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Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing.
HLA. 2020 Aug; 96(2):163-178.HLA

Abstract

Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore-based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology-based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long-range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA-LA. Called genotypes were compared with reference derived from a combination of short-read next-generation sequencing, Sanger sequence and/or single-site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost-effective long-range PCR and nanopore sequencing-based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi-sample runs to a single sample.

Authors+Show Affiliations

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.NHS Blood and Transplant, Filton, UK.NHS Blood and Transplant, Filton, UK.Queen Elizabeth Hospital Birmingham, Birmingham, UK.NHS Blood and Transplant, Filton, UK.Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32419382

Citation

Stockton, Joanne D., et al. "Rapid, Highly Accurate and Cost-effective Open-source Simultaneous Complete HLA Typing and Phasing of Class I and II Alleles Using Nanopore Sequencing." HLA, vol. 96, no. 2, 2020, pp. 163-178.
Stockton JD, Nieto T, Wroe E, et al. Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing. HLA. 2020;96(2):163-178.
Stockton, J. D., Nieto, T., Wroe, E., Poles, A., Inston, N., Briggs, D., & Beggs, A. D. (2020). Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing. HLA, 96(2), 163-178. https://doi.org/10.1111/tan.13926
Stockton JD, et al. Rapid, Highly Accurate and Cost-effective Open-source Simultaneous Complete HLA Typing and Phasing of Class I and II Alleles Using Nanopore Sequencing. HLA. 2020;96(2):163-178. PubMed PMID: 32419382.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing. AU - Stockton,Joanne D, AU - Nieto,Thomas, AU - Wroe,Elizabeth, AU - Poles,Anthony, AU - Inston,Nicholas, AU - Briggs,David, AU - Beggs,Andrew D, PY - 2020/04/14/received PY - 2020/04/30/revised PY - 2020/05/05/accepted PY - 2020/5/19/pubmed PY - 2020/5/19/medline PY - 2020/5/19/entrez KW - HLA KW - long read KW - nanopore SP - 163 EP - 178 JF - HLA JO - HLA VL - 96 IS - 2 N2 - Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore-based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology-based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long-range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA-LA. Called genotypes were compared with reference derived from a combination of short-read next-generation sequencing, Sanger sequence and/or single-site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost-effective long-range PCR and nanopore sequencing-based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi-sample runs to a single sample. SN - 2059-2310 UR - https://www.unboundmedicine.com/medline/citation/32419382/Rapid,_highly_accurate_and_cost-effective_open-source_simultaneous_complete_HLA_typing__phasing_of_Class_I__II_alleles_using_Nanopore_sequencing L2 - https://doi.org/10.1111/tan.13926 DB - PRIME DP - Unbound Medicine ER -
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