericentromeric heterochromatin boundaries, some telomeric regions and in the Y chromosome. It is also present in the centromeric region of one autosome from D. virilis and dispersed throughout several euchromatic sites in both species. We further found that DINE-TR1 is abundant at piRNA clusters, and small DINE-TR1-derived RNA transcripts (∼25 nt) are predominantly expressed in the testes and the ovaries, suggesting active targeting by the piRNA machinery. These features suggest potential piRNA-mediated regulatory roles for DINEs at local and genome-wide scales in Drosophila.

  9. Library 2000.

    ERIC Educational Resources Information Center

    Drake, Miriam A.

    In fall 1984, the Georgia Institute of Technology administration and library staff began planning for Library 2000, a project aimed at creating a showcase library to demonstrate the application of the latest information technology in an academic and research environment. The purposes of Library 2000 include: increasing awareness of students,…

  10. Library Computing.

    ERIC Educational Resources Information Center

    Dayall, Susan A.; And Others

    1987-01-01

    Six articles on computers in libraries discuss training librarians and staff to use new software; appropriate technology; system upgrades of the Research Libraries Group's information system; pre-IBM PC microcomputers; multiuser systems for small to medium-sized libraries; and a library user's view of the traditional card catalog. (EM)

  11. Library Buildings.

    ERIC Educational Resources Information Center

    Manley, Will; And Others

    1989-01-01

    The innovative designs of three libraries are described: the Tempe (Arizona) Public Library, which emphasizes services for children and students; an underground library at Park College, Missouri; and a public library located in the Vancouver (Washington) Mall. The fourth article describes the work going on to restore the Los Angeles (California)…

  12. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.

    PubMed

    Kuleshov, Maxim V; Jones, Matthew R; Rouillard, Andrew D; Fernandez, Nicolas F; Duan, Qiaonan; Wang, Zichen; Koplev, Simon; Jenkins, Sherry L; Jagodnik, Kathleen M; Lachmann, Alexander; McDermott, Michael G; Monteiro, Caroline D; Gundersen, Gregory W; Ma'ayan, Avi

    2016-07-08

    Enrichment analysis is a popular method for analyzing gene sets generated by genome-wide experiments. Here we present a significant update to one of the tools in this domain called Enrichr. Enrichr currently contains a large collection of diverse gene set libraries available for analysis and download. In total, Enrichr currently contains 180 184 annotated gene sets from 102 gene set libraries. New features have been added to Enrichr including the ability to submit fuzzy sets, upload BED files, improved application programming interface and visualization of the results as clustergrams. Overall, Enrichr is a comprehensive resource for curated gene sets and a search engine that accumulates biological knowledge for further biological discoveries. Enrichr is freely available at: http://amp.pharm.mssm.edu/Enrichr.

  13. A Microbiome DNA Enrichment Method for Next-Generation Sequencing Sample Preparation.

    PubMed

    Yigit, Erbay; Feehery, George R; Langhorst, Bradley W; Stewart, Fiona J; Dimalanta, Eileen T; Pradhan, Sriharsa; Slatko, Barton; Gardner, Andrew F; McFarland, James; Sumner, Christine; Davis, Theodore B

    2016-07-01

    "Microbiome" is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next-generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis. © 2016 by John Wiley & Sons, Inc.

  14. Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues1

    PubMed Central

    Yigit, Erbay; Hernandez, David I.; Trujillo, Joshua T.; Dimalanta, Eileen; Bailey, C. Donovan

    2014-01-01

    • Premise of the study: Variation in the distribution of methylated CpG (methyl-CpG) in genomic DNA (gDNA) across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2) to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich) vs. organellar and prokaryotic (methyl-CpG-poor) elements for genomic and metagenomic sequencing projects. • Methods: MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1) untreated gDNA, (2) a methyl-CpG-depleted fraction, and (3) a methyl-CpG-enriched fraction. • Results: Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2–11.2-fold and 3.4–11.3-fold increase in chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), respectively. Methyl-enriched fractions showed a 1.8–31.3-fold and 1.3–29.0-fold decrease in cpDNA and mtDNA, respectively. • Discussion: The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae). When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues. PMID:25383266

  15. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences

    PubMed Central

    Moran, Sebastian; Arribas, Carles; Esteller, Manel

    2016-01-01

    Aim: DNA methylation is the best known epigenetic mark. Cancer and other pathologies show an altered DNA methylome. However, delivering complete DNA methylation maps is compromised by the price and labor-intensive interpretation of single nucleotide methods. Material & methods: Following the success of the HumanMethylation450 BeadChip (Infinium) methylation microarray (450K), we report the technical and biological validation of the newly developed MethylationEPIC BeadChip (Infinium) microarray that covers over 850,000 CpG methylation sites (850K). The 850K microarray contains >90% of the 450K sites, but adds 333,265 CpGs located in enhancer regions identified by the ENCODE and FANTOM5 projects. Results & conclusion: The 850K array demonstrates high reproducibility at the 450K CpG sites, is consistent among technical replicates, is reliable in the matched study of fresh frozen versus formalin-fixed paraffin-embeded samples and is also useful for 5-hydroxymethylcytosine. These results highlight the value of the MethylationEPIC BeadChip as a useful tool for the analysis of the DNA methylation profile of the human genome. PMID:26673039

  16. Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.

    PubMed Central

    Lovett, M; Kere, J; Hinton, L M

    1991-01-01

    We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome. Images PMID:1946378

  17. Microfluidic droplet enrichment for targeted sequencing

    PubMed Central

    Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

    2015-01-01

    Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

  18. Special Libraries

    ERIC Educational Resources Information Center

    Lavendel, Giuliana

    1977-01-01

    Discusses problems involved in maintaining special scientific or engineering libraries, including budget problems, remote storage locations, rental computer retrieval systems, protecting trade secrets, and establishing a magnetic tape library. (MLH)

  19. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Exome Capture with Heterologous Enrichment in Pig (Sus scrofa).

    PubMed

    Guiatti, Denis; Pomari, Elena; Radovic, Slobodanka; Spadotto, Alessandro; Stefanon, Bruno

    2015-01-01

    The discovery of new protein-coding DNA variants related to carcass traits is very important for the Italian pig industry, which requires heavy pigs with higher thickness of subcutaneous fat for Protected Designation of Origin (PDO) productions. Exome capture techniques offer the opportunity to focus on the regions of DNA potentially related to the gene and protein expression. In this research a human commercial target enrichment kit was used to evaluate its performances for pig exome capture and for the identification of DNA variants suitable for comparative analysis. Two pools of 30 pigs each, crosses of Italian Duroc X Large White (DU) and Commercial hybrid X Large White (HY), were used and NGS libraries were prepared with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). A total of 140.2 M and 162.5 M of raw reads were generated for DU and HY, respectively. Average coverage of all the exonic regions for Sus scrofa (ENSEMBL Sus_scrofa.Sscrofa10.2.73.gtf) was 89.33X for DU and 97.56X for HY; and 35% of aligned bases uniquely mapped to off-target regions. Comparison of sequencing data with the Sscrofa10.2 reference genome, after applying hard filtering criteria, revealed a total of 232,530 single nucleotide variants (SNVs) of which 20.6% mapped in exonic regions and 49.5% within intronic regions. The comparison of allele frequencies of 213 randomly selected SNVs from exome sequencing and the same SNVs analyzed with a Sequenom MassARRAY® system confirms that this "human-on-pig" approach offers new potentiality for the identification of DNA variants in protein-coding genes.

  2. A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

    PubMed Central

    Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; Hamblin, Rachelle; Williams, Kelly P.

    2016-01-01

    When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs. PMID:28002481

  3. [Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway].

    PubMed

    Speranskii, A I; Kostyuk, S V; Kalashnikova, E A; Veiko, N N

    2016-03-01

    Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NF-kB signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNF-a in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNF-a synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.

  4. Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome.

    PubMed

    Rabinowicz, P D; Schutz, K; Dedhia, N; Yordan, C; Parnell, L D; Stein, L; McCombie, W R; Martienssen, R A

    1999-11-01

    The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required.

  5. Libraries program

    USGS Publications Warehouse

    2011-01-01

    The U.S. Congress authorized a library for the U.S. Geological Survey (USGS) in 1879. The library was formally established in 1882 with the naming of the first librarian and began with a staff of three and a collection of 1,400 books. Today, the USGS Libraries Program is one of the world's largest Earth and natural science repositories and a resource of national significance used by researchers and the public worldwide.

  6. America's Star Libraries: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  7. Preparation of PAC libraries. Final technical report

    SciTech Connect

    Pieter J. de Jong

    1997-12-31

    The goals of this project were to create P1 Artificial Chromosome (PAC) cloning vectors and use these vectors to generate, characterize, and distribute both human and mouse genomic PAC libraries to the scientific community.

  8. Library Advocacy

    ERIC Educational Resources Information Center

    Plunkett, Kate

    2010-01-01

    This paper is about the issue of advocacy. Standing at the vanguard of literacy, library media specialists have a unique role. However, it is time for media specialists to advocate their services in a proactive way. If library media specialists cannot, both individually and collectively, put advocacy at the forefront, then students will suffer the…

  9. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California…

  10. Library Research.

    ERIC Educational Resources Information Center

    Wright, Nancy Kirkpatrick

    This workbook, designed for a Library Research course at Yavapai College, provides 15 lessons in advanced library reference skills. Each lesson provides explanatory text and reinforcement exercises. After Lesson I introduces specialized dictionaries and encyclopedias (e.g., for foreign languages, medicine, music, economics, social sciences, and…

  11. Derived enriched uranium market

    SciTech Connect

    Rutkowski, E.

    1996-12-01

    The potential impact on the uranium market of highly enriched uranium from nuclear weapons dismantling in the Russian Federation and the USA is analyzed. Uranium supply, conversion, and enrichment factors are outlined for each country; inventories are also listed. The enrichment component and conversion components are expected to cause little disruption to uranium markets. The uranium component of Russian derived enriched uranium hexafluoride is unresolved; US legislation places constraints on its introduction into the US market.

  12. Making Your Own Gene Library.

    ERIC Educational Resources Information Center

    Perez-Ortin, Jose E.; Li Del Olmo, Marcel; Matallana, Emilia; Tordera, Vicente

    1997-01-01

    Presents an experiment aimed at constructing a genomic library that can be carried out over a week. Helps students learn concepts such as donor and vector DNAs, construction of recombinant DNA, host strain, and experiments in gene cloning more clearly. (PVD)

  13. Callpath Library

    SciTech Connect

    Gamblin, T.

    2013-11-09

    The "Callpath Library" is a software abstraction layer over a number of stack tracing utilities. It allows tool develoopers to conveniently represent and mNipulate call paths gathered fro U. Wisconsin's Stackwalker API and GNU Backtrace.

  14. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  15. Academic Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1970

    1970-01-01

    Building data is given for the following academic libraries: (1) Rosary College, River Forest, Illinois; (2) Abilene Christian College, Abilene, Texas; (3) University of California, San Diego, La Jolla, California. (MF)

  16. BaitFisher: A Software Package for Multispecies Target DNA Enrichment Probe Design.

    PubMed

    Mayer, Christoph; Sann, Manuela; Donath, Alexander; Meixner, Martin; Podsiadlowski, Lars; Peters, Ralph S; Petersen, Malte; Meusemann, Karen; Liere, Karsten; Wägele, Johann-Wolfgang; Misof, Bernhard; Bleidorn, Christoph; Ohl, Michael; Niehuis, Oliver

    2016-07-01

    Target DNA enrichment combined with high-throughput sequencing technologies is a powerful approach to probing a large number of loci in genomes of interest. However, software algorithms that explicitly consider nucleotide sequence information of target loci in multiple reference species for optimizing design of target enrichment baits to be applicable across a wide range of species have not been developed. Here we present an algorithm that infers target DNA enrichment baits from multiple nucleotide sequence alignments. By applying clustering methods and the combinatorial 1-center sequence optimization to bait design, we are able to minimize the total number of baits required to efficiently probe target loci in multiple species. Consequently, more loci can be probed across species with a given number of baits. Using transcript sequences of 24 apoid wasps (Hymenoptera: Crabronidae, Sphecidae) from the 1KITE project and the gene models of Nasonia vitripennis, we inferred 57,650, 120-bp-long baits for capturing 378 coding sequence sections of 282 genes in apoid wasps. Illumina reduced-representation library sequencing confirmed successful enrichment of the target DNA when applying these baits to DNA of various apoid wasps. The designed baits furthermore enriched a major fraction of the target DNA in distantly related Hymenoptera, such as Formicidae and Chalcidoidea, highlighting the baits' broad taxonomic applicability. The availability of baits with broad taxonomic applicability is of major interest in numerous disciplines, ranging from phylogenetics to biodiversity monitoring. We implemented our new approach in a software package, called BaitFisher, which is open source and freely available at https://github.com/cmayer/BaitFisher-package.git.

  17. Communities of purple sulfur bacteria in a Baltic Sea coastal lagoon analyzed by puf LM gene libraries and the impact of temperature and NaCl concentration in experimental enrichment cultures.

    PubMed

    Tank, Marcus; Blümel, Martina; Imhoff, Johannes F

    2011-12-01

    Shallow coastal waters, where phototrophic purple sulfur bacteria (PSB) regularly form massive blooms, are subjected to massive diurnal and event-driven changes of physicochemical conditions including temperature and salinity. To analyze the ability of PSB to cope with these environmental factors and to compete in complex communities we have studied changes of the environmental community of PSB of a Baltic Sea lagoon under experimental enrichment conditions with controlled variation of temperature and NaCl concentration. For the first time, changes within a community of PSB were specifically analyzed using the photosynthetic reaction center genes pufL and M by RFLP and cloning experiments. The most abundant PSB phylotypes in the habitat were found along the NaCl gradient from freshwater conditions up to 7.5% NaCl. They were accompanied by smaller numbers of purple nonsulfur bacteria and aerobic anoxygenic phototrophic bacteria. Major components of the PSB community of the brackish lagoon were affiliated to PSB genera and species known as marine, halophilic or salt-tolerant, including species of M arichromatium, H alochromatium, T hiorhodococcus, A llochromatium, T hiocapsa, T hiorhodovibrio, and T hiohalocapsa. A dramatic shift occurred at elevated temperatures of 41 and 44°C when M arichromatium gracile became most prominent which was not detected at lower temperatures.

