Transcriptome sequencing and high-resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids.

Until recently, single nucleotide polymorphism (SNP) discovery in nonmodel organisms faced many challenges, often depending upon a targeted-gene approach and Sanger sequencing of many individuals. The advent of next-generation sequencing technologies has dramatically improved discovery, but validating and testing SNPs for use in population studies remain labour intensive. Here, we detail a SNP discovery and validation pipeline that incorporates 454 pyrosequencing, high-resolution melt analysis (HRMA) and 5' nuclease genotyping. We generated 4.59×10(8) bp of redundant sequence from transcriptomes of two individual chum salmon, a highly valued species across the Pacific Rim. Nearly 26000 putative SNPs were identified--some as heterozygotes and some as homozygous for different nucleotides in the two individuals. For validation, we selected 202 templates containing single putative SNPs and conducted HRMA on 10 individuals from each of 19 populations from across the species range. Finally, 5' nuclease genotyping validated 37 SNPs that conformed to Hardy-Weinberg equilibrium expectations. Putative SNPs expressed as heterozygotes in an ascertainment individual had more than twice the validation rate of those homozygous for different alleles in the two fish, suggesting that many of the latter may have been paralogous sequence variants. Overall, this validation rate of 37/202 suggests that we have found more than 4500 templates containing SNPs for use in this population set. We anticipate using this pipeline to significantly expand the number of SNPs available for the studies of population structure and mixture analyses as well as for the studies of adaptive genetic variation in nonmodel organisms.

Short reads and nonmodel species: exploring the complexities of next-generation sequence assembly and SNP discovery in the absence of a reference genome.

How practical is gene and SNP discovery in a nonmodel species using short read sequences? Next-generation sequencing technologies are being applied to an increasing number of species with no reference genome. For nonmodel species, the cost, availability of existing genetic resources, genome complexity and the planned method of assembly must all be considered when selecting a sequencing platform. Our goal was to examine the feasibility and optimal methodology for SNP and gene discovery in the sockeye salmon (Oncorhynchus nerka) using short read sequences. SOLiD short reads (up to 50 bp) were generated from single- and pooled-tissue transcriptome libraries from ten sockeye salmon. The individuals were from five distinct populations from the Wood River Lakes and Mendeltna Creek, Alaska. As no reference genome was available for sockeye salmon, the SOLiD sequence reads were assembled to publicly available EST reference sequences from sockeye salmon and two closely related species, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Additionally, de novo assembly of the SOLiD data was carried out, and the SOLiD reads were remapped to the de novo contigs. The results from each reference assembly were compared across all references. The number and size of contigs assembled varied with the size reference sequences. In silico SNP discovery was carried out on contigs from all four EST references; however, discovery of valid SNPs was most successful using one of the two conspecific references.