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A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans am... Full description

Journal Title: Human Immunology 2010, Vol.71(10), pp.1011-1017
Main Author: Lank, Simon M
Other Authors: Wiseman, Roger W , Dudley, Dawn M , O'Connor, David H
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: ISSN: 0198-8859 ; E-ISSN: 1879-1166 ; DOI: 10.1016/j.humimm.2010.07.012
Link: https://www.sciencedirect.com/science/article/pii/S0198885910004477
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recordid: elsevier_sdoi_10_1016_j_humimm_2010_07_012
title: A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping
format: Article
creator:
  • Lank, Simon M
  • Wiseman, Roger W
  • Dudley, Dawn M
  • O'Connor, David H
subjects:
  • Pyrosequencing
  • Human Leukocyte Antigen
  • Sequence-Based Typing
  • Multiplexing
  • High-Throughput
  • Medicine
  • Biology
ispartof: Human Immunology, 2010, Vol.71(10), pp.1011-1017
description: Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.
language: eng
source:
identifier: ISSN: 0198-8859 ; E-ISSN: 1879-1166 ; DOI: 10.1016/j.humimm.2010.07.012
fulltext: fulltext
issn:
  • 0198-8859
  • 01988859
  • 1879-1166
  • 18791166
url: Link


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descriptionHuman leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.
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Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.

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