Optimal clone identifier for genomic shotgun libraries: OC Identifier tool

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray. ©FUNPEC-RP.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Abstract Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

A panel of single locus minisatellite DNA probes for application to problems in salmonid aquaculture

Eleven minisatellite DNA locus specific probes, isolated from Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) partial genomic DNA libraries, were tested for cross-hybridization to eleven other salmonid species, i.e. sockeye salmon (Oncorhynchus nerka); coho salmon (O. kisutch), chum salmon (O. keta); pink salmon (O. gorbuscha); chinook salmon (O. tshawytscha); rainbow trout (O. mykiss); brook trout (Salvelinus fontinalis); Arctic charr (S. alpinus); grayling (Thymallus thymallus); huchen (Hucho hucho); pollan (Coregonus autumnalis). Simple single locus profiles for each of these species were revealed by, from two to ten SLPs. These markers are likely to be of great value in addressing several problems in aquaculture of these species.

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.

Comparative genomic data of the Avian Phylogenomics Project.

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

“Shotgun” cloning of skin peptide precursors from skin secretion-derived cDNA libraries of the Fujian large-headed frog, Limnonectes fujianensis

Amphibian skin secretions are rich sources of biologically-active peptides and several studies involving molecular cloning of their biosynthetic precursors have revealed that many exhibit highly-conserved domain architectures with an associated high degree of primary structural conservation of the signal peptides. This conservation of primary structure is reflected at the level of nucleotide sequence — a finding that has permitted our group to design primers to these sites facilitating “shotgun” cloning using cDNA libraries from uninvestigated species. Here we describe the results of such an approach using a skin secretion-derived cDNA library from the Fujian large-headed frog, Limnonectes fujianensis, a completely unstudied species. In over 50 clones studied by this approach, 12 were found to encode peptides of different primary structure. Representatives of 5 different families of antimicrobial peptides derived from the skins of ranid frogs were found and these were brevinin-1 (n = 3), the ranatuerin-2 (n = 3), esculentin-2 (n = 1), temporin (n = 1) and chensinin (n = 1). Three clones encoded peptides that were novel with no homologues present in contemporary on-line databases. These included two related 16-mer peptides, named peptides SC-16a and b, and an unrelated 24-mer, named peptide AG-24. Preliminary biological characterisation of SC-16a has demonstrated an antimicrobial activity against Gram-negative bacteria with a minimal inhibitory concentration of 35 µM with no observable haemolysis up to 200 µM. This finding may suggest that this peptide represents a novel class of antimicrobial with little effect on eukaryotic membranes.

Microsatellite loci isolation from river buffalo using enriched partial genomic libraries

The extensive use of buffalo in agriculture, especially in developing countries, begs for genetic resources to evaluate and improve traits important to local and regional economies. Brazil presents the largest water buffalo populations in the New World, with 1 1 million heads including swamp and river types. To design rational breeding strategies for optimum utilization and conservation of available genetic variability in the Brazilian buffalo's population, it is essential to understand their genetic architecture and relationship among various breeds. This depends, in part, on the knowledge of their genetic structure based on molecular markers like microsatellites. In the present study, we developed six enriched partial genomic libraries for river buffalo using selective hybridization methods. Genomic DNA was hybridized with six different arrays of repeat motif, 5' biotinylated - (CA)(15), (CT)(15), (AGG)(8), (GAAA)(8), (GATA)(8), (AAAAC)(8) - and bound to streptavidin coated beads. The cloning process generated a total of 1920 recombinant clones. Up to date, 487 were directly sequenced for the presence of repeats, from which 13 have been positive for presence of repeats as follows: 9 for di-nucleotide repeats, 3 for tri-nucleotide repeats and 1 for tetra-nucleotide repeat. PCR primer pairs for the isolated microsatellites are under construction to determine optimum annealing temperature. These microsatellites will be useful for studies involving phylogenetic relationships, genome mapping and genetic diversity analysis within buffalo populations worldwide.

Screening of genomic libraries

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs. © Springer Science+Business Media, LLC 2013.

BioPatML : pattern sharing for the genomic sciences

Computational biology increasingly demands the sharing of sophisticated data and annotations between research groups. Web 2.0 style sharing and publication requires that biological systems be described in well-defined, yet flexible and extensible formats which enhance exchange and re-use. In contrast to many of the standards for exchange in the genomic sciences, descriptions of biological sequences show a great diversity in format and function, impeding the definition and exchange of sequence patterns. In this presentation, we introduce BioPatML, an XML-based pattern description language that supports a wide range of patterns and allows the construction of complex, hierarchically structured patterns and pattern libraries. BioPatML unifies the diversity of current pattern description languages and fills a gap in the set of XML-based description languages for biological systems. We discuss the structure and elements of the language, and demonstrate its advantages on a series of applications, showing lightweight integration between the BioPatML parser and search engine, and the SilverGene genome browser. We conclude by describing our site to enable large scale pattern sharing, and our efforts to seed this repository.

Genomic resources notes accepted 1 December 2013 - 31 January 2014

This article documents the public availability of (i) transcriptome sequence data, assembled and annotated contigs and unigenes, and BLAST hits from the Queensland fruit fly, Bactrocera tryoni; (ii) 75 single-nucleotide variants (SNVs) from 454 sequencing of reduced representation libraries for Phalangiidae harvestmen, Megabunus armatus, Megabunus vignai, Megabunus lesserti, and Rilaena triangularis; and (iii) expressed sequence tags from 454 sequencing of the lepidopterans Lymantria dispar and Lymantria monacha.

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

Polymorphic microsatellites in largemouth bronze gudgeon (Coreius guichenoti) developed from repeat-enriched libraries and cross-species amplifications

Largemouth bronze gudgeon (Coreius guichenoti) is a medium-sized fish endemic from the upper Yangtze River of China and its survival is threatened by the construction of the Three Gorges Dam. This study reports 20 new polymorphic microsatellites from a repeat-enriched genomic library with a mean number allele of 5.2, and observed and expected heterozygosities ranging from 0.035 to 1, and from 0.13 to 0.917, respectively. In a cross-species amplification test, nine of the 37 tested loci were found to be also polymorphic in a congeneric species, brass gudgeon (C. heterodon). In addition, other four loci from common carp (Cyprinus carpio) were also polymorphic in C. guichenoti. Out of these 24 polymorphic microsatellites, only three loci significantly deviated from Hardy-Weinberg equilibrium in the sampled population (P < 0.0025), and all pairwise tests for linkage disequilibrium among loci were nonsignificant after applying sequential Bonferroni correction (P > 0.0026). These novel microsatellites provide sufficient levels of polymorphism for studies on population genetics and conservation in C. guichenoti and its related species.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.