High-throughput next-generation sequencing to genotype six classical HLA loci from 96 donors in a single MiSeq run.HLA. 2017 11; 90(5):284-291.HLA
Next generation sequencing (NGS) methods have been established as an efficient approach for HLA typing because unlike traditional Sanger sequencing, they provide unambiguous results at a reasonable cost. We previously developed a multi-locus index method to genotype four HLA loci (A, B, C, and DRB1) on the Illumina MiSeq platform. We have now expanded this method to include two additional loci, HLA-DPB1 and DQB1. Contiguous full-length amplicons from 5'UTR through 3'UTR regions were generated using one long-range PCR reaction per locus for each of the six loci from 96 individuals of different ethnicities. The six amplicons from each donor were pooled, enzymatically fragmented and given a donor-specific index. This approach enabled sequencing of 576 loci from 96 individuals in a single MiSeq run. Donor-specific sequence reads were demultiplexed, and allele calls were generated from FASTQ files using commercially available software. Comparison to HLA genotypes generated from Sanger sequence-based typing (SBT) identified no discordances among any of the alleles analyzed in this study. Importantly, this method was able to resolve 22 DPB1 and 20 DQB1 alleles that were ambiguous with the SBT method. Furthermore, a novel allele in each of these two loci was identified, with the DQB1*05:01:24 allele having a frequency of greater than five percent. This method was subsequently validated against a blinded panel of 22 samples from the 17th International HLA and Immunogenetics Workshop. The flexibility of the method is further highlighted by successful genotyping of eight loci comprising all classical HLA loci for a subset of the samples. We now present a high-throughput, high-resolution, scalable NGS HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci.