Evaluation of GenDx Protocol for HLA NGS Genotyping Using the Ion Torrent Sequencing Platform

Abstract Background: The Human Major Histocompatibility Complex (HLA) is the most polymorphic region of the human genome and encodes molecules with a central role in antigen-specific immune responses. Class I and class II HLA genes have an important role in bone marrow and solid organ transplantations. HLA typing by Next Generation Sequencing (NGS) provides unambiguous second field or allelic level resolution and, therefore, better matching of donor and recipient and a higher chance of survival for transplanted patients. The aim of this study was to validate GenDx NGS HLA typing protocol for 6 HLA loci (HLA-A, -B, -C, DRB1, -DQB1 and DPB1). Materials and methods: 240 samples of both bone marrow healthy donors from the Bulgarian Bone Marrow Donors Registry (BBMDR) and bone marrow recipients were sequenced on the Ion Torrent System using the GenDx NGS HLA typing kit, compared to 65 samples from the BBMDR analyzed by Holotype HLA kit (Omixon) and sequenced on the Illumina platform MiniSeq. A number of metrics including allele balance, read length, mappability, coverage and ambiguity were assessed in order to evaluate the GenDx NGS HLA typing protocol. We also investigated haplotype and allele frequencies of class I (HLA-A, -B, -C) and class II (HLA-DRB1, -DQB1 and -DPB1) alleles. Results: The results demonstrate the accuracy of this protocol, eliminating almost all ambiguities, providing a reasonable read length of 180, 76% mappability and 1267 depth of coverage on average for all 6 HLA loci. We found that the most frequent alleles for class I are HLA-A*02:01:01 (27,5%), B*51:01:01 (16,5%), C*07:01:01 (16,5%) and class II – DRB1*11:04:01 (14,5%), DQB1*03:01:01 (26,9%) and DPB1*04:01:01 (33,2%) which corresponds with other investigations of the research team on HLA diversity in the Bulgarian population. Conclusion: The main advantages of GenDx NGS HLA typing protocol are the shorter time for DNA library preparation and the shorter sequencing time. Phasing ambiguities are also liminated, which is a significant advantage in the clinical environment. The presented data show the suitability of this NGS protocol for clinical practice, especially in HLA typing of donors from Bone Marrow Donors registries.