Date: Saturday, January 14, 2012
Time: 1:55 PM
Time: 1:55 PM
Room: Royal Palm Salons 3-4
Subtractive suppressive hybridization (SSH) libraries are used to generate de novo transcriptomic information associated with a contrasting biological condition in aquaculture species, including those in which genomic information is unavailable. In order to isolate EST’s from genes up/down regulated during spermatogenesis (meiosis, cell cycle control, and gamete development), we obtained two libraries from the scallop Nodipecten subnodosus after maturing testis vs. inactive gonad were reciprocally subtracted. The original conception of the SSH libraries workflow ends with the cloning of a variety of cDNA fragments followed by its sequencing by Sanger capillary electrophoresis. We obtained 564 EST’s by this sequencing method. Additionally and with the same cost invested in Sanger sequences, we also used next-generation sequencing technology. Unique amplicon-library adapters for 454-FLX (Roche) sequencing were added by tailing-PCR to the cDNA fragments obtained in each library after subtraction, allowing for both libraries to be sequenced in a single run. Under similar sequencing costs, 64x more high-quality reads were obtained by next-generation sequencing (454-FLX) in comparison with Sanger. Sequencing with 454-FLX yielded a total of 31,786 reads, with 29,186 reads longer than 50 bp (after extensive low quality and poly(A) trimming). From the 31,786 reads, only 4,953 were redundant, indicating that the subtraction was effective in isolating spermatogenesis differentially expressed genes. Deep sequencing by 454-FLX under a tailing-PCR and multiplex strategy provides cost-effective, deeper coverage than traditional Sanger sequencing for the isolation of EST's in SSH libraries.