High-throughput sequence analysis of turbot (Scophthalmus maximus L.) transcriptome using 454-pyrosequencing for discovery genes involved in antiviral immune response.

Background: Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is not too much 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: Several 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 with an average read length of 297 bp. Singletons were ruled out and reads were assembled into 55,404 contigs that were subjected to annotation steps against NCBI Swissprot, NCBI Metazoan Refseq and NCBI non-redundant databases. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 37.94% of the annotated sequences showed similarity to fish sequences. Moreover, only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. The 25 most frequently detected sequences in a BLASTx top-hit revealed a high number of immune-related genes. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. Conclusions: To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Our results provide a rich source of data 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.