Performance of a multiplexed amplicon-based next-generation sequencing assay for HLA typing

Abstract

Background Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplantrelevant HLA genes in one PCR reaction. Here, we report the performance of this unique workflow evaluated using 218 genetically diverse samples. Methods Five whole genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/ DRB345/DQB1/DPB1; excluding exon 1 and part of intron 1) were amplified in a multiplexed, long-range PCR. Manual library preparation was performed per manufacturer's protocol, followed by template preparation and chip loading on the Ion Chef, and sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and repeat testing were followed; technologists were blinded to the reference results. The concordance between AllType and reference results was determined at 2-field resolution. We also describe the ranges of input DNA and library concentrations, read number per sample and per locus, and key health metrics in relation to typing results. Results The concordance rates were 98.6%, 99.8% and 99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, respectively. Three genotypes were discordant, all of which shared the same G group typing results with the reference. Most ambiguous genotypes (116 out of 144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and library

HLA genetic polymorphism in patients with Coronavirus Disease 2019 in Midwestern United States

“…Genomic DNA was extracted from remnant peripheral blood specimens using the EZ1 DNA Blood 350 μl Kit (Qiagen, Hilden, Germany). A total of 192 samples were typed by the AllType assay (One Lambda, West Hills, CA) on the Ion Chef/S5 Ion Torrent platform 25 . A total of 42 samples were amplifed using the NGS LR kit (One Lambda, West Hills, CA) and sequenced following the SQK‐LSK109 protocol on the R10.3 MinION flow cells (FLO‐MIN111, Oxford Nanopore Technologies) 26 .…”

“…A total of 192 samples were typed by the AllType assay (One Lambda, West Hills, CA) on the Ion Chef/S5 Ion Torrent platform. 25 A total of 42 samples were amplifed using the NGS LR kit (One Lambda, West Hills, CA) and sequenced following the SQK-LSK109 protocol on the R10.3 MinION flow cells (FLO-MIN111, Oxford Nanopore Technologies). 26 Genotypes of HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3/4/5 genes were assigned based on keyexon sequences (G groups) and limited to the 2-field resolution.…”

A novel B*07 variant allele, B*07:416, identified by next‐generation sequencing

“…Genomic DNA was extracted from a whole blood specimen. HLA typing was performed using the AllType assay (One Lambda, West Hills, CA) with the Ion Torrent S5 sequencing platform, which has been validated as a lab‐developed test 3 . Data analysis and allele assignment were performed using the TypeStream Visual software (version 2.0) and the IPD‐IMGT/HLA Database version 3.40.0 4 .…”

“…HLA typing was performed using the AllType assay (One Lambda, West Hills, CA) with the Ion Torrent S5 sequencing platform, which has been validated as a lab-developed test. 3 Data analysis and allele assignment were performed using the TypeStream Visual software (version 2.0) and the IPD-IMGT/HLA Database version 3.40.0. 4 The software identified HLA-B*07:02 as the closest allele to the novel allele, and the result was flagged due to a mismatch, or variant base, in exon 3.…”

Identification of a single nucleotide deletion in the novel HLA‐DQB1*06:379N allele, detected by Polymerase Chain Reaction‐Sequence Based Typing but not by Next Generation Sequencing

“…The high throughput of NGS also allows multiplexed sequencing of DNA enriched from multiple genes and many samples in one run, which maximizes the efficiency and lowers the cost per sample. [11][12][13][14][15][16] In this report we describe the characterization of a novel null HLA-DQB1 allele, detected by PCR-SBT but not by NGS, and identified during the routine typing of a family group, looking for a fully matched hematopoietic stem cell donor for an Italian patient with acute myeloid leukemia. The new allele was first detected in the patient's peripheral blood sample and in order to exclude Francesco Ingrassia and Alice Pecoraro contributed equally to this study.…”