  18. A First Insight into the Genome of the Filter-Feeder Mussel Mytilus galloprovincialis

    PubMed Central

    Murgarella, Maria; Puiu, Daniela; Novoa, Beatriz; Figueras, Antonio; Posada, David; Canchaya, Carlos

    2016-01-01

    Mussels belong to the phylum Mollusca, one of the largest and most diverse taxa in the animal kingdom. Despite their importance in aquaculture and in biology in general, genomic resources from mussels are still scarce. To broaden and increase the genomic knowledge in this family, we carried out a whole-genome sequencing study of the cosmopolitan Mediterranean mussel (Mytilus galloprovincialis). We sequenced its genome (32X depth of coverage) on the Illumina platform using three pair-end libraries with different insert sizes. The large number of contigs obtained pointed out a highly complex genome of 1.6 Gb where repeated elements seem to be widespread (~30% of the genome), a feature that is also shared with other marine molluscs. Notwithstanding the limitations of our genome sequencing, we were able to reconstruct two mitochondrial genomes and predict 10,891 putative genes. A comparative analysis with other molluscs revealed a gene enrichment of gene ontology categories related to multixenobiotic resistance, glutamate biosynthetic process, and the maintenance of ciliary structures. PMID:26977809

  19. A First Insight into the Genome of the Filter-Feeder Mussel Mytilus galloprovincialis.

    PubMed

    Murgarella, Maria; Puiu, Daniela; Novoa, Beatriz; Figueras, Antonio; Posada, David; Canchaya, Carlos

    2016-01-01

    Mussels belong to the phylum Mollusca, one of the largest and most diverse taxa in the animal kingdom. Despite their importance in aquaculture and in biology in general, genomic resources from mussels are still scarce. To broaden and increase the genomic knowledge in this family, we carried out a whole-genome sequencing study of the cosmopolitan Mediterranean mussel (Mytilus galloprovincialis). We sequenced its genome (32X depth of coverage) on the Illumina platform using three pair-end libraries with different insert sizes. The large number of contigs obtained pointed out a highly complex genome of 1.6 Gb where repeated elements seem to be widespread (~30% of the genome), a feature that is also shared with other marine molluscs. Notwithstanding the limitations of our genome sequencing, we were able to reconstruct two mitochondrial genomes and predict 10,891 putative genes. A comparative analysis with other molluscs revealed a gene enrichment of gene ontology categories related to multixenobiotic resistance, glutamate biosynthetic process, and the maintenance of ciliary structures.

  20. Personal Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2004-01-01

    Virtual libraries are becoming more and more common. Most states have a virtual library. A growing number of public libraries have a virtual presence on the Web. Virtual libraries are a growing addition to school library media collections. The next logical step would be personal virtual libraries. A personal virtual library (PVL) is a collection…

  1. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  2. Preparation of high-molecular weight DNA and metagenomic libraries from soils and hot springs.

    PubMed

    Reigstad, Laila J; Bartossek, Rita; Schleper, Christa

    2011-01-01

    Metagenomics has become an important tool for the characterization of microorganisms, as it is independent of their enrichment or cultivation in the laboratory. Its application has led to the discovery of metabolisms from widespread, yet uncharacterized organisms such as the ammonia-oxidizing archaea. Different approaches ranging from the generation of short sequence reads by direct use of high-throughput sequencing technologies to the construction and sequencing of large-insert DNA libraries are being employed. For these purposes, DNA of high quality needs to be prepared from an environmental sample, which is a particular challenge for soils and sediments. Here we describe the methods used for the isolation of high-molecular weight (hmw) DNA from soil and hot spring samples, the subsequent production of large-insert metagenomic libraries, and the analysis of the resulting genomic fragments. Detailed step-by-step procedures include (1) how to isolate good-quality hmw DNA from soils and mud; (2) how to prepare the DNA for cloning; (3) how to efficiently establish, grow, pick, replicate, and store the large-insert metagenomic fosmid library; and finally, (4) how to screen the library for genes of interest.

  3. Underground Libraries.

    ERIC Educational Resources Information Center

    Fuhlrott, Rolf

    1986-01-01

    Discussion of underground buildings constructed primarily during last two decades for various reasons (energy conservation, density of environment, preservation of landscape and historic buildings) notes advantages, disadvantages, and psychological and design considerations. Examples of underground libraries, built mainly in United States, are…

  4. Minnesota Zoological Garden Library.

    ERIC Educational Resources Information Center

    Norell, Angela

    1988-01-01

    Describes the history and functions of the Minnesota Zoological Garden library. Topics covered include the library collections; library services, including online search capabilities; and the various groups of users served by the library. (three references) (CLB)

  5. Metadata Harvesting in Regional Digital Libraries in the PIONIER Network

    ERIC Educational Resources Information Center

    Mazurek, Cezary; Stroinski, Maciej; Werla, Marcin; Weglarz, Jan

    2006-01-01

    Purpose: The paper aims to present the concept of the functionality of metadata harvesting for regional digital libraries, based on the OAI-PMH protocol. This functionality is a part of regional digital libraries platform created in Poland. The platform was required to reach one of main objectives of the Polish PIONIER Programme--to enrich the…

  6. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    PubMed

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  7. Principles and application of antibody libraries for infectious diseases.

    PubMed

    Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

    2014-12-01

    Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

  8. Turkish Libraries: Historical Context.

    ERIC Educational Resources Information Center

    Cakin, Irfan

    1984-01-01

    Summary of the development of libraries in Turkey highlights the existence of libraries in the ninth century, the Shamssaddin Altunaba Medrese library in Konya, libraries established during the Ottoman era, reports to reform libraries (1869-70, 1909), and reports and library developments attributed to the Republican Era beginning in 1923. (EJS)

  9. Library Research and Statistics.

    ERIC Educational Resources Information Center

    Lynch, Mary Jo; St. Lifer, Evan; Halstead, Kent; Fox, Bette-Lee; Miller, Marilyn L.; Shontz, Marilyn L.

    2001-01-01

    These nine articles discuss research and statistics on libraries and librarianship, including libraries in the United States, Canada, and Mexico; acquisition expenditures in public, academic, special, and government libraries; price indexes; state rankings of public library data; library buildings; expenditures in school library media centers; and…

  10. Methods for combinatorial and parallel library design.

    PubMed

    Schnur, Dora M; Beno, Brett R; Tebben, Andrew J; Cavallaro, Cullen

    2011-01-01

    Diversity has historically played a critical role in design of combinatorial libraries, screening sets and corporate collections for lead discovery. Large library design dominated the field in the 1990s with methods ranging anywhere from purely arbitrary through property based reagent selection to product based approaches. In recent years, however, there has been a downward trend in library size. This was due to increased information about the desirable targets gleaned from the genomics revolution and to the ever growing availability of target protein structures from crystallography and homology modeling. Creation of libraries directed toward families of receptors such as GPCRs, kinases, nuclear hormone receptors, proteases, etc., replaced the generation of libraries based primarily on diversity while single target focused library design has remained an important objective. Concurrently, computing grids and cpu clusters have facilitated the development of structure based tools that screen hundreds of thousands of molecules. Smaller "smarter" combinatorial and focused parallel libraries replaced those early un-focused large libraries in the twenty-first century drug design paradigm. While diversity still plays a role in lead discovery, the focus of current library design methods has shifted to receptor based methods, scaffold hopping/bio-isostere searching, and a much needed emphasis on synthetic feasibility. Methods such as "privileged substructures based design" and pharmacophore based design still are important methods for parallel and small combinatorial library design. This chapter discusses some of the possible design methods and presents examples where they are available.

  11. Now's the Time: Online Library Orientations

    ERIC Educational Resources Information Center

    Farrell, Sandy L.; Driver, Carol; Weathers, Anita

    2011-01-01

    Increasingly at West Kentucky Community and Technical College (WKCTC), English 101, English 102, and Enrichment 091 are taught online, allowing students more work availability and time with family, while decreasing commute time and expense. In order to meet the library orientation needs of these online students and provide other options for…

  12. Advances in clinical next-generation sequencing: target enrichment and sequencing technologies.

    PubMed

    Ballester, Leomar Y; Luthra, Rajyalakshmi; Kanagal-Shamanna, Rashmi; Singh, Rajesh R

    2016-01-01

    The huge parallel sequencing capabilities of next generation sequencing technologies have made them the tools of choice to characterize genomic aberrations for research and diagnostic purposes. For clinical applications, screening the whole genome or exome is challenging owing to the large genomic area to be sequenced, associated costs, complexity of data, and lack of known clinical significance of all genes. Consequently, routine screening involves limited markers with established clinical relevance. This process, referred to as targeted genome sequencing, requires selective enrichment of the genomic areas comprising these markers via one of several primer or probe-based enrichment strategies, followed by sequencing of the enriched genomic areas. Here, the authors review current target enrichment approaches and next generation sequencing platforms, focusing on the underlying principles, capabilities, and limitations of each technology along with validation and implementation for clinical testing.

  13. Enrichment Activities for Geometry.

    ERIC Educational Resources Information Center

    Usiskin, Zalman

    1983-01-01

    Enrichment activities that teach about geometry as they instruct in geometry are given for some significant topics. The facets of geometry included are tessellations, round robin tournaments, geometric theorems on triangles, and connections between geometry and complex numbers. (MNS)

  14. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on art libraries, librarianship, and documentation presented at the 1982 International Federation of Library Associations (IFLA) conference include: (1) "The Tyranny of Distance: Art Libraries in Canada," a description by Mary F. Williamson of Canada's regional art libraries which serve both art students and the general public;…

  15. Strengthening State Library Administrative Agency (Territorial Library).

    ERIC Educational Resources Information Center

    Nieves M. Flores Memorial Library, Agana, Guam.

    This document describes the Basic State Plan Amendments for the Library Services and Construction Act in Guam and the regulations promulgated thereunder. The major projects described under the plan are: Strengthening State Library Administrative Agency; Staff Development; Library Collections, Extention Services, Institutional Libraries; and…

  16. Isolation of cDNA and genomic DNA clones encoding type II collagen.

    PubMed Central

    Young, M F; Vogeli, G; Nunez, A M; Fernandez, M P; Sullivan, M; Sobel, M E

    1984-01-01

    A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs. Images PMID:6203098

  17. 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.

  18. 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

  19. Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

    NASA Technical Reports Server (NTRS)

    Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

    2003-01-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

  20. Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

    PubMed Central

    Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

    2003-01-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years. PMID:12676695

  1. Graph Library

    SciTech Connect

    Schulz, Martin; Arnold, Dorian

    2007-06-12

    GraphLib is a support library used by other tools to create, manipulate, store, and export graphs. It provides a simple interface to specifS’ arbitrary directed and undirected graphs by adding nodes and edges. Each node and edge can be associated with a set of attributes describing size, color, and shape. Once created, graphs can be manipulated using a set of graph analysis algorithms, including merge, prune, and path coloring operations. GraphLib also has the ability to export graphs into various open formats such as DOT and GML.

  2. Cell Libraries

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  3. Modification of the GS LT Paired-end Library Protocol for Constructing Longer Insert Size Libraries

    SciTech Connect

    Peng, Ze; Peng, Ze; Hamilton, Matthew; Ting, Sara; Tu, Hank; Goltsman, Eugene; Lapidus, Alla; Lucas, Susan; Cheng, Jan-Fang

    2008-05-22

    Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

  4. State Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2003-01-01

    Virtual library? Electronic library? Digital library? Online information network? These all apply to the growing number of Web-based resource collections managed by consortiums of state library entities. Some, like "INFOhio" and "KYVL" ("Kentucky Virtual Library"), have been available for a few years, but others are just starting. Searching for…

  5. Library Directions in 1988.

    ERIC Educational Resources Information Center

    DeCandido, GraceAnne A.

    1989-01-01

    Reviews major library issues and events of 1988, including: (1) the Federal Bureau of Investigation's Library Awareness Program; (2) cooperation with USSR libraries; (3) library finance; (4) preservation; and (5) special programing. News about a number of prominent library professionals is included in a sidebar. (MES)

  6. Genealogy and Libraries.

    ERIC Educational Resources Information Center

    Carothers, Diane Foxhill; And Others

    1983-01-01

    Ten articles describe genealogical collections of the National Archives and Library of Congress, state archives programs, academic research libraries, Allen County Public Library (Indiana), Newberry Library, Church of Jesus Christ of Latter-Day Saints genealogical library, and New England Historic Genealogical Society, and approaches to Black…

  7. Marketing the Virtual Library

    ERIC Educational Resources Information Center

    Fagan, Jody Condit

    2009-01-01

    Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

  8. Library Handbook for Faculty.

    ERIC Educational Resources Information Center

    Martinez, Angelina, Ed.

