Data from: Genotyping-in-Thousands by sequencing (GT-seq): a cost effective SNP genotyping method based on custom amplicon sequencing

Genotyping-in-Thousands by sequencing (GT-seq) is a novel method that uses next generation sequencing of multiplexed PCR products to generate genotypes from small to moderate panels (50-500) of targeted SNPs for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabeled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci, addition of sequencing adapters, and dual barcode tagging, enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is done using a simple perl script which counts amplicon specific sequences for each allele and uses allele ratios to determine genotypes. We demonstrate this technique by genotyping 2,068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 157 SNP markers in a single library and compare genotype data to previously collected TaqMan™ genotypes at the same loci. Results indicated greater than 99.9% concordance between genotypes generated with the GT-seq method relative to Taqman™ assays with call rates averaging over 90.0% for GT-seq. This novel amplicon sequencing method greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.