OR36 Defining KIR and HLA class I alleles at highest resolution using next generation sequencing

Abstract

Aim Combinatorial diversity of KIR and HLA class I alleles directly affects NK cell activity and impacts the outcome of human immune responses to infections, and success in reproduction and transplantation. Therefore it is important to identify genetic variation in these highly polymorphic genes at high resolution. Here we have used our high throughput capture/NGS method to target KIR and HLA class I genes. Methods Libraries were prepared using fragmented genomic DNA samples from 1000 European individuals, 97 IHWG cell lines and 31 family trios from West Africa. After capture of fragments with oligonucleotide probes, the entire KIR locus and HLA class I genes were sequenced to high depth (100×) using Illumina paired-end technology. We also developed a bioinformatics pipeline (PING: Pushing Immunogenetics to the Next Generation) specifically to extract and analyze the KIR-specific reads from any NGS data source. PING attributes sequencing reads to specific KIR genes, determines their copy number by read depth and virtual sequence probes, and then calls high-resolution genotypes. PING also returns information on newly-identified SNPs and recombinant alleles. PING is available as an R package at https://github.com/ . Results The method was validated using data from the 97 IHWG cell lines. KIR gene copy numbers were independently verified by complete correlation with real time PCR, and the allele calls using pyrosequencing and Sanger sequencing. We determined the composition of all the KIR haplotypes represented in the 97 IHWG cells, which are available for reference. PING was further validated by analyzing the 1000 genomes data set for 3DL1/S1 and 3DL2, where we identified and validated 100 novel alleles. This analysis of just two KIR genes doubled the number of known 3DL2 alleles, and showed that 22% of the 1000 Genomes subjects have a novel allele or a structural variant. Subsequently in analyzing the European and African we find few novel HLA class I alleles, but in contrast we find one novel KIR allele per ten individuals. Conclusions The KIR locus is likely to be as diverse as HLA. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations, matching transplant donors with recipients, and defining the precise HLA and KIR factors associated with disease.