Genotyping in Thousands (GTseq): A Cost Effective SNP Genotyping Method Based on Custom Amplicon Sequencing

GTseq is a method that uses massively parallel sequencing of multiplex amplified 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 in a single library.  Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences.  Then, a simple perl script counts amplicon specific sequences for each allele and genotypes are generated using ratios of allele counts.  Testing of this method showed greater than 99% concordance between genotypes generated with the GTseq method relative to existing Taqman™ assays with call rates >97% for GTseq.   This novel amplicon sequencing method greatly reduces the cost of small panel SNP genotyping relative to other methods by utilizing a simple library preparation method and a massive efficiency of scale.