{"doi":"10.1371/journal.pone.0232050","title":"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 transplant-relevant 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 concentrations were tolerated. Per sample read numbers were adequate for accurate genotyping. Per locus read numbers showed some inter-lot variations, and a trend toward improved inter-locus balance was observed with later lots of reagents. CONCLUSION: The AllType assay on the Ion Chef/Ion S5 platform offers a robust and efficient workflow for clinical HLA typing at the 2-field resolution. The multiplex PCR strategy simplifies the laboratory procedure without compromising the typing accuracy.","journal":"PLoS ONE","year":2020,"id":75346,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":25,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9545,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":394257,"name":"Brian Duffy","orcid":null,"position":1,"is_corresponding":false},{"id":393263,"name":"Eric T. Weimer","orcid":"0000-0003-3677-5724","position":2,"is_corresponding":false},{"id":394258,"name":"Maureen C. Montgomery","orcid":null,"position":3,"is_corresponding":false},{"id":394259,"name":"Jo-Ellen Jennemann","orcid":null,"position":4,"is_corresponding":false},{"id":393264,"name":"Rachel Hill","orcid":"0000-0001-6111-355X","position":5,"is_corresponding":false},{"id":394260,"name":"D. Phelan","orcid":null,"position":6,"is_corresponding":false},{"id":394261,"name":"Lindsay Lay","orcid":null,"position":7,"is_corresponding":false},{"id":393265,"name":"Bijal A. Parikh","orcid":"0000-0003-2490-8294","position":8,"is_corresponding":false},{"id":393262,"name":"Chang Liu","orcid":"0000-0001-6624-1454","position":0,"is_corresponding":true}],"reference_count":34,"raw_metadata":null,"created_at":"2026-07-18T21:46:33.313355Z","pmid":"32324777","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}