title: 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).
abstract: BACKGROUND
  Genomic 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.
  
  
  RESULTS
  Expressed 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.
authors.forename: Laia
authors.initials: L
authors.lastname: Ribas
authors.forename: Belén G
authors.initials: BG
authors.lastname: Pardo
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Alvarez-Diós
authors.forename: Antonio
authors.initials: A
authors.lastname: Gómez-Tato
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Quiroga
authors.forename: Josep V
authors.initials: JV
authors.lastname: Planas
authors.forename: Ariadna
authors.initials: A
authors.lastname: Sitjà-Bobadilla
authors.forename: Paulino
authors.initials: P
authors.lastname: Martínez
authors.forename: Francesc
authors.initials: F
authors.lastname: Piferrer
mesh_terms: D000818:Animals; D001947:Breeding / Q000379:methods*; D030541:Databases, Genetic; D005393:Fish Diseases / Q000517:prevention & control*; D005412:Flatfishes / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D005819:Genetic Markers / Q000235:genetics; D023281:Genomics; D006801:Humans; D058977:Molecular Sequence Annotation; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012098:Reproduction / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D005819:Genetic Markers; D016372:RNA, Antisense; D012333:RNA, Messenger