    Discussions of library resources, services and related activities as well as library materials selection and acquisition are provided for faculty to facilitate and enhance their use of the library. Included in the library resources section are books, periodicals, microforms, and special collections and archives. Instruction in library use,…

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

    PubMed

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

    2012-11-01

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

  10. Identification of novel cancer therapeutic targets using a designed and pooled shRNA library screen

    PubMed Central

    Oliver, David; Ji, Hao; Liu, Piaomu; Gasparian, Alexander; Gardiner, Ellen; Lee, Samuel; Zenteno, Adrian; Perinskaya, Lillian O.; Chen, Mengqian; Buckhaults, Phillip; Broude, Eugenia; Wyatt, Michael D.; Valafar, Homayoun; Peña, Edsel; Shtutman, Michael

    2017-01-01

    Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities specifically affecting neoplastic cells without similarly affecting normal cells. Here we performed sequencing-based screening of an shRNA library on a panel of cancer cells of different origins as well as normal cells. The shRNA library was designed to target a subset of genes previously identified using a whole genome screening approach. This focused shRNA library was infected into cells followed by analysis of enrichment and depletion of the shRNAs over the course of cell proliferation. We developed a bootstrap likelihood ratio test for the interpretation of the effects of multiple shRNAs over multiple cell line passages. Our analysis identified 44 genes whose depletion preferentially inhibited the growth of cancer cells. Among these genes ribosomal protein RPL35A, putative RNA helicase DDX24, and coatomer complex I (COPI) subunit ARCN1 most significantly inhibited growth of multiple cancer cell lines without affecting normal cell growth and survival. Further investigation revealed that the growth inhibition caused by DDX24 depletion is independent of p53 status underlining its value as a drug target. Overall, our study establishes a new approach for the analysis of proliferation-based shRNA selection strategies and identifies new targets for the development of cancer therapeutics. PMID:28223711

  11. Identification of novel cancer therapeutic targets using a designed and pooled shRNA library screen.

    PubMed

    Oliver, David; Ji, Hao; Liu, Piaomu; Gasparian, Alexander; Gardiner, Ellen; Lee, Samuel; Zenteno, Adrian; Perinskaya, Lillian O; Chen, Mengqian; Buckhaults, Phillip; Broude, Eugenia; Wyatt, Michael D; Valafar, Homayoun; Peña, Edsel; Shtutman, Michael

    2017-02-22

    Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities specifically affecting neoplastic cells without similarly affecting normal cells. Here we performed sequencing-based screening of an shRNA library on a panel of cancer cells of different origins as well as normal cells. The shRNA library was designed to target a subset of genes previously identified using a whole genome screening approach. This focused shRNA library was infected into cells followed by analysis of enrichment and depletion of the shRNAs over the course of cell proliferation. We developed a bootstrap likelihood ratio test for the interpretation of the effects of multiple shRNAs over multiple cell line passages. Our analysis identified 44 genes whose depletion preferentially inhibited the growth of cancer cells. Among these genes ribosomal protein RPL35A, putative RNA helicase DDX24, and coatomer complex I (COPI) subunit ARCN1 most significantly inhibited growth of multiple cancer cell lines without affecting normal cell growth and survival. Further investigation revealed that the growth inhibition caused by DDX24 depletion is independent of p53 status underlining its value as a drug target. Overall, our study establishes a new approach for the analysis of proliferation-based shRNA selection strategies and identifies new targets for the development of cancer therapeutics.

  12. Rapid plasmid library screening using RecA-coated biotinylated probes.

    PubMed

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M

    1986-12-01

    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  13. Laser and gas centrifuge enrichment

    SciTech Connect

    Heinonen, Olli

    2014-05-09

    Principles of uranium isotope enrichment using various laser and gas centrifuge techniques are briefly discussed. Examples on production of high enriched uranium are given. Concerns regarding the possibility of using low end technologies to produce weapons grade uranium are explained. Based on current assessments commercial enrichment services are able to cover the global needs of enriched uranium in the foreseeable future.

  14. MTTE: an innovative strategy for the evaluation of targeted/exome enrichment efficiency

    PubMed Central

    Klonowska, Katarzyna; Handschuh, Luiza; Swiercz, Aleksandra; Figlerowicz, Marek; Kozlowski, Piotr

    2016-01-01

    Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects. PMID:27572310

  15. Transposase mediated construction of RNA-seq libraries.

    PubMed

    Gertz, Jason; Varley, Katherine E; Davis, Nicholas S; Baas, Bradley J; Goryshin, Igor Y; Vaidyanathan, Ramesh; Kuersten, Scott; Myers, Richard M

    2012-01-01

    RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (∼1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

  16. Homecoming for Library Symbol.

    ERIC Educational Resources Information Center

    Egan, Bessie

    1987-01-01

    Discusses the significance and development of the library symbol and the history of its acceptance by the American Library Association (ALA) and the Canadian Library Association (CLA). Suggestions are made for its use. (CLB)

  17. Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment.

    PubMed

    Hewson, Ian; Moisander, Pia H; Morrison, Amanda E; Zehr, Jonathan P

    2007-05-01

    We investigated diazotrophic bacterioplankton assemblage composition in the Heron Reef lagoon (Great Barrier Reef, Australia) using culture-independent techniques targeting the nifH fragment of the nitrogenase gene. Seawater was collected at 3 h intervals over a period of 72 h (i.e. over diel as well as tidal cycles). An incubation experiment was also conducted to assess the impact of phosphate (PO(4)3*) availability on nifH expression patterns. DNA-based nifH libraries contained primarily sequences that were most similar to nifH from sediment, microbial mat and surface-associated microorganisms, with a few sequences that clustered with typical open ocean phylotypes. In contrast to genomic DNA sequences, libraries prepared from gene transcripts (mRNA amplified by reverse transcription-polymerase chain reaction) were entirely cyanobacterial and contained phylotypes similar to those observed in open ocean plankton. The abundance of Trichodesmium and two uncultured cyanobacterial phylotypes from previous studies (group A and group B) were studied by quantitative-polymerase chain reaction in the lagoon samples. These were detected as transcripts, but were not detected in genomic DNA. The gene transcript abundance of these phylotypes demonstrated variability over several diel cycles. The PO(4)3* enrichment experiment had a clearer pattern of gene expression over diel cycles than the lagoon sampling, however PO(4)3* additions did not result in enhanced transcript abundance relative to control incubations. The results suggest that a number of diazotrophs in bacterioplankton of the reef lagoon may originate from sediment, coral or beachrock surfaces, sloughing into plankton with the flooding tide. The presence of typical open ocean phylotype transcripts in lagoon bacterioplankton may indicate that they are an important component of the N cycle of the coral reef.

  18. Comprehensive long-span paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes.

    PubMed

    Hillmer, Axel M; Yao, Fei; Inaki, Koichiro; Lee, Wah Heng; Ariyaratne, Pramila N; Teo, Audrey S M; Woo, Xing Yi; Zhang, Zhenshui; Zhao, Hao; Ukil, Leena; Chen, Jieqi P; Zhu, Feng; So, Jimmy B Y; Salto-Tellez, Manuel; Poh, Wan Ting; Zawack, Kelson F B; Nagarajan, Niranjan; Gao, Song; Li, Guoliang; Kumar, Vikrant; Lim, Hui Ping J; Sia, Yee Yen; Chan, Chee Seng; Leong, See Ting; Neo, Say Chuan; Choi, Poh Sum D; Thoreau, Hervé; Tan, Patrick B O; Shahab, Atif; Ruan, Xiaoan; Bergh, Jonas; Hall, Per; Cacheux-Rataboul, Valère; Wei, Chia-Lin; Yeoh, Khay Guan; Sung, Wing-Kin; Bourque, Guillaume; Liu, Edison T; Ruan, Yijun

    2011-05-01

    Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.

  19. Science Student Enrichment Opportunities.

    ERIC Educational Resources Information Center

    California State Dept. of Education, Sacramento.

    This document was developed with the intention of increasing California public school students' awareness of and participation in science-related enrichment activities. Some of the activities are intended for participation by individuals, while others are meant for teams of students. These annual events are listed in chronological order for a…

  20. Generation of bioactive peptides by biological libraries.

    PubMed

    Mersich, Christa; Jungbauer, Alois

    2008-01-15

    Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.

  1. Virtual Library on Genetics from Oak Ridge National Laboratory

    DOE Data Explorer

    The World Wide Web (WWW) Virtual Library is a collaborative effort to provide topic indices that break down into many subtopics guiding users to vast resources of information around the world. ORNL hosts the Virtual Library on Genetics as part of the WWWVL's Biosciences topic area. The VL on Genetics is also a collection of links to information resources that supported the DOE Human Genome Project. That project has now evolved into Genomics: GTL. GTL is DOE's next step in genomics--builds on data and resources from the Human Genome Project, the Microbial Genome Program, and systems biology. GTL will accelerate understanding of dynamic living systems for solutions to DOE mission challenges in energy and the environment. The section of the Virtual Library on Genetics that is titled Organisms guides users to genetic information resources and gene sequences for animals, insects, microbes, and plant life.

  2. Shearing DNA for genomic library construction.

    PubMed

    Hengen, P N

    1997-07-01

    Methods and reagents is a unique monthly column that highlights current discussion in the newsgroup bionet.molibio.methds-reagnts, available on the internet. This month's column discusses the pros and cons of various techniques used to shear DNA for shotgun cloning. For details on how to partake in the newsgroup, see the accompanying box.

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

    SciTech Connect

    Phung, Wilson; Hack, Christopher; Shapiro, Harris; Lucas, Susan; Cheng, Jan-Fang

    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 of 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.

  4. Rhipicephalus (Boophilus) microplus strain Deutsch, whole genome shotgun sequencing project first submission of genome sequence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence difficult. Cot filtration/selection techniques were used to reduce the repetitive fraction of the tick genome and enrich for the fraction of DNA with gene-containing regions. The Cot-selected ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods.

    PubMed

    Lindner, Thomas; Kolmar, Harald; Haberkorn, Uwe; Mier, Walter

    2011-02-15

    Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  7. Automated recycling of chemistry for virtual screening and library design.

    PubMed

    Vainio, Mikko J; Kogej, Thierry; Raubacher, Florian

    2012-07-23

    An early stage drug discovery project needs to identify a number of chemically diverse and attractive compounds. These hit compounds are typically found through high-throughput screening campaigns. The diversity of the chemical libraries used in screening is therefore important. In this study, we describe a virtual high-throughput screening system called Virtual Library. The system automatically "recycles" validated synthetic protocols and available starting materials to generate a large number of virtual compound libraries, and allows for fast searches in the generated libraries using a 2D fingerprint based screening method. Virtual Library links the returned virtual hit compounds back to experimental protocols to quickly assess the synthetic accessibility of the hits. The system can be used as an idea generator for library design to enrich the screening collection and to explore the structure-activity landscape around a specific active compound.

  8. The New Library Professional

    ERIC Educational Resources Information Center

    Wilder, Stanley

    2007-01-01

    This article discusses what the growing generation gap among library employees mean for academic research libraries and for the profession. Viewed collectively, the members of the under-35 cohort are a harbinger of a new kind of academic library professional, one whose traits bear directly on the ability of libraries to thrive amid the continuing…

  9. Growing Competition for Libraries.

    ERIC Educational Resources Information Center

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  10. California: Library Information Technologies.

    ERIC Educational Resources Information Center

    Will, Barbara, Ed.

    1996-01-01

    Describes six information technology projects in California libraries, including Internet access in public libraries; digital library developments at the University of California, Berkeley; the World Wide Web home page for the state library; Pacific Bell's role in statewide connectivity; state government initiatives; and services of the state…

  11. The "Integrated Library System."

    ERIC Educational Resources Information Center

    Dowlin, Kenneth E.

    1985-01-01

    Reviews internal and external dimensions of library environment that must be taken into account by library managers when choosing an integrated library system. The selection, acquisition, and implementation stages of Maggie III--a computerized library system sensitive to the internal and external organizational environment--are described. (MBR)

  12. Libraries, Ebooks, and Competition

    ERIC Educational Resources Information Center

    Hellman, Eric

    2010-01-01

    People keep writing articles about how valuable libraries are, even with ebooks and the Internet. What people are overlooking is that the reason libraries are having such fits dealing with a changing environment is not that libraries are unrecognized as fountains of value, it's that libraries are so valuable that they attract voracious new…

  13. Libraries and Learning

    ERIC Educational Resources Information Center

    Rainie, Lee

    2016-01-01

    The majority of Americans think local libraries serve the educational needs of their communities and families pretty well and library users often outpace others in learning activities. But many do not know about key education services libraries provide. This report provides statistics on library usage and presents key education services provided…

  14. Chromatin enrichment for proteomics

    PubMed Central

    Kustatscher, Georg; Wills, Karen L. H.; Furlan, Cristina; Rappsilber, Juri

    2015-01-01

    During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a ‘snapshot’ of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses. PMID:25101823

  15. 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).

  16. Genetic engineering of syringyl-enriched lignin in plants

    DOEpatents

    Chiang, Vincent Lee; Li, Laigeng

    2004-11-02

    The present invention relates to a novel DNA sequence, which encodes a previously unidentified lignin biosynthetic pathway enzyme, sinapyl alcohol dehydrogenase (SAD) that regulates the biosynthesis of syringyl lignin in plants. Also provided are methods for incorporating this novel SAD gene sequence or substantially similar sequences into a plant genome for genetic engineering of syringyl-enriched lignin in plants.

  17. Microchip method for the enrichment of specific DNA sequences

    DOEpatents

    Mirzabekov, A.D.; Lysov, Y.P.; Shick, V.V.; Dubiley, S.A.

    1998-12-22

    A method for enriching specific genetic material sequences is provided, whereby oligonucleotide molecules complementary to the desired genetic material is first used to isolate the genetic material from a first source of genomic material. Then the genetic material is used as a label to isolate similar genetic sequences from other sources. 4 figs.

  18. Microchip method for the enrichment of specific DNA sequences

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Shick, Valentine Vladimirovich; Dubiley, Svetlana Alekseevna

    1998-01-01

    A method for enriching specific genetic material sequences is provided, whereby oligonucleotide molecules complementary to the desired genetic material is first used to isolate the genetic material from a first source of genomic material. Then the genetic material is used as a label to isolate similar genetic sequences from other sources.

  19. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

  20. Differentiating between monozygotic twins through next-generation mitochondrial genome sequencing.

    PubMed

    Wang, Zheng; Zhu, Ruxin; Zhang, Suhua; Bian, Yinnan; Lu, Daru; Li, Chengtao

    2015-12-01

    Monozygotic (MZ) twins, considered to be genetically identical, cannot be distinguished from one another by standard forensic DNA testing. A recent study employed whole genome sequencing to identify extremely rare mutations and reported that mutation analysis could be used to differentiate between MZ twins. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has higher mutation rates; therefore, minor differences theoretically exist in MZ twins' mitochondrial genome (mtGenome). However, conventional Sanger-type sequencing (STS) is neither amenable to, nor feasible for, the detection of low-level sequence variants. The recent introduction of massively parallel sequencing (MPS) has the capability to sequence many targeted regions of multiple samples simultaneously with desirable depth of coverage. Thus, the aim of this study was to assess whether full mtGenome sequencing analysis can be used to differentiate between MZ twins. Ten sets of MZ twins provided blood samples that underwent extraction, quantification, mtDNA enrichment, library preparation, and ultra-deep sequencing. Point heteroplasmies were observed in eight sets of MZ twins, and a single nucleotide variant (nt15301) was detected in five sets of MZ twins. Thus, this study demonstrates that ultra-deep mtGenome sequencing could be used to differentiate between MZ twins.

