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L’any 1977 Frederick Sanger va desenvolupar un mètode per a determinar l’ordre de les bases dels fragments de DNA. Aquesta tecnologia encara s’utilitza actualment i ha sigut clau per a aconseguir fites tan importants com la primera seqüenciació completa del genoma humà. L’aparició d’una nova generació de tecnologies de seqüenciació del DNA, tecnologies d’NGS (de l’anglès next generation sequencing), més la gran explosió d’eines computacionals per a analitzar-lo, ha permès seqüenciar de manera ràpida, econòmica i amb una elevada precisió genomes de microbis, plantes i animals. Durant l’última dècada, hi ha hagut una gran expansió de plataformes d’NGS: primer van sorgir les tecnologies d’NGS de cadena curta i, més endavant, les de cadena llarga. Tot i que les tecnologies d’NGS de lectures llargues prometien grans avenços per a resoldre genomes complexos, les freqüències d’error d’aquestes tecnologies són elevades. Això ha fet que, en els últims anys, hagin aparegut una sèrie de mètodes de seqüenciació complementaris per a resoldre les deficiències de les tecnologies d’NGS de lectures curtes i llargues., In 1977, Frederick Sanger developed a method for determining the order of the bases of DNA fragments. This technology still works today and has been crucial in achieving such important milestones as the first complete sequencing of the human genome. The emergence of the new generation of DNA sequencing technologies (NGS) plus the great explosion of computer tools for their analysis has become a matter of routine and allows the sequencing of the genomes of microbes, plants and animals in a way that is quick, relatively cheap and highly precise. There has been a great expansion of NGS sequencing platforms over the last decade, first involving short-read and later longread NGS sequencing technologies. Although long-read NGS sequencing promised great advances in solving complex genomes, the error rates of these technologies are high. This has led to the appearance in recent years of a number of complementary sequencing methods to address the shortcomings of NGS sequencing of short and long readings.

Background: 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. Conclusions/Significance: 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 eno

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 sequenced RNA from immunestimulated R. philippinarum hemocytes by 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 the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. 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 discovery of new 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