Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing.

Background: The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we subjected the R. philippinarum stimulated hemocyte transcriptome to 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings: High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and singletons were excluded to improve the quality and reliability of our results. The 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently expressed sequences included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways. Conclusions: This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The enrichment in immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.