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Hum Immunol. 2014 Oct;75(10):1040-6. doi: 10.1016/j.humimm.2014.08.206. Epub 2014 Aug 27.

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors.

Author information

1
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
2
Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA 98109, United States.
3
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA 98109, United States.
4
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States. Electronic address: geraghty@fhcrc.org.
5
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA 98109, United States. Electronic address: jhansen@fhcrc.org.

Abstract

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.

KEYWORDS:

Genotyping; HLA-class II; High resolution; Multiplex reaction PCR; Next generation sequencing

PMID:
25167774
DOI:
10.1016/j.humimm.2014.08.206
[Indexed for MEDLINE]

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