Low-Cost Genotyping by Next Generation Sequencing: Next Generation Genotyping

The utility of mass genotyping has been tempered by both the expense and time requirements of existing technologies, particularly for larger scale projects. We have demonstrated the ability to use next generation sequencing technologies to genotype thousands of DNA samples for hundreds of loci – a next generation genotyping technology (NGG). The power and economy of NGG is that it focuses sequence read generation on the loci specified to be genotyped. NGG is a ligation-dependent PCR that uses allele barcodes contained within the ligation probes as well as sample barcodes added by PCR. A library suitable for short next generation sequence data generation on a single GAIIx lane on hundreds of loci in thousands of samples can be created in 24 hours. The barcoded sequence on the reads are binned and counted. The bins provide the sample, locus, and allele identification. The read counts are used to infer genotypes. The cost per sample is highly competitive. The sample barcodes allow different assays to be mixed for sequence data generation. The most cumbersome step is DNA extraction. The NGG assay has been used to interrogate simple and poly allelic SNPs, INDELs and STR. In NGG experiments on cattle parentage with 95 loci on 1080 Bos taurus samples, we have obtained 99.1% concordance and 96.1% call rate with genotype data previously identified using the Illumina BovineSNP50 bead chip.