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      A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus)

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          Abstract

          BackgroundGenomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry.ResultsExpressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database (“Turbot 2 database”) was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences (“Turbot 3 database”), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50–90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified.ConclusionsThe combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.

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          Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.

          DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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            KEGG: kyoto encyclopedia of genes and genomes.

             S. Goto,  M Kanehisa (2000)
            KEGG (Kyoto Encyclopedia of Genes and Genomes) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher order functional information. The genomic information is stored in the GENES database, which is a collection of gene catalogs for all the completely sequenced genomes and some partial genomes with up-to-date annotation of gene functions. The higher order functional information is stored in the PATHWAY database, which contains graphical representations of cellular processes, such as metabolism, membrane transport, signal transduction and cell cycle. The PATHWAY database is supplemented by a set of ortholog group tables for the information about conserved subpathways (pathway motifs), which are often encoded by positionally coupled genes on the chromosome and which are especially useful in predicting gene functions. A third database in KEGG is LIGAND for the information about chemical compounds, enzyme molecules and enzymatic reactions. KEGG provides Java graphics tools for browsing genome maps, comparing two genome maps and manipulating expression maps, as well as computational tools for sequence comparison, graph comparison and path computation. The KEGG databases are daily updated and made freely available (http://www. genome.ad.jp/kegg/).
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              Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists

              Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
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                Author and article information

                Affiliations
                [1 ]Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, 08003, Spain
                [2 ]Departamento de Genética. Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, Lugo, 27002, Spain
                [3 ]Departamento de Matemática Aplicada, Facultad de Matemáticas, Universidad de Santiago de Compostela, Santiago de Compostela, 15781, Spain
                [4 ]Departamento de Geometría y Topología, Facultad de Matemáticas, Universidad de Santiago de Compostela, Santiago de Compostela, 15782, Spain
                [5 ]Departamento de Ciencias Clínicas Veterinarias. Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, Lugo, 27002, Spain
                [6 ]Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain
                [7 ]Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, 12595, Spain
                Contributors
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2013
                15 March 2013
                : 14
                : 180
                23497389
                3700835
                1471-2164-14-180
                10.1186/1471-2164-14-180
                Copyright ©2013 Ribas et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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