Dataset Information

Multiplexed Nanopore Sequencing of HLA-B Locus in Maori 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 M?ori 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 M?ori and Pacific Island people.

SUBMITTER: Ton KNT

PROVIDER: S-EPMC5936980 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

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

Ton Kim N T KNT Cree Simone L SL Gronert-Sum Sabine J SJ Merriman Tony R TR Stamp Lisa K LK Kennedy Martin A MA

Frontiers in genetics 20180430

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 improve ...[more]

PMID: 29760718

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Project description:BACKGROUND:Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction. Here, we report the performance of this unique workflow evaluated using 218 genetically diverse samples. METHODS:Five whole genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/DRB345/DQB1/DPB1; excluding exon 1 and part of intron 1) were amplified in a multiplexed, long-range PCR. Manual library preparation was performed per manufacturer's protocol, followed by template preparation and chip loading on the Ion Chef, and sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and repeat testing were followed; technologists were blinded to the reference results. The concordance between AllType and reference results was determined at 2-field resolution. We also describe the ranges of input DNA and library concentrations, read number per sample and per locus, and key health metrics in relation to typing results. RESULTS:The concordance rates were 98.6%, 99.8% and 99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, respectively. Three genotypes were discordant, all of which shared the same G group typing results with the reference. Most ambiguous genotypes (116 out of 144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and library concentrations were tolerated. Per sample read numbers were adequate for accurate genotyping. Per locus read numbers showed some inter-lot variations, and a trend toward improved inter-locus balance was observed with later lots of reagents. CONCLUSION:The AllType assay on the Ion Chef/Ion S5 platform offers a robust and efficient workflow for clinical HLA typing at the 2-field resolution. The multiplex PCR strategy simplifies the laboratory procedure without compromising the typing accuracy.

Dataset Information

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

Publications

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

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