Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

Abstract

BackgroundUnambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA.MethodsMultiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing.ResultsUsing this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%).ConclusionLong-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Evaluation of the AllType kit for HLA typing using the Ion Torrent S5 XL platform

“…These were easily detected due to non‐common linkage disequilibrium. Most studies have reported very low allele dropout frequencies (<1%), whereas others have reported frequencies as high as 10%, especially for the HLA‐DQB1 locus . Therefore, the need for alternative methods to confirm apparently homozygous HLA alleles is a matter of debate.…”

Next generation sequencing characterizes HLA diversity in a registry population from the Netherlands

“…These algorithms base their predictions on the frequency of haplotypes in the unrelated donor population. The increased utilization of DNA sequencing for typing registry populations has provided higher resolution HLA assignments, speeding donor selection. The application of next generation DNA sequencing to compile full length sequences of HLA genes has improved the ability to interpret sequencing results .…”

A novel HLA‐A null allele, HLA‐A*66:28N

“…This DNA sample was typed for HLA loci‐A, ‐B, ‐C, ‐DRB1, and ‐DQB1 using our routine NGS‐based HLA typing protocol covering full length of HLA‐A, ‐B, and ‐C, and exons 2 and 3 for HLA‐DRB1 and HLA‐DQB1 . Briefly, genomic DNA was extracted from a buccal swab sample, amplified, and sequenced on MiSeq (Illumina, SanDiego, California).…”

“…This DNA sample was typed for HLA loci-A, -B, -C, -DRB1, and -DQB1 using our routine NGS-based HLA typing protocol covering full length of HLA-A, -B, and -C, and exons 2 and 3 for HLA-DRB1 and HLA-DQB1. 2 Briefly, genomic DNA was extracted from a buccal swab sample, amplified, and sequenced on MiSeq (Illumina, SanDiego, California). The results showed the HLA typing of this individual to be HLA-A*24:02:01, HLA-A*66new; HLA-B*18:01:01, HLA-B*44:03:01; HLA-C*04:01:01, HLA-C*07:01:01; HLA-DQB1*06:02, HLA-DQB1*06:02; and HLA-DRB1*11:04, HLA-DRB1*15:01 analyzed using NGSengine v2.4.0 (GenDx, Utrecht, The Netherlands) and HLA Twin v1.0.7 (Omixon, Budapest, Hungary).…”