Logo image
Back
P050 Rapid, high resolution HLA genotyping using nanopore sequencing
Journal article   Peer reviewed

P050 Rapid, high resolution HLA genotyping using nanopore sequencing

Peter M. Clark, Deborah Ferriola and Dimitri S. Monos
Human immunology, v 78, pp 90-90
Sep 2017
DOI: https://doi.org/10.1016/j.humimm.2017.06.110

Abstract

Despite the significant advancement of high resolution HLA genotyping by next generation sequencing (NGS), the full promise of NGS has yet to be fully realized. Paired-end Illumina sequencing of HLA amplicons results in lengthy turn around times often approaching 72h and remains unable to resolve genotyping ambiguities within class II HLA genes. We aim to address both of these issues by performing rapid, single molecule sequencing of full length HLA amplicons on the Oxford Nanopore sequencing platform. HLA amplicons were generated at 11 loci (HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5) using the Omixon Holotype kit for five individuals with ambiguous HLA-DPB1 genotyping (as assessed by the analysis of paired-end sequencing data). HLA amplicons were used to generate 2D sequencing libraries that were sequenced on the Oxford Nanopore MinION platform with real-time base calling. A custom bioinformatics pipeline was developed to generate phased, full-length HLA allele consensus sequences and perform high resolution genotyping for each amplified locus. Sequencing reads were found to have a high insertion/deletion error rate (particulary around homopolymer stretches) and an observed average basecall error rate of ∼4%. However, with an average read depth greater than 200X per locus, our analytical pipeline was able to generate highly accurate (99.99%), full-length consensus sequences and unambiguous HLA genotyping results for each HLA locus analyzed that were 100% consistent with genotyping results obtained from the analysis of paired-end sequencing data. All steps of the process, from DNA extraction to high resolution HLA genotyping for each sample was performed within ∼24h. Our results demonstrate the utility of the Oxford Nanopore MinION sequencing platform in combination with our developed bioinformatics pipeline to generate full-length gene consensus sequences and unambiguous high resolution HLA genotyping results. As the platform chemistry and flow-cell continue to be optimized, we hope that turn around time and read accuracy will further improve, facilitating unambiguous, rapid high resolution HLA genotyping within Immunogenetics laboratories addressing currently unmet needs.

Metrics

13 Record Views
1 citations in Web of Science

Details

InCites Highlights

Data related to this publication, from InCites Benchmarking & Analytics tool:

Web of Science research areas
Immunology
Logo image