  1. America's Star Libraries, 2010: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  2. Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells

    PubMed Central

    2012-01-01

    Background The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. Results Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. Conclusions HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known. PMID:22458358

  3. JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC.

    PubMed

    Cui, Libin; Chung, Tzu Hung; Tan, Darany; Sun, Xueguang; Jia, Xi-Yu

    2014-11-01

    We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and next-generation sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylated in vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5h from DNA inputs as low as 50ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling.

  4. Development of bacterial artificial chromosome library resources for parasitoid Hymenoptera (Nasonia vitripennis and Nasonia giraulti: Pteromalidae).

    PubMed

    Muñoz-Torres, M C; Saski, C; Blackmon, B; Romero-Severson, J; Werren, J H

    2010-02-01

    The species of the genus Nasonia possess qualities that make them excellent candidates for genetic and genomic studies. To increase the wealth of genomic resources for the genus we constructed publicly available bacterial artificial chromosome (BAC) libraries for Nasonia vitripennis and Nasonia giraulti. Libraries have 36 864 clones each, empty-vector contents of approximately 2% and average insert sizes of 113.1 and 97.7 Kb, respectively, representing 12 and 11 genome equivalents. The N. vitripennis library was used for genome sequence assembly and in efforts at positional cloning of a developmental gene. The genome assembly of N. vitripennis is currently composed on 6181 un-joined scaffolds. These BAC libraries can be used to identify and close regions between scaffolds of the genome assemblies of both species.

  5. Construction and characterization of two bacterial artificial chromosome libraries of grass carp.

    PubMed

    Jang, Songhun; Liu, Hang; Su, Jianguo; Dong, Feng; Xiong, Feng; Liao, Lanjie; Wang, Yaping; Zhu, Zuoyan

    2010-06-01

    Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4 x haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3 x haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.

  6. Summer library reading programs.

    PubMed

    Fiore, Carole D

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore summarizes some of the research in the field and relates it to library programs and usage by students. Several traditional and innovative programs from U.S. and Canadian libraries are described. She concludes with a call for further research related to summer library reading programs.

  7. 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.

  8. BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

    PubMed Central

    2011-01-01

    Background Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing. PMID:21595870

  9. Library Advocacy Now! Library Advocate's Handbook. [Videotape.

    ERIC Educational Resources Information Center

    American Library Association Video/Library Video Network, Towson, MD.

    Libraries are one of the world's greatest assets. Changes in the political, social, and economic climate in the U.S. mean that people cannot take public access to information for granted. Intense competition for public, private, and institutional dollars makes it more crucial than ever that policymakers understand that libraries--public, school,…

  10. The Virtual Library: Computers in Libraries Canada.

    ERIC Educational Resources Information Center

    Saunders, Laverna

    1992-01-01

    Reports on a postconference workshop on virtual libraries that was presented at the third annual Computers in Libraries Canada Conference. Highlights include networks; technological change; administrative issues, including organizational structure, staff training, strategic planning, and financial resources; a case study of the University of…

  11. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on art libraries and information services for the arts, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) "'I See All': Information Technology and the Universal Availability of Images" by Philip Pacey (United Kingdom); (2) "Online Databases in the Fine Arts"…

  12. Administrative Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Six papers on the theme, "Administrative Libraries in a Technological World: Past, Present, and Future--A General Stocktaking and a State of the Art Report" focus on West German telecommunications and information technology and the use of data processing and other technological aids in West German administrative libraries. These papers,…

  13. Multipurpose Transposon-Insertion Libraries in Yeast.

    PubMed

    Kumar, Anuj

    2016-06-01

    Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds.

  14. Advances in plant chromosome genomics.

    PubMed

    Doležel, Jaroslav; Vrána, Jan; Cápal, Petr; Kubaláková, Marie; Burešová, Veronika; Simková, Hana

    2014-01-01

    Next generation sequencing (NGS) is revolutionizing genomics and is providing novel insights into genome organization, evolution and function. The number of plant genomes targeted for sequencing is rising. For the moment, however, the acquisition of full genome sequences in large genome species remains difficult, largely because the short reads produced by NGS platforms are inadequate to cope with repeat-rich DNA, which forms a large part of these genomes. The problem of sequence redundancy is compounded in polyploids, which dominate the plant kingdom. An approach to overcoming some of these difficulties is to reduce the full nuclear genome to its individual chromosomes using flow-sorting. The DNA acquired in this way has proven to be suitable for many applications, including PCR-based physical mapping, in situ hybridization, forming DNA arrays, the development of DNA markers, the construction of BAC libraries and positional cloning. Coupling chromosome sorting with NGS offers opportunities for the study of genome organization at the single chromosomal level, for comparative analyses between related species and for the validation of whole genome assemblies. Apart from the primary aim of reducing the complexity of the template, taking a chromosome-based approach enables independent teams to work in parallel, each tasked with the analysis of a different chromosome(s). Given that the number of plant species tractable for chromosome sorting is increasing, the likelihood is that chromosome genomics - the marriage of cytology and genomics - will make a significant contribution to the field of plant genetics.

  15. Sequencing technologies and genome sequencing.

    PubMed

    Pareek, Chandra Shekhar; Smoczynski, Rafal; Tretyn, Andrzej

    2011-11-01

    The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.

  16. Technology for Libraries.

    ERIC Educational Resources Information Center

    Phenix, Katharine; And Others

    1990-01-01

    Five articles discuss information technology in libraries: (1) "Software for Libraries" (Katharine Phenix); (2) "Online Update: European Online Services" (Martin Kesselman); (3) "Connect Time: Online Pricing Breakthroughs" (Barbara Quint); (4) "Microcomputing: Micro Biology Computer Viruses" (James LaRue);…

  17. Community, Library, and Revolution

    ERIC Educational Resources Information Center

    Freiser, Leonard H.

    1970-01-01

    Discusses the need for greatly expanded community library services, for librarians to accept social responsibility for education and information, and for the library to become a program for getting information to where it is needed. (JS)

  18. Selecting Library Furniture & Equipment.

    ERIC Educational Resources Information Center

    Media & Methods, 1997

    1997-01-01

    Offers suggestions for selecting school library furniture and equipment. Describes various models of computer workstations; reading tables and chairs; and shelving. Sidebar lists names and addresses of library furniture manufactures and distributors. (AEF)

  19. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 2003

    2003-01-01

    Describes the designs of the Ferris State University Library for Information, Technology and Education (FLITE), and the Meyer Library and Information Technology Center at Southwest Missouri State University. Includes photographs. (EV)

  20. Small Libraries, Big Technology

    ERIC Educational Resources Information Center

    Roberts,Gary

    2005-01-01

    Small libraries don't have the resources to adopt every new technology. It is important that small libraries operate strategically, adopting only those technologies that are the most beneficial to their patrons.

  1. Genomics of hybrid poplar (Populus trichocarpax deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar.

    PubMed

    Ralph, Steven; Oddy, Claire; Cooper, Dawn; Yueh, Hesther; Jancsik, Sharon; Kolosova, Natalia; Philippe, Ryan N; Aeschliman, Dana; White, Rick; Huber, Dezene; Ritland, Carol E; Benoit, François; Rigby, Tracey; Nantel, André; Butterfield, Yaron S N; Kirkpatrick, Robert; Chun, Elizabeth; Liu, Jerry; Palmquist, Diana; Wynhoven, Brian; Stott, Jeffrey; Yang, George; Barber, Sarah; Holt, Robert A; Siddiqui, Asim; Jones, Steven J M; Marra, Marco A; Ellis, Brian E; Douglas, Carl J; Ritland, Kermit; Bohlmann, Jörg

    2006-04-01

    As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full-length cDNA libraries, we generated 139,007 3'- or 5'-end sequenced ESTs, representing more than one-third of the c. 385,000 publicly available Populus ESTs. Clustering and assembly of 107,519 3'-end ESTs resulted in 14,451 contigs and 20,560 singletons, altogether representing 35,011 putative unique transcripts, or potentially more than three-quarters of the predicted c. 45,000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15,496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up-regulated, compared to only 537 down-regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1-aminocyclopropane-1-carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (-)-germacrene D synthase] and transcriptional regulation [e.g. leucine-rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defences in a woody perennial providing a solid platform for

  2. New York Zoological Society Library.

    ERIC Educational Resources Information Center

    Johnson, Steven P.

    1988-01-01

    Describes the institutional setting, history, and services of the New York Zoological Society Library. Topics covered include clientele; library collections and special collections; library staffing and organizational structure; computer applications; and relationships with other libraries. (11 references) (CLB)

  3. Thermal breeder fuel enrichment zoning

    DOEpatents

    Capossela, Harry J.; Dwyer, Joseph R.; Luce, Robert G.; McCoy, Daniel F.; Merriman, Floyd C.

    1992-01-01

    A method and apparatus for improving the performance of a thermal breeder reactor having regions of higher than average moderator concentration are disclosed. The fuel modules of the reactor core contain at least two different types of fuel elements, a high enrichment fuel element and a low enrichment fuel element. The two types of fuel elements are arranged in the fuel module with the low enrichment fuel elements located between the high moderator regions and the high enrichment fuel elements. Preferably, shim rods made of a fertile material are provided in selective regions for controlling the reactivity of the reactor by movement of the shim rods into and out of the reactor core. The moderation of neutrons adjacent the high enrichment fuel elements is preferably minimized as by reducing the spacing of the high enrichment fuel elements and/or using a moderator having a reduced moderating effect.

  4. Advanced Neutron Source enrichment study

    SciTech Connect

    Bari, R.A.; Ludewig, H.; Weeks, J.R.

    1994-12-31

    A study has been performed of the impact on performance of using low enriched uranium (20% {sup 235}U) or medium enriched uranium (35% {sup 235}U) as an alternative fuel for the Advanced Neutron Source, which is currently designed to use uranium enriched to 93% {sup 235}U. Higher fuel densities and larger volume cores were evaluated at the lower enrichments in terms of impact on neutron flux, safety, safeguards, technical feasibility, and cost. The feasibility of fabricating uranium silicide fuel at increasing material density was specifically addressed by a panel of international experts on research reactor fuels. The most viable alternative designs for the reactor at lower enrichments were identified and discussed. Several sensitivity analyses were performed to gain an understanding of the performance of the reactor at parametric values of power, fuel density, core volume, and enrichment that were interpolations between the boundary values imposed on the study or extrapolations from known technology.

  5. A Century of Change: The Evolution of School Library Resources, 1915-2015

    ERIC Educational Resources Information Center

    Lamb, Annette

    2015-01-01

    School libraries have been in existence since at least the eighth century. However, it wasn't until the twentieth century that the school library was seen primarily as "a source of enrichment for the curriculum, and a means of developing reading and study habits in the pupils" (Clyde 1981, 263). While the formats available and tools for…

  6. Library Community Services.

    ERIC Educational Resources Information Center

    Correy, Therese; And Others

    Services to the elderly, institutionalized, and physically and mentally exceptional, who are unable to use the public library in its traditional form, are described in this guide to programs at the Inglewood (California) Public Library. Topics include: (1) overall library goals and activities; (2) functional and organizational structure of…

  7. Art for Libraries' Sake.

    ERIC Educational Resources Information Center

    Lugo, Mark-Elliot

    1999-01-01

    Illustrates the benefits of an aggressive library program of regularly scheduled and professionally curated art exhibitions and related events. Describes the Visual Arts Program at the Pacific Beach branch library (San Diego). A sidebar by Debra Wilcox Johnson discusses libraries' development of cultural programming for adults. (AEF)

  8. School Library Policy Statement.

    ERIC Educational Resources Information Center

    Manitoba Dept. of Education, Winnipeg. Instructional Resources Branch.

    The School Library Policy Statement for Manitoba schools begins with the mission statement of Manitoba Education and Training and the Goals of Learning for Manitoba. Statements of Manitoba's School Library Policy and the Philosophy of the School Library Program are also provided, together with an outline of the responsibilities of both Manitoba's…

  9. Virtual Libraries: Service Realities.

    ERIC Educational Resources Information Center

    Novak, Jan

    2002-01-01

    Discussion of changes in society that have resulted from information and communication technologies focuses on changes in libraries and a new market for library services with new styles of clients. Highlights client service issues to be considered when transitioning to a virtual library situation. (Author/LRW)

  10. Technostress and Library Values.

    ERIC Educational Resources Information Center

    Gorman, Michael

    2001-01-01

    Discusses information overload and society's and libraries' responses to technology. Considers eight values that libraries should focus on and how they relate to technology in libraries: democracy, stewardship, service, intellectual freedom, privacy, rationalism, equity of access, and building harmony and balance. (LRW)

  11. The Occasional Library Newsletter

    ERIC Educational Resources Information Center

    Gustafson, Chris

    2004-01-01

    An important tool for communication and collaboration is the library newsletter, which the library media specialist writes to the staff, teachers and library media specialist from other schools. The newsletter includes upcoming events teachers can sign up for, information on web sites to be used for projects, curriculum ideas, introduction of a…

  12. Library Studies I Workbook.

    ERIC Educational Resources Information Center

    Frost, William J.

    Developed for use in the Library Studies I component of the Library Studies Program at Bloomsburg University (Pennsylvania), this self-paced workbook is intended to acquaint students with the Harvey A. Andruss Library and help them develop information-seeking skills. The workbook is designed to be used in conjunction with an exercise book, and…

  13. PAL: Positional Astronomy Library

    NASA Astrophysics Data System (ADS)

    Jenness, T.; Berry, D. S.

    2016-06-01

    The PAL library is a partial re-implementation of Pat Wallace's popular SLALIB library written in C using a Gnu GPL license and layered on top of the IAU's SOFA library (or the BSD-licensed ERFA) where appropriate. PAL attempts to stick to the SLA C API where possible.

