title: Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing.
abstract: High-resolution human leukocyte antigen (HLA) matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplant. Sanger sequencing has been the gold standard for HLA typing since 1996. However, given the increasing number of new HLA alleles identified and the complexity of the HLA genes, clinical HLA typing by Sanger sequencing requires several rounds of additional testing to provide allele-level resolution. Although next-generation sequencing (NGS) is routinely used in molecular genetics, few clinical HLA laboratories use the technology. The performance characteristics of NGS HLA typing using TruSight HLA were determined using Sanger sequencing as the reference method. In total, 211 samples were analyzed with an overall accuracy of 99.8% (2954/2961) and 46 samples were analyzed for precision with 100% (368/368) reproducibility. Most discordant alleles were because of technical error rather than assay performance. More important, the ambiguity rate was 3.5% (103/2961). Seventy-four percentage of the ambiguities were within the DRB1 and DRB4 loci. HLA typing by NGS saves approximately $6000 per run when compared to Sanger sequencing. Thus, TruSight HLA assay enables high-throughput HLA typing with an accuracy, precision, ambiguity rate, and cost savings that should facilitate adoption of NGS technology in clinical HLA laboratories.
authors.affiliation: Human Leukocyte Antigen Laboratory, McLendon Clinical Laboratories, University of North Carolina Hospitals, Chapel Hill, North Carolina; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Electronic address: eric_weimer@med.unc.edu.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Weimer
authors.affiliation: Human Leukocyte Antigen Laboratory, McLendon Clinical Laboratories, University of North Carolina Hospitals, Chapel Hill, North Carolina.
authors.forename: Maureen
authors.initials: M
authors.lastname: Montgomery
authors.affiliation: Human Leukocyte Antigen Laboratory, McLendon Clinical Laboratories, University of North Carolina Hospitals, Chapel Hill, North Carolina.
authors.forename: Rosanne
authors.initials: R
authors.lastname: Petraroia
authors.affiliation: Human Leukocyte Antigen Laboratory, McLendon Clinical Laboratories, University of North Carolina Hospitals, Chapel Hill, North Carolina.
authors.forename: John
authors.initials: J
authors.lastname: Crawford
authors.affiliation: Human Leukocyte Antigen Laboratory, McLendon Clinical Laboratories, University of North Carolina Hospitals, Chapel Hill, North Carolina; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina.
authors.forename: John L
authors.initials: JL
authors.lastname: Schmitz
mesh_terms: D003362:Cost-Benefit Analysis; D005784:Gene Amplification; D015723:Gene Library; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods / Q000592:standards; D006650:Histocompatibility Testing / Q000191:economics / Q000379:methods* / Q000592:standards; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards
chemical_list: D006680:HLA Antigens