P037 Genotyping of six classical HLA loci using next generation sequencing

Abstract

Aim The Multi-locus Individual Tagging – Next Generation Sequencing (MIT-NGS) method was previously shown to efficiently genotype four HLA loci (A, B, C, and DRB1) from 96 donors while maintaining phase, and resolving ambiguities associated with traditional sequence based HLA typing (Ehrenberg et al., BMC Genomics, 2014). Here we expanded MIT-NGS to six loci to include DPB1 and DQB1, and evaluated HLA genotypes for all loci against those obtained from both the HLA sequence-based typing (SBT) method and the four loci MIT-NGS method. Methods Full-length genes of HLA-A, B, C, DPB1, DQB1 and DRB1 loci from 96 donors of Caucasian, African and Asian ethnicities were amplified using long-range PCR. Individual donor pools, each consisting of equimolar inputs corresponding to the target size of the six HLA loci amplicons were enzymatically fragmented and uniquely indexed. This 6-plex approach enabled sequencing of 576 loci from 96 individuals in a single Illumina MiSeq run. Locus specific sequence reads were demultiplexed, aligned, and genotyped using commercially available software and an in-house pipeline. Results There were no observed discordances between the 6-plex MIT-NGS method and the combined SBT or 4-plex NGS methods for all 1152 alleles analyzed in this study. Furthermore, this method was able to resolve 20 DQBI and 22 DPB1 genotypes that were ambiguous with SBT. These alleles included DPB1 ∗ 104:01,DPB1 ∗ 105:01, DPB1 ∗ 107:01, DPB1 ∗ 131:01, DPB1 ∗ 135:01, DPB1 ∗ 296:01, DPB1 ∗ 414:01, DQB1 ∗ 02:02, DQB1 ∗ 03:19 and DQB1 ∗ 06:01:03. All of these ambiguous changes affected the protein coding sequence. Conclusions We have developed a six-locus high resolution HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci while maintaining phase, and resolving ambiguities associated with conventional sequence based HLA typing.