  14. Tomorrow's Library Today.

    ERIC Educational Resources Information Center

    Penniman, W. David

    1987-01-01

    Presents a model for predicting and shaping the impact of information technologies on libraries and the library role which is based on the effects of computerization in various business applications. The library network at AT&T Bell Laboratories is described as an example of a successful implementation of the model. (CLB)

  15. Place as Library?

    ERIC Educational Resources Information Center

    Davenport, Nancy

    2006-01-01

    Digital technology is redrawing the library's blueprint. Planners are thinking in new ways about how to design libraries as places for learning rather than primarily as storehouses of information. This thinking has given rise to much discussion--and to many publications--about the "library as place." In this article, the author asks why not also…

  16. Worthington Libraries, OH

    ERIC Educational Resources Information Center

    Berry, John N., III

    2007-01-01

    Worthington, Ohio, has deep library roots. A library has been part of its history since the planning by settlers before the city's birth in 1803. Among the treasures brought by James Kilbourne and the Scioto Company from Connecticut to the new, planned community they built was a collection of books for their new subscription library. The books…

  17. California Library Laws, 2009

    ERIC Educational Resources Information Center

    Smith, Paul G., Ed.

    2009-01-01

    California Library Laws 2009 is a selective guide to state laws and related materials that most directly affect the everyday operations of public libraries and organizations that work with public libraries. It is intended as a convenient reference, not as a replacement for the annotated codes or for legal advice. The guide is organized as follows.…

  18. California Library Laws, 2008

    ERIC Educational Resources Information Center

    Smith, Paul G., Ed.

    2008-01-01

    "California Library Laws 2008" is a selective guide to state laws and related materials that most directly affect the everyday operations of public libraries and organizations that work with public libraries. It is intended as a convenient reference, not as a replacement for the annotated codes or for legal advice. The guide is organized…

  19. Digital Libraries: An Overview.

    ERIC Educational Resources Information Center

    Schwartz, Candy

    2000-01-01

    Provides an overview of the basic components of a digital library. Highlights include library collections; metadata; services, including information seeking and retrieval, reference query fulfillment, and user training; user interaction with digital libraries, including searching, browsing, and navigation; economic support; maintenance;…

  20. Marketing Academic Libraries

    ERIC Educational Resources Information Center

    Mallon, Melissa, Ed.

    2013-01-01

    Ask any academic librarian if marketing their library and its services is an important task, and the answer will most likely be a resounding "yes!" Particularly in economically troubled times, librarians are increasingly called upon to promote their services and defend their library's worth. Since few academic libraries have in-house marketing…

  1. Spanish Museum Libraries Network.

    ERIC Educational Resources Information Center

    Lopez de Prado, Rosario

    This paper describes the creation of an automated network of museum libraries in Spain. The only way in which the specialized libraries in the world today can continue to be active and to offer valid information is to automate the service they offer, and create network libraries with cooperative plans. The network can be configured with different…

  2. LIBRARY SYSTEMS ANALYSIS.

    ERIC Educational Resources Information Center

    HIEBER, CAROLINE E.; TAYLOR, ROBERT S.

    STEPS THAT THE LIBRARY CAN TAKE IN PREPARING FOR A CHANGE TO AN AUTOMATED SYSTEM WERE INVESTIGATED. THE METHOD DESCRIBED WAS BASED ON AN ANALYSIS OF CLERICAL AND OFFICE WORK CALLED INTEGRATED PROCEDURES OF CONTROL (IPC). THE APPLICATION OF LIBRARY SYSTEMS ANALYSIS (LISA) TO THE LEHIGH UNIVERSITY LIBRARY IS DESCRIBED. THIS PROCESS INVOLVED (1)…

  3. Library Journal Classics.

    ERIC Educational Resources Information Center

    Crunden, Frederick; And Others

    1993-01-01

    Includes four articles originally published in the 1890s. Highlights include a discussion of freedom of speech, political opinions, and the role of the public library in social reform; the establishment of a congressional or national library; censorship in libraries; and a criticism of a librarian by his assistant. (LRW)

  4. Library Journal Classics.

    ERIC Educational Resources Information Center

    Berninghausen, David; And Others

    1993-01-01

    Provides excerpts from David Berninghausens's "Social Responsibility vs. the Library Bill of Rights" and responses that appeared in "Library Journal" in 1972 and 1973 because of the continuing debate over the role of the American Library Association (ALA) Social Responsibilities Round Table and whether or not ALA should take…

  5. The Library Morphs

    ERIC Educational Resources Information Center

    Waters, John K.

    2008-01-01

    As campus renovation projects go, the Ohio State University's plan to turn its main library into "a library for the 21st century" is ambitious. The author describes the decade-long, $109 million transformation of the William Oxley Thompson Memorial Library. The overhaul calls for a complete replacement of all mechanical and electrical…

  6. Library Building and Design.

    ERIC Educational Resources Information Center

    Jones, David J.; Gordon, Heather; Caddy, Julie; Kahlert, Maureen; Johnson, Carolyn; Holdstock, Fiona

    1997-01-01

    More frequently, community connections are being expressed in library design briefs and reflected in the completed buildings. This collection of brief articles discusses community involvement in library design and services and describes library construction projects in Australia and Malaysia. Also, discusses community art programs, integrating…

  7. Libraries and the Environment.

    ERIC Educational Resources Information Center

    LaRue, James; And Others

    1991-01-01

    Three articles address issues that relate to libraries and the environment. Highlights include recycling projects; buying recycled paper products and other ecology-minded purchasing ideas; energy-efficient libraries; indoor pollution problems; a list of environmental information sources; designing library buildings; and activities that libraries…

  8. Summer Library Reading Programs

    ERIC Educational Resources Information Center

    Fiore, Carole D.

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore…

  9. Changing State Digital Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2006-01-01

    Research has shown that state virtual or digital libraries are evolving into websites that are loaded with free resources, subscription databases, and instructional tools. In this article, the author explores these evolving libraries based on the following questions: (1) How user-friendly are the state digital libraries?; (2) How do state digital…

  10. Concepts of Library Goodness.

    ERIC Educational Resources Information Center

    Buckland, Michael K.

    1982-01-01

    Orr's schema for measuring the quality and value of libraries is described and discussed with respect to three paradoxes--the evaluation of catalogs, optimal library size, and Lenin's view of public libraries in the U.S. Seven references are cited. (CHC)

  11. Public Libraries Going Green

    ERIC Educational Resources Information Center

    Miller, Kathryn

    2010-01-01

    Going green is now a national issue, and patrons expect their library to respond in the same way many corporations have. Libraries are going green with logos on their Web sites, programs for the public, and a host of other initiatives. This is the first book to focus strictly on the library's role in going green, helping you with: (1) Collection…

  12. A Truly Bookless Library

    ERIC Educational Resources Information Center

    Kolowich, Steve

    2011-01-01

    The difference between the University of Texas at San Antonio's Applied Engineering and Technology Library and other science-focused libraries is not that its on-site collection is also available electronically. It is that its on-site collection is only available electronically. The idea of libraries with no bound books has been a recurring theme…

  13. School Libraries and Innovation

    ERIC Educational Resources Information Center

    McGrath, Kevin G.

    2015-01-01

    School library programs have measured success by improved test scores. But how do next-generation school libraries demonstrate success as they strive to be centers of innovation and creativity? These libraries offer solutions for school leaders who struggle to restructure existing systems built around traditional silos of learning (subjects and…

  14. 16. VIEW OF THE ENRICHED URANIUM RECOVERY SYSTEM. ENRICHED URANIUM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. VIEW OF THE ENRICHED URANIUM RECOVERY SYSTEM. ENRICHED URANIUM RECOVERY PROCESSED RELATIVELY PURE MATERIALS AND SOLUTIONS AND SOLID RESIDUES WITH RELATIVELY LOW URANIUM CONTENT. URANIUM RECOVERY INVOLVED BOTH SLOW AND FAST PROCESSES. (4/4/66) - Rocky Flats Plant, General Manufacturing, Support, Records-Central Computing, Southern portion of Plant, Golden, Jefferson County, CO

  15. Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.

    PubMed

    Howard, Thomas P; Hayward, Andrew P; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A; Tohme, Joe; Kausch, Albert P; Mottinger, John P; Dellaporta, Stephen L

    2014-01-01

    Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.

  16. Assessing Library Automation and Virtual Library Development in Four Academic Libraries in Oyo, Oyo State, Nigeria

    ERIC Educational Resources Information Center

    Gbadamosi, Belau Olatunde

    2011-01-01

    The paper examines the level of library automation and virtual library development in four academic libraries. A validated questionnaire was used to capture the responses from academic librarians of the libraries under study. The paper discovers that none of the four academic libraries is fully automated. The libraries make use of librarians with…

  17. Flight Software Math Library

    NASA Technical Reports Server (NTRS)

    McComas, David

    2013-01-01

    The flight software (FSW) math library is a collection of reusable math components that provides typical math utilities required by spacecraft flight software. These utilities are intended to increase flight software quality reusability and maintainability by providing a set of consistent, well-documented, and tested math utilities. This library only has dependencies on ANSI C, so it is easily ported. Prior to this library, each mission typically created its own math utilities using ideas/code from previous missions. Part of the reason for this is that math libraries can be written with different strategies in areas like error handling, parameters orders, naming conventions, etc. Changing the utilities for each mission introduces risks and costs. The obvious risks and costs are that the utilities must be coded and revalidated. The hidden risks and costs arise in miscommunication between engineers. These utilities must be understood by both the flight software engineers and other subsystem engineers (primarily guidance navigation and control). The FSW math library is part of a larger goal to produce a library of reusable Guidance Navigation and Control (GN&C) FSW components. A GN&C FSW library cannot be created unless a standardized math basis is created. This library solves the standardization problem by defining a common feature set and establishing policies for the library s design. This allows the libraries to be maintained with the same strategy used in its initial development, which supports a library of reusable GN&C FSW components. The FSW math library is written for an embedded software environment in C. This places restrictions on the language features that can be used by the library. Another advantage of the FSW math library is that it can be used in the FSW as well as other environments like the GN&C analyst s simulators. This helps communication between the teams because they can use the same utilities with the same feature set and syntax.

  18. Partition enrichment of nucleotide sequences (PINS)--a generally applicable, sequence based method for enrichment of complex DNA samples.

    PubMed

    Kvist, Thomas; Sondt-Marcussen, Line; Mikkelsen, Marie Just

    2014-01-01

    The dwindling cost of DNA sequencing is driving transformative changes in various biological disciplines including medicine, thus resulting in an increased need for routine sequencing. Preparation of samples suitable for sequencing is the starting point of any practical application, but enrichment of the target sequence over background DNA is often laborious and of limited sensitivity thereby limiting the usefulness of sequencing. The present paper describes a new method, Probability directed Isolation of Nucleic acid Sequences (PINS), for enrichment of DNA, enabling the sequencing of a large DNA region surrounding a small known sequence. A 275,000 fold enrichment of a target DNA sample containing integrated human papilloma virus is demonstrated. Specifically, a sample containing 0.0028 copies of target sequence per ng of total DNA was enriched to 786 copies per ng. The starting concentration of 0.0028 target copies per ng corresponds to one copy of target in a background of 100,000 complete human genomes. The enriched sample was subsequently amplified using rapid genome walking and the resulting DNA sequence revealed not only the sequence of a the truncated virus, but also 1026 base pairs 5' and 50 base pairs 3' to the integration site in chromosome 8. The demonstrated enrichment method is extremely sensitive and selective and requires only minimal knowledge of the sequence to be enriched and will therefore enable sequencing where the target concentration relative to background is too low to allow the use of other sample preparation methods or where significant parts of the target sequence is unknown.

  19. Integration of Library Services in the System of Digital Libraries

    NASA Astrophysics Data System (ADS)

    Bezdushnyj, A. N.; Kalenov, N. E.; Kouriv, P. M.; Serebryakov, V. A.

    The article is devoted to the functional extension of the Integrated System of Information Resources of RAS (ISIR RAS - RFBR 99-07-90139 project). This extension (Library Subsystem) provides integration of library services in ISIR RAS electronic libraries system. Library Subsystem implements standard library services such as information about free and ordered copies of editions, creation of the library orders for edition etc. The Library Subsystem is an integrated solution; it's based on ISIR data model and technologies.

  20. Library Resources for Bac End Sequencing. Final Technical Report

    SciTech Connect

    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 been constructed. RPCI-23 contain 576 plates (approx 210,000 clones) and represents approximately 11-fold coverage of the mouse genome.

