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      High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

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          Turbot ( Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.

          Methodology/Principal Findings

          Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways.


          To our knowledge , this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

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          Most cited references 80

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          A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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              We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process. Blast2GO is freely available via Java Web Start at -> Evaluation.

                Author and article information

                [1 ]Instituto de Investigaciones Marinas, IIM, CSIC, Vigo, Spain
                [2 ]Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona i Institut de Biomedicina de la Universitat de Barcelona, IBUB, Barcelona, Spain
                [3 ]Centros Científicos y Tecnológicos de la UB, CCiT-UB, Universitat de Barcelona, Edifici Clúster, Parc Científic de Barcelona, Barcelona, Spain
                Auburn University, United States of America
                Author notes

                Conceived and designed the experiments: BN AF. Performed the experiments: PP. Wrote the paper: PP. Conducted the 454 sequencing and the bioinformatic work: SB BF GF-C. Performed functional annotation analyses: PP PB. Deposited the sequences in databases: PP GF-C. Involved in the construction and annotation of the immune cascades: AR SD. Revised the study and manuscript: JP PB BN AF. Edited, read and approved the manuscript: AF PP PB AR SD GF-C BF JP SB BN.

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                18 May 2012
                : 7
                : 5
                Pereiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Pages: 17
                Research Article
                Fish Farming
                Marine Biology
                Veterinary Science
                Veterinary Diseases
                Veterinary Pathology



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