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 method that uses
next-generation sequencing of multiplexed PCR products to generate
genotypes from relatively small panels (50–500) of targeted
single-nucleotide polymorphisms (SNPs) for thousands of individuals in a
single Illumina HiSeq lane. This method uses only unlabelled oligos and
PCR master mix in two thermal cycling steps for amplification of targeted
SNP loci. During this process, sequencing adapters and dual barcode
sequence tags are incorporated into the amplicons 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
performed with a simple perl script which counts amplicon-specific
sequences for each allele, and allele ratios are used to determine the
genotypes. We demonstrate this technique by genotyping 2068 individual
steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP
markers in a single library sequenced in a single Illumina HiSeq lane.
Genotype data were 99.9% concordant to previously collected TaqMan™
genotypes at the same 192 loci, but call rates were slightly lower with
GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were
genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped
in <70% of samples. This study demonstrates amplicon sequencing
with GT-seq 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.