  1. Characterization of the genome of bald cypress

    PubMed Central

    2011-01-01

    Background Bald cypress (Taxodium distichum var. distichum) is a coniferous tree of tremendous ecological and economic importance. It is a member of the family Cupressaceae which also includes cypresses, redwoods, sequoias, thujas, and junipers. While the bald cypress genome is more than three times the size of the human genome, its 1C DNA content is amongst the smallest of any conifer. To learn more about the genome of bald cypress and gain insight into the evolution of Cupressaceae genomes, we performed a Cot analysis and used Cot filtration to study Taxodium DNA. Additionally, we constructed a 6.7 genome-equivalent BAC library that we screened with known Taxodium genes and select repeats. Results The bald cypress genome is composed of 90% repetitive DNA with most sequences being found in low to mid copy numbers. The most abundant repeats are found in fewer than 25,000 copies per genome. Approximately 7.4% of the genome is single/low-copy DNA (i.e., sequences found in 1 to 5 copies). Sequencing of highly repetitive Cot clones indicates that most Taxodium repeats are highly diverged from previously characterized plant repeat sequences. The bald cypress BAC library consists of 606,336 clones (average insert size of 113 kb) and collectively provides 6.7-fold genome equivalent coverage of the bald cypress genome. Macroarray screening with known genes produced, on average, about 1.5 positive clones per probe per genome-equivalent. Library screening with Cot-1 DNA revealed that approximately 83% of BAC clones contain repetitive sequences iterated 103 to 104 times per genome. Conclusions The BAC library for bald cypress is the first to be generated for a conifer species outside of the family Pinaceae. The Taxodium BAC library was shown to be useful in gene isolation and genome characterization and should be an important tool in gymnosperm comparative genomics, physical mapping, genome sequencing, and gene/polymorphism discovery. The single/low-copy (SL) component of

  2. Genomic organization and characterization of a three-gene rat adult beta-globin haplotype.

    PubMed

    Au, D M; Wong, W M; Tam, J W; Cheng, L Y; Lam, V M

    1995-11-20

    The isolation and detailed characterization of a three-beta-globin gene (GloB) haplotype in the Sprague-Dawley (S-D) rat is described. An enriched library, lambda SDHelib, was screened with a human GloB probe, humbg44, and from which a beta minor gene, Rathbbz, was isolated, sequenced and characterized. A S-D rat GloB-specific probe, Ratbgze12, derived from the Rathbbz gene, was then used to screen a S-D rat genomic library, lambda SDglib. The clone T1510 was isolated and identified to include the entire Rathbbz gene and part of another GloB gene, Rathbby, which was 5' upstream from Rathbbz. Chromosomal walking upstream using the riboprobe, rnaT71, led to the isolation of an overlapping clone, Ta49, which was shown to include two full-length GloB genes; the most 5' was Rathbbx followed by Rathbby. Sequence data suggests that Rathbbx is a beta major gene, whereas Rathbby is a hybrid gene of Rathbbx and Rathbbz. Genomic hybridization confirmed this particular three-gene haplotype in the S-D rat. This haplotype, a1, may be the prototype of the GloB cluster in rat.

  3. Combining Quantitative Genetic Footprinting and Trait Enrichment Analysis to Identify Fitness Determinants of a Bacterial Pathogen

    PubMed Central

    Wiles, Travis J.; Norton, J. Paul; Russell, Colin W.; Dalley, Brian K.; Fischer, Kael F.; Mulvey, Matthew A.

    2013-01-01

    Strains of Extraintestinal Pathogenic Escherichia c oli (ExPEC) exhibit an array of virulence strategies and are a major cause of urinary tract infections, sepsis and meningitis. Efforts to understand ExPEC pathogenesis are challenged by the high degree of genetic and phenotypic variation that exists among isolates. Determining which virulence traits are widespread and which are strain-specific will greatly benefit the design of more effective therapies. Towards this goal, we utilized a quantitative genetic footprinting technique known as transposon insertion sequencing (Tn-seq) in conjunction with comparative pathogenomics to functionally dissect the genetic repertoire of a reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection models, we tracked changes in the abundance of ExPEC variants within saturated transposon mutant libraries following selection within distinct host niches. Nine hundred and seventy bacterial genes (18% of the genome) were found to promote pathogen fitness in either a niche-dependent or independent manner. To identify genes with the highest therapeutic and diagnostic potential, a novel Trait Enrichment Analysis (TEA) algorithm was developed to ascertain the phylogenetic distribution of candidate genes. TEA revealed that a significant portion of the 970 genes identified by Tn-seq have homologues more often contained within the genomes of ExPEC and other known pathogens, which, as suggested by the first axiom of molecular Koch's postulates, is considered to be a key feature of true virulence determinants. Three of these Tn-seq-derived pathogen-associated genes—a transcriptional repressor, a putative metalloendopeptidase toxin and a hypothetical DNA binding protein—were deleted and shown to independently affect ExPEC fitness in zebrafish and mouse models of infection. Together, the approaches and observations reported herein provide a resource for future pathogenomics-based research and highlight the diversity of

  4. New generation enrichment monitoring technology for gas centrifuge enrichment plants

    SciTech Connect

    Ianakiev, Kiril D; Alexandrov, Boian S.; Boyer, Brian D.; Hill, Thomas R.; Macarthur, Duncan W.; Marks, Thomas; Moss, Calvin E.; Sheppard, Gregory A.; Swinhoe, Martyn T.

    2008-06-13

    The continuous enrichment monitor, developed and fielded in the 1990s by the International Atomic Energy Agency, provided a go-no-go capability to distinguish between UF{sub 6} containing low enriched (approximately 4% {sup 235}U) and highly enriched (above 20% {sup 235}U) uranium. This instrument used the 22-keV line from a {sup 109}Cd source as a transmission source to achieve a high sensitivity to the UF{sub 6} gas absorption. The 1.27-yr half-life required that the source be periodically replaced and the instrument recalibrated. The instrument's functionality and accuracy were limited by the fact that measured gas density and gas pressure were treated as confidential facility information. The modern safeguarding of a gas centrifuge enrichment plant producing low-enriched UF{sub 6} product aims toward a more quantitative flow and enrichment monitoring concept that sets new standards for accuracy stability, and confidence. An instrument must be accurate enough to detect the diversion of a significant quantity of material, have virtually zero false alarms, and protect the operator's proprietary process information. We discuss a new concept for advanced gas enrichment assay measurement technology. This design concept eliminates the need for the periodic replacement of a radioactive source as well as the need for maintenance by experts. Some initial experimental results will be presented.

  5. Somatic cell hybrid and long-range physical mapping of 11p13 microdissected genomic clones.

    PubMed Central

    Davis, L M; Senger, G; Lüdecke, H J; Claussen, U; Horsthemke, B; Zhang, S S; Metzroth, B; Hohenfellner, K; Zabel, B; Shows, T B

    1990-01-01

    Microdissection and microcloning of banded human metaphase chromosomes have been used to construct a genomic library of 20,000 clones that is highly enriched for chromosome 11p13 DNA sequences. Clones from this library have been mapped on a panel of human-rodent somatic cell hybrids that divides the region from distal p12 to proximal p14 into seven physical intervals, A total of 1500 clones has been isolated, 250 clones have been characterized, and 58 clones have been mapped. Six of the clones were used to complete a long-range physical map of 7.5 megabases through the region. Two of the clones are localized to the Wilms tumor (WT) region, three are localized to the aniridia (AN2) region, and two are localized to the region between WT and AN2. The library represents DNA sequences spanning a distance of approximately 13 x 10(6) base pairs, with an average density of one clone per 37,000 base pairs. Images PMID:2169618

  6. Hydrogen-enriched fuels

    SciTech Connect

    Roser, R.

    1998-08-01

    NRG Technologies, Inc. is attempting to develop hardware and infrastructure that will allow mixtures of hydrogen and conventional fuels to become viable alternatives to conventional fuels alone. This commercialization can be successful if the authors are able to achieve exhaust emission levels of less than 0.03 g/kw-hr NOx and CO; and 0.15 g/kw-hr NMHC at full engine power without the use of exhaust catalysts. The major barriers to achieving these goals are that the lean burn regimes required to meet exhaust emissions goals reduce engine output substantially and tend to exhibit higher-than-normal total hydrocarbon emissions. Also, hydrogen addition to conventional fuels increases fuel cost, and reduces both vehicle range and engine output power. Maintaining low emissions during transient driving cycles has not been demonstrated. A three year test plan has been developed to perform the investigations into the issues described above. During this initial year of funding research has progressed in the following areas: (a) a cost effective single-cylinder research platform was constructed; (b) exhaust gas speciation was performed to characterize the nature of hydrocarbon emissions from hydrogen-enriched natural gas fuels; (c) three H{sub 2}/CH{sub 4} fuel compositions were analyzed using spark timing and equivalence ratio sweeping procedures and finally; (d) a full size pick-up truck platform was converted to run on HCNG fuels. The testing performed in year one of the three year plan represents a baseline from which to assess options for overcoming the stated barriers to success.

  7. Academic Libraries, 2000. E.D. Tabs.

    ERIC Educational Resources Information Center

    Carey, Nancy; Justh, Natalie M.; Williams, Jeffrey W.

    This report discusses the state of academic libraries in 2000. It defines "academic library" as well as discusses library services, library collections, library staff, library expenditures, and electronic services. (Author/AMT)

  8. A Directory of Library Instruction Programs in Indiana Academic Libraries.

    ERIC Educational Resources Information Center

    Skinner, Jane, Comp.; Violette, Judith, Comp.

    A survey of 40 reporting academic libraries in Indiana developed this directory of library user instruction programs which provides an index profile of library resources and types of programs within the state. Entries are alphabetical in outline form and provide a library contact person, capsule information about the library instruction program,…

  9. Standards for Medical Library Technicians, Medical Library Association.

    ERIC Educational Resources Information Center

    Medical Library Association, Chicago, IL.

    A medical library technician is a semiprofessional library employee whose duties require knowledge and skill based on a minimum of two years' general college education that includes library instruction beyond the clerical level. The medical library technician must have a practical knowledge of library functions and services, an understanding of…

  10. libdrdc: software standards library

    NASA Astrophysics Data System (ADS)

    Erickson, David; Peng, Tie

    2008-04-01

    This paper presents the libdrdc software standards library including internal nomenclature, definitions, units of measure, coordinate reference frames, and representations for use in autonomous systems research. This library is a configurable, portable C-function wrapped C++ / Object Oriented C library developed to be independent of software middleware, system architecture, processor, or operating system. It is designed to use the automatically-tuned linear algebra suite (ATLAS) and Basic Linear Algebra Suite (BLAS) and port to firmware and software. The library goal is to unify data collection and representation for various microcontrollers and Central Processing Unit (CPU) cores and to provide a common Application Binary Interface (ABI) for research projects at all scales. The library supports multi-platform development and currently works on Windows, Unix, GNU/Linux, and Real-Time Executive for Multiprocessor Systems (RTEMS). This library is made available under LGPL version 2.1 license.

  11. Drug discovery with DNA-encoded chemical libraries.

    PubMed

    Buller, Fabian; Mannocci, Luca; Scheuermann, Jörg; Neri, Dario

    2010-09-15

    DNA-encoded chemical libraries represent a novel avenue for the facile discovery of small molecule ligands against target proteins of biological or pharmaceutical importance. Library members consist of small molecules covalently attached to unique DNA fragments that serve as amplifiable identification barcodes. This encoding allows the in vitro selection of ligands at subpicomolar concentrations from large library populations by affinity capture on a target protein of interest, in analogy to established technologies for the selection of binding polypeptides (e.g., antibodies). Different library formats have been explored by various groups, allowing the construction of chemical libraries comprising up to millions of DNA-encoded compounds. Libraries before and after selection have been characterized by PCR amplification of the DNA codes and subsequent relative quantification of library members using high-throughput sequencing. The most enriched compounds have then been further analyzed in biological assays, in the presence or in the absence of linked DNA. This article reviews experimental strategies used for the construction of DNA-encoded chemical libraries, revealing how selection, decoding, and hit validation technologies have been used for drug discovery programs.

  12. Environmental enrichment for aquatic animals.

    PubMed

    Corcoran, Mike

    2015-05-01

    Aquatic animals are the most popular pets in the United States based on the number of owned pets. They are popular display animals and are increasingly used in research settings. Enrichment of captive animals is an important element of zoo and laboratory medicine. The importance of enrichment for aquatic animals has been slower in implementation. For a long time, there was debate over whether or not fish were able to experience pain or form long-term memories. As that debate has reduced and the consciousness of more aquatic animals is accepted, the need to discuss enrichment for these animals has increased.

  13. DEFOG: Discrete Enrichment of Functionally Organized Genes†

    PubMed Central

    Wittkop, Tobias; Berman, Ari E.; Fleisch, K. Mathew; Mooney, Sean D.

    2012-01-01

    High-throughput biological experiments commonly result in a list of genes or proteins of interest. In order to understand the observed changes of the genes and to generate new hypotheses, one needs to understand the functions and roles of the genes and how those functions relate to the experimental conditions. Typically, statistical tests are performed in order to detect enriched Gene Ontology categories or Pathways, i.e. the categories are observed in the genes of interest more often than is expected by chance. Depending on the number of genes and the complexity and quantity of functions in which they are involved, such an analysis can easily result in hundreds of enriched terms. To this end we developed DEFOG, a web-based application that facilitates the functional analysis of gene sets by hierarchically organizing the genes into functionally related modules. Our computational pipeline utilizes three powerful tools to achieve this goal: (1) GeneMANIA creates a functional consensus network of the genes of interest based on gene-list-specific data fusion of hundreds of genomic networks from publicly available sources; (2) Transitivity Clustering organizes those genes into a clear hierarchy of functionally related groups, and (3) Ontologizer performs a Gene Ontology enrichment analysis on the resulting gene clusters. DEFOG integrates this computational pipeline within an easy-to-use web interface, thus allowing for a novel visual analysis of gene sets that aids in the discovery of potentially important biological mechanisms and facilitates the creation of new hypotheses. DEFOG is available at http://www.mooneygroup.org/defog. PMID:22706384

  14. 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.

  15. Improving mammalian genome scaffolding using large insert mate-pair next-generation sequencing

    PubMed Central

    2013-01-01

    Background Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses. Results Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly. Conclusions We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes. PMID:23590730

  16. Integrated circuit cell library

    NASA Technical Reports Server (NTRS)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  17. NEIC Library Services

    EPA Pesticide Factsheets

    The National Enforcement Investigation Center (NEIC) Environmental Forensic Library partners with NEIC's forensic scientists to retrieve, validate and deliver information to develop methods, defensible regulations, and environmental measurements.

  18. Automated Library System Specifications.

    DTIC Science & Technology

    1986-06-01

    AD-A78 95 AUTOMATED LIBRARY SYSTEM SPECIFICATIONS(U) ARMY LIBRARY /i MANAGEMENT OFFICE ALEXANDRIA VA ASSISTANT CHIEF OF STAFF FOR INFORMATION... MANAGEMENT M B BONNETT JUN 86 UNCLASSIFIED F/G 9/2 NLEElIIhllEEEEE IllEEEEEllllEI .1lm lliml * ~I fI.L25 MI, [OCM RL,;OCLUTO fl. ’N k~ AUTOMATED LIBRARY...SYSTEM SPECIFICATIONS .,I Prepared by Mary B. Bonnett ARMY LIBRARY MANAGEMENT OFFICE OFFICE OF THE ASSISTANT CHIEF OF STAFF FOR INFORMATION MANAGEMENT Lij

  19. Enriched Title-Based Keyword Index Generation Using dBase II.

    ERIC Educational Resources Information Center

    Rajendran, P. P.

