Pooled cDNA Illumina 76bp paired-end sequence database for Litopenaeus vannamei
To enable the identification and development of genome-wide Type I SNPs, high-quality total RNA was extracted from the uropod tissue of 30 L. vannamei individuals representing prominent industry domesticated lines, using TRIZOL Reagent (Life Technologies). RNA from each individual were pooled together in equimolar amounts before being converted to double stranded cDNA using the Mint cDNA synthesis kit (Evrogen), and normalised using the Trimmer cDNA normalisation kit (Evrogen) using manufacturers recommendations. Normalised cDNA was then sequenced using an Illumina GA-IIX at 76 bp paired-end resulting in approximately 25 gigabases of paired-end EST sequence data (~10x genome coverage). Sequence adaptors and primers were screened and removed using the software Seqclean. MOTHUR was used to remove sequences with an average quality score less than 15 (window size = 10 bp) and / or shorter than 50 bp. The cleaned sequence data was assembled using Velvet V1.0 and OASES. Assembly parameters consisted of no extra gap penalty with all other options at default or recommended settings. Transcript assemblies were conducted at kmer lengths of k39, k41, k43, k45, k47, k49, k51 and k53 before being clustered together at a 90% sequence identify threshold using the software CD-HIT. Where multiple transcript sequences were identified, only the longest sequence was retained. Assembly of the cleaned-up sequence data produced 76,963 contigs. The N50 of the assembly was 2,375 bp, the average contig length was 1,429 bp and median contig length was 955. Transcript redundancy removal was undertaken, since it is a requirement for SNP discovery.