1Affymetrix, 3420 Central Expressway, Santa Clara, CA, USA
The utility of high sample throughput of mid- to low- SNP-plex genotyping has been tempered by both the expense and time requirements of existing technologies, particularly for large scale projects. Many fields in agriculture need an affordable high throughput method to provide customizable genotyping for interrogation of tens to thousands of SNPs per sample. Eureka™ genotyping solution utilizes next generation sequencing to enable genotyping of thousands of DNA samples for tens to thousands of loci. The Eureka genotyping assay is a ligation-dependent PCR reaction, which uses interrogation site bar codes contained within the ligation probes, as well as sample index bar codes added during the amplification step. Next generation sequencing libraries can be created for thousands of DNA samples within 24 hours. Short cycle sequence data is generated from the prepared libraries and software is used to tabulate the number of reads that contain each combination of sample, locus and allele bar code (as appropriate). The genotype of each sample for each locus is inferred from statistical analysis of the tabulated reads. The Eureka genotyping assay has been used to interrogate simple and poly allelic SNPs, small to large INDELs, tetra-ploids and for the detection of low percent contamination in a large background of non-target DNA . The Eureka genotyping solution is flexible, accurate, specific, affordable and robust. Custom Eureka genotyping panels that have been created to date include barley, corn, soy, cattle and sheep.