    1986-01-01

    Describes the use of a database management system (DBMS)--dBaseII--to create an enriched title-based keyword index for a collection of news items at the Renewable Energy Resources Information Center of the Asian Institute of Technology. The use of DBMSs in libraries in developing countries is emphasized. (Author/LRW)

  20. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

    PubMed

    Le Paslier, M C; Pierce, R J; Merlin, F; Hirai, H; Wu, W; Williams, D L; Johnston, D; LoVerde, P T; Le Paslier, D

    2000-04-15

    A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

  1. Houston Cole Library Policy Manual.

    ERIC Educational Resources Information Center

    Henderson, William Abbot, Ed.

    This policy manual of Jacksonville State University's Houston Cole Library serves as a statement of the library's mission, goals, and objectives; an interpretive guide for library faculty, staff, and students; and informational document for library patrons concerning the library's programs and policies. The contents include the following:…

  2. Library Latchkey Children. ERIC Digest.

    ERIC Educational Resources Information Center

    Dowd, Frances Smardo

    This digest discusses ways in which public libraries deal with latchkey children who spend their after-school hours at public libraries while their parents are at work. Research conducted in 1990 of 110 public libraries revealed that almost all libraries encountered unattended children after school hours, and that most libraries surveyed were not…

  3. FCLib: The Feature Characterization Library.

    SciTech Connect

    Gentile, Ann C.; Doyle, Wendy S. K.; Kegelmeyer, W. Philip,; Ulmer, Craig D.

    2008-11-01

    The Feature Characterization Library (FCLib) is a software library that simplifies the process of interrogating, analyzing, and understanding complex data sets generated by finite element applications. This document provides an overview of the library, a description of both the design philosophy and implementation of the library, and examples of how the library can be utilized to extract understanding from raw datasets.

  4. Future-Proof Your Library

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2008-01-01

    This article presents an excerpt of Library Journal's (LJ's) Movers & Shakers ideas on how to ensure a vital library for the future. Several identify a disconnect between what librarians and libraries do and what users think they do. Mending this gap, they say, would transform library recruitment and library support. While specific insights on…

  5. Wisconsin Library Service Record, 1978.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison. Div. of Library Services.

    Narrative description and updated statistical tables cover all types of libraries and library related activities in the state. Major areas reported on are the Wisconsin library network, including planning, media services, and special services for children, the institutionalized, and handicapped; library and media organizations; library education,…

  6. Wisconsin Library Service Record, 1974.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison. Div. of Library Services.

    In a record of library activities in Wisconsin in 1974, the activities of the Division for Library Services are described along with the Wisconsin Library Network and the state and regional library networks, services, and resources. Lists of library and media organizations and education programs are followed by a directory of academic, public,…

  7. A Tale of Two Libraries.

    ERIC Educational Resources Information Center

    Martinez, Ed

    1999-01-01

    Reviews the profiles of a typical public library, the Redondo Beach Public Library (California), and its nearby community college library, the Schauerman Library at El Camino College in Torrance, California. Discusses similarities between staff in public and academic libraries; differences between the two environments; and like missions of both…

  8. Central Statistical Libraries in Europe.

    ERIC Educational Resources Information Center

    Kaiser, Lisa

    The paper tries to clarify the position special governmental libraries hold in the system of libraries of today by investigating only one specific type of library mainly from a formal and historical point of view. Central statistical libraries in Europe were first regarded as administrative and archival libraries. Their early holdings of foreign…

  9. Suggestions for Library Network Design.

    ERIC Educational Resources Information Center

    Salton, Gerald

    1979-01-01

    Various approaches to the design of automatic library systems are described, suggestions for the design of rational and effective automated library processes are posed, and an attempt is made to assess the importance and effect of library network systems on library operations and library effectiveness. (Author/CWM)

  10. Public Relations in Special Libraries.

    ERIC Educational Resources Information Center

    Rutkowski, Hollace Ann; And Others

    1991-01-01

    This theme issue includes 11 articles on public relations (PR) in special libraries. Highlights include PR at the Special Libraries Association (SLA); sources for marketing research for libraries; developing a library image; sample PR releases; brand strategies for libraries; case studies; publicizing a consortium; and a bibliography of pertinent…

  11. Genome Radio Project: Quarterly report

    SciTech Connect

    1997-08-01

    The process of conducting background research for the programs of the Genome Radio Project is continuing. The most developed of the program ``backgrounders`` have been reviewed by series and program advisors from various fields. Preliminary and background interviews have been conducted with dozens of potential program participants and advisors. Structurally, efforts are being directed toward developing and formalizing the project and series advisor relationships so that the best use can be made of those experts who have offered to assist the project in its presentation of program content. The library of research materials has been expanded considerably, creating a useful resource library for the producers.

  12. Enrichment of statistical power for genome-wide association studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The inheritance of most human diseases and agriculturally important traits is controlled by many genes with small effects. Identifying these genes, while simultaneously controlling false positives, is challenging. Among available statistical methods, the mixed linear model (MLM) has been the most fl...

  13. Libraries in Mauritius: Ideal Size for a National Library Service.

    ERIC Educational Resources Information Center

    Harrison, K. C.

    1978-01-01

    A UNESCO consultant describes services provided by academic, public, and school libraries in Mauritius, and discusses levels of professionalism and salaries of librarians, opportunities for library education, and the possibility of developing a national library service. (JAB)

  14. piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice.

    PubMed

    Xu, Chunlong; Qi, Xiaolan; Du, Xuguang; Zou, Huiying; Gao, Fei; Feng, Tao; Lu, Hengxing; Li, Shenglan; An, Xiaomeng; Zhang, Lijun; Wu, Yuanyuan; Liu, Ying; Li, Ning; Capecchi, Mario R; Wu, Sen

    2017-01-24

    CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

  15. STAR Library Education Network: a hands-on learning program for libraries and their communities

    NASA Astrophysics Data System (ADS)

    Dusenbery, P.

    2010-12-01

    Science and technology are widely recognized as major drivers of innovation and industry (e.g. Rising above the Gathering Storm, 2006). While the focus for education reform is on school improvement, there is considerable research that supports the role that out-of-school experiences can play in student achievement and public understanding of STEM disciplines. Libraries provide an untapped resource for engaging underserved youth and their families in fostering an appreciation and deeper understanding of science and technology topics. Designed spaces, like libraries, allow lifelong, life-wide, and life-deep learning to take place though the research basis for learning in libraries is not as developed as other informal settings like science centers. The Space Science Institute’s National Center for Interactive Learning (NCIL) in partnership with the American Library Association (ALA), the Lunar and Planetary Institute (LPI), and the National Girls Collaborative Project (NGCP) have received funding from NSF to develop a national education project called the STAR Library Education Network: a hands-on learning program for libraries and their communities (or STAR-Net for short). STAR stands for Science-Technology, Activities and Resources. The overarching goal of the project is to reach underserved youth and their families with informal STEM learning experiences. This project will deepen our knowledge of informal/lifelong learning that takes place in libraries and establish a learning model that can be compared to the more established free-choice learning model for science centers and museums. The project includes the development of two STEM hands-on exhibits on topics that are of interest to library staff and their patrons: Discover Earth and Discover Tech. In addition, the project will produce resources and inquiry-based activities that libraries can use to enrich the exhibit experience. Additional resources will be provided through partnerships with relevant

  16. Genome resource for the Indonesian coelacanth, Latimeria menadoensis.

    PubMed

    Danke, Joshua; Miyake, Tsutomu; Powers, Thomas; Schein, Jacqueline; Shin, Heesun; Bosdet, Ian; Erdmann, Mark; Caldwell, Roy; Amemiya, Chris T

    2004-03-01

    We have generated a BAC library from the Indonesian coelacanth, Latimeria menadoensis. This library was generated using genomic DNA of nuclei isolated from heart tissue, and has an average insert size of 171 kb. There are a total of 288 384-well microtiter dishes in the library (110,592 clones) and its genomic representation is estimated to encompass > or = 7X coverage based on the amount of DNA presumably cloned in the library as well as via hybridization with probes to a small set of single copy genes. This genomic resource has been made available to the public and should prove useful to the scientific community for many applications, including comparative genomics, molecular evolution and conservation genetics.

  17. Human BAC library: construction and rapid screening.

    PubMed

    Asakawa, S; Abe, I; Kudoh, Y; Kishi, N; Wang, Y; Kubota, R; Kudoh, J; Kawasaki, K; Minoshima, S; Shimizu, N

    1997-05-20

    We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

  18. Genome sequencing of methanogenic Archaea Methanosarcina mazei TUC01 strain isolated from an Amazonian Flooded Area

    NASA Astrophysics Data System (ADS)

    Baraúna, R. A.; Graças, D. A.; Ramos, R. T.; Carneiro, A. R.; Lopes, T. S.; Lima, A. R.; Zahlouth, R. L.; Pellizari, V. H.; Silva, A.

    2013-05-01

    Methanosarcina mazei is a strictly anaerobic methanogen from the Methanosarcinales order. This species is known for its broad catabolic range among methanogens and is widespread throughout diverse environments. The draft genome of a strain cultivated from the sediment of the Tucuruí hydroelectric power station, the fourth largest hydroelectric dam in the world, is described here. Approximately 80% of methane is produced by biogenic sources, such as methanogenic archaea from M. mazei species. Although the methanogenesis pathway is well known, some aspects of the core genome, genome evolution and shared genes are still unclear. A sediment sample from the Tucuruí hydropower station reservoir was inoculated in mineral media supplemented with acetate and methanol. This media was maintained in an H2:CO2 (80:20) atmosphere to enrich and cultivate M. mazei. The enrichment was conducted at 30°C under standard anaerobic conditions. After several molecular and cellular analyses, total DNA was extracted from a non-pure culture of M. mazei, amplified using phi29 DNA polymerase (BioLabs) and finally used as a source template for genome sequencing. The draft genome was obtained after two rounds of sequencing. First, the genome was sequenced using a SOLiD System V3 with a mate-paired library, which yielded 24,405,103 and 24,399,268 reads (50 bp) for the R3 and F3 tags, respectively. The second round of sequencing was performed using the SOLiD 5500 XL platform with a mate-paired library, resulting in a total of 113,588,848 reads (60 bp) for each tag (F3 and R3). All reads obtained by this procedure were filtered using Quality Assessment software, whereby reads with an average quality score below Phred 20 were removed. Velvet and Edena were used to assemble the reads, and Simplifier was used to remove the redundant sequences. After this, a total of 16,811 contigs were obtained. M. mazei GO1 (AE008384) genome was used to map the contigs and generate the scaffolds. We used the

  19. The Genomic Scrapheap Challenge; Extracting Relevant Data from Unmapped Whole Genome Sequencing Reads, Including Strain Specific Genomic Segments, in Rats

    PubMed Central

    van der Weide, Robin H.; Simonis, Marieke; Hermsen, Roel; Toonen, Pim; Cuppen, Edwin; de Ligt, Joep

    2016-01-01

    Unmapped next-generation sequencing reads are typically ignored while they contain biologically relevant information. We systematically analyzed unmapped reads from whole genome sequencing of 33 inbred rat strains. High quality reads were selected and enriched for biologically relevant sequences; similarity-based analysis revealed clustering similar to previously reported phylogenetic trees. Our results demonstrate that on average 20% of all unmapped reads harbor sequences that can be used to improve reference genomes and generate hypotheses on potential genotype-phenotype relationships. Analysis pipelines would benefit from incorporating the described methods and reference genomes would benefit from inclusion of the genomic segments obtained through these efforts. PMID:27501045

  20. The Genomic Scrapheap Challenge; Extracting Relevant Data from Unmapped Whole Genome Sequencing Reads, Including Strain Specific Genomic Segments, in Rats.

    PubMed

    van der Weide, Robin H; Simonis, Marieke; Hermsen, Roel; Toonen, Pim; Cuppen, Edwin; de Ligt, Joep

    2016-01-01

    Unmapped next-generation sequencing reads are typically ignored while they contain biologically relevant information. We systematically analyzed unmapped reads from whole genome sequencing of 33 inbred rat strains. High quality reads were selected and enriched for biologically relevant sequences; similarity-based analysis revealed clustering similar to previously reported phylogenetic trees. Our results demonstrate that on average 20% of all unmapped reads harbor sequences that can be used to improve reference genomes and generate hypotheses on potential genotype-phenotype relationships. Analysis pipelines would benefit from incorporating the described methods and reference genomes would benefit from inclusion of the genomic segments obtained through these efforts.

  1. Principal Angle Enrichment Analysis (PAEA): Dimensionally Reduced Multivariate Gene Set Enrichment Analysis Tool.

    PubMed

    Clark, Neil R; Szymkiewicz, Maciej; Wang, Zichen; Monteiro, Caroline D; Jones, Matthew R; Ma'ayan, Avi

    2015-11-01

    Gene set analysis of differential expression, which identifies collectively differentially expressed gene sets, has become an important tool for biology. The power of this approach lies in its reduction of the dimensionality of the statistical problem and its incorporation of biological interpretation by construction. Many approaches to gene set analysis have been proposed, but benchmarking their performance in the setting of real biological data is difficult due to the lack of a gold standard. In a previously published work we proposed a geometrical approach to differential expression which performed highly in benchmarking tests and compared well to the most popular methods of differential gene expression. As reported, this approach has a natural extension to gene set analysis which we call Principal Angle Enrichment Analysis (PAEA). PAEA employs dimensionality reduction and a multivariate approach for gene set enrichment analysis. However, the performance of this method has not been assessed nor its implementation as a web-based tool. Here we describe new benchmarking protocols for gene set analysis methods and find that PAEA performs highly. The PAEA method is implemented as a user-friendly web-based tool, which contains 70 gene set libraries and is freely available to the community.

