Multiplexed Nanopore Sequencing of HLA-B Locus in Māori and Pacific Island Samples

Abstract

The human leukocyte antigen (HLA) system encodes the human major histocompatibility complex (MHC). HLA-B is the most polymorphic gene in the MHC class I region and many HLA-B alleles have been associated with adverse drug reactions (ADRs) and disease susceptibility. The frequency of such HLA-B alleles varies by ethnicity, and therefore it is important to understand the prevalence of such alleles in different population groups. Research into HLA involvement in ADRs would be facilitated by improved methods for genotyping key HLA-B alleles. Here, we describe an approach to HLA-B typing using next generation sequencing (NGS) on the MinION™ nanopore sequencer, combined with data analysis with the SeqNext-HLA software package. The nanopore sequencer offers the advantages of long-read capability and single molecule reads, which can facilitate effective haplotyping. We developed this method using reference samples as well as individuals of New Zealand Māori or Pacific Island descent, because HLA-B diversity in these populations is not well understood. We demonstrate here that nanopore sequencing of barcoded, pooled, 943 bp polymerase chain reaction (PCR) amplicons of 49 DNA samples generated ample read depth for all samples. HLA-B alleles were assigned to all samples at high-resolution with very little ambiguity. Our method is a scaleable and efficient approach for genotyping HLA-B and potentially any other HLA locus. Finally, we report our findings on HLA-B genotypes of this cohort, which adds to our understanding of HLA-B allele frequencies among Māori and Pacific Island people.

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

“…One of the first platforms used for HLA typing was the 454/GS FLX sequencer (Roche), which has been progressively supplanted by Illumina and Ion Torrent technologies . Advanced technologies are also used, such as Pacific Biosciences's single‐molecule real‐time sequencing and Oxford Nanopore single‐cell DNA template strand sequencing …”

“…[21][22][23][24] Advanced technologies are also used, such as Pacific Biosciences's single-molecule real-time sequencing [25][26][27][28][29][30] and Oxford Nanopore single-cell DNA template strand sequencing. [31][32][33] We recently implemented an NGS method for HLA typing using the AllType NGS kit from One Lambda and Thermo Fisher Scientific reagents on the Ion S5 XL platform. The objective of this study was to evaluate the performance of this method by systematic analysis of warnings given by the analysis software and extensive comparison of HLA typing results obtained via other methods and/or knowledge of common HLA associations as a consequence of linkage disequilibrium.…”

Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes

“…The discrepancies were focused to allotypes B * 07 and B * 35. Compared to the previous studies using MinION in the sequencing of HLA-B (Ton et al, 2018;De Santis et al, 2020), our ONT RNA-Seq method did reach as high concordance with the validation method. This might be due to methodological differences or differences in data analysis since both of the previous studies were based on the analysis of genomic data.…”

HLA RNAseq reveals high allele-specific variability in mRNA expression

“…Since genomic ONT data have been shown to be successful in HLA-typing [18,21], we explored the accuracy of ONT RNAseq data in HLA allele calling. The 2D reads from the full-length sequencing of HLA amplicons with MinION resulted in a good accordance with the Luminex reference methods at the 2-field resolution level, suggesting that HLA typing can be performed from targeted ONT RNAseq data.…”