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.
Highlights
The human leukocyte antigen (HLA) locus contains a large family of genes encoding the human major histocompatibility complex (MHC) proteins
Five individuals were included from a local study on adverse drug reactions (ADRs) called Understanding ADRs or responses Using Genome Sequencing (UDRUGS) (Maggo et al, 2017), which was approved by the Southern Health and Disability Ethics Committee (New Zealand), with written informed consent from all subjects
Each polymerase chain reaction (PCR) product was amplified with barcode primers, at which point all 49 PCR products were tagged with barcodes, increasing their length to 1,063 bp (Figure 1)
Summary
The human leukocyte antigen (HLA) locus contains a large family of genes encoding the human major histocompatibility complex (MHC) proteins. It is located on chromosome 6p21 and divided into three classes: class I, class II, and class III. HLA molecules are extremely variable due to their peptide-binding function and are associated with autoimmune diseases and adverse drug reactions (ADRs) (Tiwari and Terasaki, 1985; Bharadwaj et al, 2010). Previous studies have identified particular HLA-B alleles as risk factors for drug-induced hypersensitivity reactions (Alfirevic and Pirmohamed, 2010; Sukasem et al, 2014).
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call