  2. Global Warming's Library Challenge

    ERIC Educational Resources Information Center

    Meyer, Jennifer

    2008-01-01

    Like every institution that uses energy, consumes resources, and engages in construction or renovation, libraries have an impact on the environment and on the critical problem of climate change. Taking action to protect library collections is not only an idealistic professional goal but also a very practical one. Disaster preparation measures and…

  3. Merchandising Techniques and Libraries.

    ERIC Educational Resources Information Center

    Green, Sylvie A.

    1981-01-01

    Proposes that libraries employ modern booksellers' merchandising techniques to improve circulation of library materials. Using displays in various ways, the methods and reasons for weeding out books, replacing worn book jackets, and selecting new books are discussed. Suggestions for learning how to market and 11 references are provided. (RBF)

  4. Library Media Services Handbook.

    ERIC Educational Resources Information Center

    Winnipeg School Div. Number 1, Manitoba (Canada).

    This 1991 edition of the handbook for Winnipeg School Division Number 1 school library media programs presents detailed program guidelines, objectives, philosophies, and goals in five major sections: (1) Library Policy and Practices (statement of the division's educational philosophy and goals, philosophy and goals of the division's library…

  5. Planning for Library Excellence.

    ERIC Educational Resources Information Center

    Virginia State Library and Archives, Richmond.

    Intended to be used by librarians, this guide will also be useful to boards of trustees, governing officials, members of funding agencies, and community support groups involved in planning on a local level and within the context of regional and state library service. It provides information to help libraries plan and evaluate their services and…

  6. The Jimmy Carter Library.

    ERIC Educational Resources Information Center

    Schewe, Donald B.

    1989-01-01

    Reviews past approaches to the preservation of Presidential papers, the development of the system of Presidential Libraries, and the development of the Jimmy Carter Library. The National Archives' approach to computerization to deal with the increasing volume of materials is detailed and the PresNet computerized information system is described.…

  7. Homelessness in Public Libraries

    ERIC Educational Resources Information Center

    Wong, Yi Ling

    2009-01-01

    This paper takes a theoretical and practical approach in defining the "problem" of homelessness in libraries. The author examines three fundamental problems on homelessness. The three fundamental questions are: (a) Who are the homeless? (b) Why are they homeless? (c) What are their information needs in libraries? These questions are important in…

  8. Trotsky's Vision of Libraries.

    ERIC Educational Resources Information Center

    Baker, William; Hurych, Jitka

    1991-01-01

    Discusses Leon Trotsky's views on libraries, librarians, and librarianship in Soviet society in the 1920s as expressed in a speech, "Leninism and Library Work." Topics discussed include role of librarian in promoting literacy, including the use of maps and reference books to explain newspaper stories; the selection of books; and fighting…

  9. Financing Public Library Construction.

    ERIC Educational Resources Information Center

    Rohlf, Robert H.; Stoffel, Lester L.

    1987-01-01

    Reviews financing options available to Illinois public libraries for construction or expansion, including general obligation bonds, mortgage funds, building reserve funds, building fund levies, lease back arrangements, sale of air or ground development rights, interest on special funds, gift funds and grants, Library Service and Construction Act…

  10. Improving Library Research Skills.

    ERIC Educational Resources Information Center

    Holley, Catherine W.

    This report describes a program to advance library research skills in two sixth grade science classes. The problem was assessed through a survey, questionnaire, and worksheet, and by direct observation. Analysis of probable cause data revealed that students displayed a lack of research skills related to library research. Some of the causes were…

  11. Image-analysis library

    NASA Technical Reports Server (NTRS)

    1980-01-01

    MATHPAC image-analysis library is collection of general-purpose mathematical and statistical routines and special-purpose data-analysis and pattern-recognition routines for image analysis. MATHPAC library consists of Linear Algebra, Optimization, Statistical-Summary, Densities and Distribution, Regression, and Statistical-Test packages.

  12. Library Technician Skill Standards.

    ERIC Educational Resources Information Center

    Highline Community Coll., Des Moines, WA.

    This document presents skill standards for library technicians. Introductory sections describe the industry and the job, what skill standards are, how the library technician skill standards were developed, employability skills and critical competencies, and the SCANS (Secretary's Commission on Achieving Necessary Skills) foundation skills profile.…

  13. Stress in the Library.

    ERIC Educational Resources Information Center

    Bunge, Charles

    1987-01-01

    Reports results of a survey of 800 library staff members on aspects of library work that they find stressful. The findings include sources of satisfaction and stress for all participants, and sources of stress specific to public services librarians, technical services librarians, and support staff members. (CLB)

  14. Online Library Videos

    ERIC Educational Resources Information Center

    Power, June

    2010-01-01

    One of the challenges of access services is letting patrons know what one has to offer at his or her library, and then communicating how they may avail themselves of those services. Increasingly, libraries are doing this through more than the traditional handouts and newsletters, but also through blogs, Facebook pages, podcasts, and videos. This…

  15. Academic Libraries in Japan

    ERIC Educational Resources Information Center

    Cullen, Rowena; Nagata, Haruki

    2008-01-01

    Academic libraries in Japan are well resourced by international standards, and support Japan's internationally recognized research capability well, but there are also ways in which they reflect Japan's strong bureaucratic culture. Recent changes to the status of national university libraries have seen a new interest in customer service, and…

  16. Revolution and the Library.

    ERIC Educational Resources Information Center

    Gorniak-Kocikowska, Krystyna

    2001-01-01

    Considers the impact that the computer revolution has had on college and university libraries. Discusses the historical background of changes that resulted from the invention of the printing press; the development of national languages; knowledge acquisition; the historical role of religion; content control of libraries; and changes in students.…

  17. Libraries of the Future.

    ERIC Educational Resources Information Center

    McKenzie, Jamie

    1996-01-01

    Predicts the effects of information technology (IT) on libraries of the future. Discusses negative implications for school libraries. Describes four emerging roles for media specialists: (1) showing students and staff how to navigate information networks; (2) teaching users how to evaluate and select information; (3) designing information systems;…

  18. Keeping Libraries Open.

    ERIC Educational Resources Information Center

    Franklin, Hardy R.

    Major social institutions repeatedly reinforce the deprivation of the urban disadvantaged. Libraries, as the "Universities of the People," must commit themselves to the promotion of a better standard of life for all people, especially the poor. A close look at priorities is especially important if libraries are to pursue their mission in…

  19. An Online Library Catalogue.

    ERIC Educational Resources Information Center

    Alloro, Giovanna; Ugolini, Donatella

    1992-01-01

    Describes the implementation of an online catalog in the library of the National Institute for Cancer Research and the Clinical and Experimental Oncology Institute of the University of Genoa. Topics addressed include automation of various library functions, software features, database management, training, and user response. (10 references) (MES)

  20. Library Connection Development.

    ERIC Educational Resources Information Center

    Berry, John N., III

    1990-01-01

    The John Wiley and Sons publishing company has made a commitment to publishing more tradebooks for the retail and library marketplace. As part of this expansion, Wiley has strengthened its library sales and promotion efforts and launched new publications and promotions to reach librarians. (MES)

  1. Library Laws of Texas.

    ERIC Educational Resources Information Center

    Seidenberg, Ed, Ed.

    Compiled to provide a central reference point for all legislative information pertaining to libraries in the state of Texas, this publication includes all pertinent legislation as amended through the 66th Legislature, Regular Session, 1979. It contains articles dealing specifically with archives, buildings and property, city libraries, non-profit…

  2. Libraries at the Ready

    ERIC Educational Resources Information Center

    Celano, Donna C.; Neuman, Susan B.

    2016-01-01

    Because English language learners enter kindergarten at a distinct disadvantage, Celano and Neuman examine the role public libraries can play in rallying around these young children to better prepare them for school. The authors document a new program called Every Child Ready to Read, which recently launched in 4,000 public libraries across the…

  3. Guidelines on Library Twinning.

    ERIC Educational Resources Information Center

    Doyle, Robert P., Comp.; Scarry, Patricia, Comp.

    This document serves as an overview of the many forms of library twinning, or formal exchanges of materials and/or staff between two institutions. Twinning can be reciprocal, but often involves a one-way arrangement whereby a library in a developing country receives a visit from a librarian of a developed country. Either way, both institutions can…

  4. The Animated Library

    ERIC Educational Resources Information Center

    Brewer, Jim; Dyal, Donald H.; Sweet, Robert

    2009-01-01

    Libraries have always been poised at the crossroads of access tools and content. Librarians, their personnel, and supporters have worked for generations to create tools to store and utilize content for the benefit of patrons. Libraries house materials and the tools to unlock them; their staffers teach patrons to use the materials and associated…

  5. Privacy and Library Records

    ERIC Educational Resources Information Center

    Bowers, Stacey L.

    2006-01-01

    This paper summarizes the history of privacy as it relates to library records. It commences with a discussion of how the concept of privacy first originated through case law and follows the concept of privacy as it has affected library records through current day and the "USA PATRIOT Act."

  6. Integrated library systems.

    PubMed Central

    Goldstein, C M

    1983-01-01

    The development of integrated library systems is discussed. The four major discussion points are (1) initial efforts; (2) network resources; (3) minicomputer-based systems; and (4) beyond library automation. Four existing systems are cited as examples of current systems. PMID:6354321

  7. Library Instruction Program Proposal.

    ERIC Educational Resources Information Center

    Cassata, Mary B.

    A number of studies cited in the literature show a positive correlation between the students scholastic grade point average and his library usage. The case is made by a growing cult of librarians who are engaged in "teaching the library" that the librarian so engaged makes a direct contribution to the student's success at his studies. Although…

  8. Prison Libraries: Bibliography.

    ERIC Educational Resources Information Center

    Gillespie, David M.

    An Alphabetically arranged bibliography, listing 485 entries representing 518 citations taken from "Poole's Index to Periodical Literature" 1802-1906, "Cannon's Bibliography of Library Economy," and "Library Literature" 1921 to mid-1970. Other sources used include: The Index to the Journal of Correctional Education; ten bibliographies taken from…

  9. "Greening" up the Library

    ERIC Educational Resources Information Center

    Woodland, Jean

    2010-01-01

    This paper looks at the library's ability to spread environmental conservation efforts throughout the community. The paper suggests various ways library staff can explore environmentally friendly procedures within the context of the media center as well as ways to promote community interest and support. It identifies areas for improvement, ideas…

  10. Library Technology International

    ERIC Educational Resources Information Center

    Breeding, Marshall

    2008-01-01

    The author has been extremely fortunate over the last few years to have had the opportunity to travel to many different parts of the world and speak and work with librarians in many countries and to have the chance to see first-hand some incredible libraries that demonstrate creative approaches to library services, innovative uses of technology,…

  11. Centennial State Libraries, 1998.

    ERIC Educational Resources Information Center

    Parent, Kathleen D., Ed.

    1998-01-01

    This document consists of 12 consecutive issues of the monthly "Centennial State Libraries" newsletter, of the Colorado Department of Education, State Library and Adult Education Office. The issues cover the year 1998. Each issue of the newsletter--except the August issue which is an Annual Report--includes some or all of the following…

  12. XML in Libraries.

    ERIC Educational Resources Information Center

    Tennant, Roy, Ed.

    This book presents examples of how libraries are using XML (eXtensible Markup Language) to solve problems, expand services, and improve systems. Part I contains papers on using XML in library catalog records: "Updating MARC Records with XMLMARC" (Kevin S. Clarke, Stanford University) and "Searching and Retrieving XML Records via the…

  13. Iranian Special Library Standards.

    ERIC Educational Resources Information Center

    Harvey, John F.

    The purposes of these standards are: (1) to suggest the steps necessary for the establishment of special libraries; (2) for those recently established, to suggest the steps necessary to achieve satisfactory performance levels in all areas, and finally, (3) for well established libraries, to suggest the steps necessary to achieve excellence. These…

  14. The Amazing Library Computer.

    ERIC Educational Resources Information Center

    Anderson, Eric S.

    1983-01-01

    Identifies computer software designed specifically for library management tasks in three distinct categories: library utility programs, and circulation and catalog programs for floppy disk and for hard disk. Company name and address, program, application, hardware, cost, and comments are provided in each category. (EJS)

  15. Guide to Library Automation.

    ERIC Educational Resources Information Center

    Toohill, Barbara G.

    Directed toward librarians and library administrators who wish to procure automated systems or services for their libraries, this guide offers practical suggestions, advice, and methods for determining requirements, estimating costs and benefits, writing specifications procuring systems, negotiating contracts, and installing systems. The advice…

  16. Dumping the "Library."

    ERIC Educational Resources Information Center

    Crowley, Bill

    1998-01-01

    Discusses an alternative to abandoning the word "library" for "information" in graduate education. Recommends patient, consistent effort by library- and information-science educators to convince academic librarians that if they accept the standards of the teaching/research faculty, including the need to earn a Ph.D., it will raise the prestige of…

  17. Weeding Library Collections.

    ERIC Educational Resources Information Center

    Slote, Stanley J.

    This work, based on two recent research projects in the weeding of library collections and the identification of core collections, provides a comprehensive summary of the literature and research on these topics. It also presents practical guidance in weeding for the professional librarian or for the library school student. The book is divided into…

  18. Small Public Library Management

    ERIC Educational Resources Information Center

    Pearlmutter, Jane; Nelson, Paul

    2012-01-01

    Anyone at the helm of a small public library knows that every little detail counts. But juggling the responsibilities that are part and parcel of the job is far from easy. Finally, here's a handbook that includes everything administrators need to keep a handle on library operations, freeing them up to streamline and improve how the organization…

  19. Disaster Planning in Libraries

    ERIC Educational Resources Information Center

    Wong, Yi Ling; Green, Ravonne

    2006-01-01

    Disaster preparedness is an important issue in library management today. This article presents a general overview of the theoretical aspects of disaster planning in libraries. The stages of disaster planning are a circular process of planning, prevention, response, recovery, preparedness, and training.

  20. Libraries and Lenin.

    ERIC Educational Resources Information Center

    Lynn, Karen

    This instructional unit combines a study of the Soviet leader V. I. Lenin with a study of libraries. Lenin was selected as the focus because of his support of books and libraries and because he oversaw a revolution that altered the political and social structure of Russia and the balance of power throughout the world. Included are lesson plan…