Single-Tube Eight-Plex Full-Length PCR-NGS for High-Resolution HLA Typing: Assay Validation and Exploratory ADO-Risk Quality Control
Year: 2026
Type: article
Abstract: Background: High-resolution typing of human leukocyte antigen (HLA) genes is a core technology for dissecting genetic polymorphism in the MHC region of the human genome and has important clinical value in hematopoietic stem cell transplantation, solid organ transplantation, and tumor immunotherapy. Although full-length HLA typing methods based on next-generation sequencing (NGS) can achieve high-resolution results, long-range PCR amplification carries the risk of allelic dropout (ADO). Single nucleotide polymorphisms (SNPs) in primer-binding regions may cause the amplification failure of a given allele, leading to heterozygotes being misclassified as homozygotes and resulting in HLA typing errors. Such mistyping during donor selection for hematopoietic stem cell transplantation may lead to HLA matching errors between donors and recipients, increasing the risk of graft-versus-host disease (GVHD) or transplant rejection. Existing NGS approaches generally rely on a single set of amplification primers and lack an independent verification mechanism. Methods: This study designed a single-tube 8-plex full-length PCR-NGS HLA typing assay and performed systematic methodological validation. The assay simultaneously co-amplifies eight classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1, -DPA1, and -DQA1) in a single tube, with primers anchored in the conserved 5′UTR and 3′UTR regions to capture the complete gene sequences, and high-resolution typing was performed using the HLA-HD software. Sanger sequencing-based typing (SBT) and PacBio/ONT third-generation long-read sequencing served as dual gold standards, and multi-level validation was completed using 92 anonymous peripheral blood genomic DNA samples from the Chinese population and the MDA-MB-231 standard cell line. During primer testing, we found that two samples exhibited allele loss at the HLA-DRB1 locus in the context of SNPs in the primer-binding region, indicating that ADO risk exists in practice; after adjusting the primers to more conserved positions, ADO in these samples was resolved, but the limitations of PCR technology and the high polymorphism of HLA dictate that ADO cannot be fundamentally eliminated by primer adjustment. Accordingly, we further designed, in an exploratory manner, a 6-plex QC assay whose primer-binding regions do not overlap at all with those of the 8-plex system, for proportional spot-checking verification in large-scale sample testing. Typing results were reported and used for consistency statistics primarily at 3-field resolution; the assay can read up to 4-field resolution, but given that the accuracy of 4-field interpretation cannot be independently verified by existing gold standards, 4-field data were retained only as exploratory supplementary results. Results: (1)Validation of the 8-plex system: For the 10 SBT reference samples, the 2-field typing concordance rate was 100%, and 4-field high-resolution results could be output; the 8-plex typing of 10 blinded samples matched the PacBio third-generation sequencing 3-field results 100%; the MDA-MB-231 standard cell line typing showed no discrepancies compared with the official Cellosaurus data. (2) Discovery of ADO risk: During primer testing, two samples that were heterozygous by SBT were misclassified as homozygous by the 8-plex assay; sequence alignment analysis revealed high-frequency SNPs in the primer-binding region of the 8-plex assay for these two samples, suggesting that primer binding may have been affected. After adjusting the primers to more conserved positions, the 8-plex typing results were consistent with SBT, confirming heterozygosity. (3) Exploration of 6-plex QC: Stratified validation included 31 samples in total, comprising 10 samples dually validated by the 8-plex assay and third-generation sequencing, one standard cell line, and 20 randomly sampled clinical samples; the 8-plex and 6-plex typing results were completely consistent for all these samples; the allele depth ratio (ADR) was mostly ≤2.0 across loci in the 6-plex assay. (4) Performance evaluation: The ADR was mostly ≤2.0 across all samples; the ranking of polymorphism abundance at each locus was consistent with the trends in published Chinese population cohorts; the rare allele HLA-DPB1*744:01 was successfully captured and verified by third-generation sequencing (ONT). Conclusions: This study designed and validated a single-tube 8-plex full-length PCR-NGS HLA typing assay, which showed stable amplification, accurate typing, strong rare-allele capture capability, short turnaround time, and controllable batch testing costs in 92 Chinese population samples. During validation, ADO risk was found to exist in practice; adjusting primers for known SNP sites can correct specific ADO events but cannot fundamentally eliminate the risk. The 6-plex independent QC assay designed on this basis showed complete concordance with the 8-plex results in 31 samples, but its ability to correct real ADO events remains a theoretical inference and an indirect validation, and further evaluation through long-term clinical accumulation or large-scale parallel validation is still needed. This method is expected to provide an accurate, economical, and practical new approach for clinical high-resolution HLA typing.
Source: Biology
Institution: Donghua University
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $3,258
OpenAlex ID: W7225799219