OR22 Towards clinical NGS HLA-typing: A performance comparison of next-generation sequencing technologies for DNA HLA typing in a clinical diagnostic environment

Abstract

Next generation sequencing (NGS) offers a new paradigm in HLA genotyping that will fundamentally change the way clinical laboratories report patient genotypes. The technique reveals entire gene sequences with ultra-high base resolution and can resolve phase ambiguities for multiple samples in a single assay. While implementation of NGS is challenging in clinical diagnostics, the definite advantages and new diagnostic possibilities make the switch to the technology inevitable. Recently a number of commercially available NGS-based HLA kits have come on the market. On behalf of the Canadian HLA network, we present a comparative study of 4 of these. HLA genotyping requires three basic steps; PCR, sequencing of amplicon library, and allele assignment. The relative performance of each step was assessed. We evaluated three commercially available NGS HLA typing kits; Illumina’s TruSight, Omixon’s Holotype HLA and GenDx’s NGSgo kit. We also had early access to OneLambda’s kit. All amplicon libraries were sequenced using either the Illumina MiSeq or Ion Torrent PGM platform. Each of the commercial kits included the necessary proprietary allele assignment software. All methods provided targeted capturing of the classical class I (HLA-A, B, C) and class II HLA genes (HLA-DRB1, DQA1, DQB1, DPB1) as a minimum. All calling algorithms provided allele-calling to at least three-field resolution. A validation panel of 48 clinical and proficiency testing samples were analyzed using all 4 methods and performed by the same technician. All samples had known HLA alleles obtained by LABType SSO/SSP typing (4 digit). A total of 1680 loci were analyzed. An overview of each protocol with our experience on sequence performance efficiencies, read depth uniformity and ambiguity analysis will be presented. Briefly, targeted NGS HLA typing kits were found to be easy to use, flexible to the need of the clinical laboratory and priced comparable to HD SSO. Results were highly congruous with standard SSO/SSP typing. Our studies show that NGS is fully feasible for routine use, and offers precise, ultra-high resolution, complete sequence, cost-efficient high-throughput HLA DNA typing which provides informative data and improved HLA matching for medical research, transplantation medicine, and HLA-related disease diagnosis.