Allele-Level Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations

The killer-cell immunoglobulin-like receptor () genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported variants repeatedly in independent samples, underscoring the large diversity of the region that awaits discovery. This cost-efficient high-resolution genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution genotyping, like population genetics and disease association studies.