title: Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors.
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.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Anajane G
authors.initials: AG
authors.lastname: Smith
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Chul-Woo
authors.initials: CW
authors.lastname: Pyo
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Wyatt
authors.initials: W
authors.lastname: Nelson
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Edward
authors.initials: E
authors.lastname: Gow
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Ruihan
authors.initials: R
authors.lastname: Wang
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Shu
authors.initials: S
authors.lastname: Shen
authors.affiliation: Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA 98109, United States.
authors.forename: Maggie
authors.initials: M
authors.lastname: Sprague
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA 98109, United States.
authors.forename: Shalini E
authors.initials: SE
authors.lastname: Pereira
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States. Electronic address: geraghty@fhcrc.org.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Geraghty
authors.affiliation: 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.
authors.forename: John A
authors.initials: JA
authors.lastname: Hansen
mesh_terms: D015331:Cohort Studies; D005091:Exons / Q000235:genetics; D005838:Genotype; D059867:HLA-DP alpha-Chains / Q000235:genetics; D059868:HLA-DP beta-Chains / Q000235:genetics; D059848:HLA-DQ alpha-Chains / Q000235:genetics; D059866:HLA-DQ beta-Chains / Q000235:genetics; D006684:HLA-DR Antigens / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D000949:Histocompatibility Antigens Class II / Q000737:chemistry*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D019520:Living Donors; D015203:Reproducibility of Results; D012984:Software
chemical_list: D059867:HLA-DP alpha-Chains; D059868:HLA-DP beta-Chains; C071172:HLA-DPA1 antigen; C071170:HLA-DPB1 antigen; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C069050:HLA-DQA1 antigen; C069051:HLA-DQB1 antigen; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II
keywords: Genotyping; HLA-class II; High resolution; Multiplex reaction PCR; Next generation sequencing