OR28 High-throughput KIR sequencing by NGS: 500,000 registry samples genotyped at allelic resolution

Aim The human killer-cell immunoglobulin-like receptor (KIR) family of genes is a key regulator of natural killer cell activity. Several studies have indicated that KIR genotypes affect hematopoietic stem cell transplantation outcome and the evidence is rising that the extensive allelic diversity discovered in KIR genes may be an important component to consider. However, high-resolution characterization of KIR genotypes has so far been applied only to smaller cohorts. Therefore, we developed a KIR genotyping approach based on NGS that would be cost-effective and amendable for high-throughput application. Methods We amplified KIR exons 3, 4, 5, 7, 8 and 9, targeting one to two exons per PCR reaction but multiplexed across all KIR genes. Amplicons of 2 × 3760 samples were combined for sequencing on Illumina HiSeq 2500 instruments together with amplicons for HLA and blood groups. The reads were mapped against the described KIR alleles (IPD-KIR Release 2.6). Based on the read coverage, we estimated the genomic copy number of each sequence feature. These sequence copy calculations enabled precise allele calling and in addition empowered us to determine gene copy numbers. Results After successful validation, we applied this workflow to genotype 500,000 registry samples. Despite the focus on throughput and cost efficiency, we achieved mostly allotype (3 digit) resolution with the exception of certain allotypes differing only in the short non-targeted exon regions. In addition, certain phasing ambiguities remained. We report the observed allele diversity and the relative abundance of alleles for a mainly Caucasian cohort. Conclusions We demonstrate that high-resolution KIR genotyping is feasible using a very cost-efficient workflow. This approach enables KIR genotyping for population genetics, disease association studies and other applications requiring large cohorts. As of October 2016 we have been applying this workflow to the analysis of all volunteer samples registering with DKMS as potential donors for hematopoietic stem cell transplantation. High-resolution KIR genotyping results will thus become available to search coordinators and may soon be used as an additional selection criterion to improve transplantation outcome.