Determining performance characteristics of an NGS‐based HLA typing method for clinical applications

Abstract

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

Somatic HLA mutations expose the role of class I–mediated autoimmunity in aplastic anemia and its clonal complications

“…The diagnosis and severity aAA were determined by standard criteria 37,39,40 ; congenital bone marrow failure syndromes were excluded as previously described 38 . The 66 aAA patients in this study included 16 aAA patients from our previous WES cohort 36 .…”

Role of major histocompatibility complex variation in graft-versus-host disease after hematopoietic cell transplantation

“…Serologic methods were the mainstay of tissue typing early in the history of the field but since have been supplanted by molecular methods. Today, next-generation sequencing (NGS) platforms designed to comprehensively characterize large segments of HLA genes and in some cases establish the cis-relationship between markers provide investigators with an unprecedented view of sequence diversity and the organization of variation on haplotypes 13– 16 . Use of molecular tools for HLA genes has led to the recognition of over 3,830 HLA-A, 4,647 HLA-B, 3,382 HLA-C, 2,011 HLA-DRB1, 1,054 HLA-DQB1, and 740 HLA-DPB1 alleles, just to name the six classical loci currently considered in the selection of HCT donors 17 .…”

Killer Immunoglobulin-Like Receptor Allele Determination Using Next-Generation Sequencing Technology

“…Due to the high degree of KIR polymorphisms and the fact that NGS technology generates a lot of sequencing reads, three different algorithms were evaluated to increase the reliability of KIR allele assignment as reported for NGS-based HLA typing (50). KIR allele assignment was first done manually and then confirmed using both BiRD pipeline and Profiler software.…”