Optimized Multiplex PCR-NGS for Comprehensive HLA Genotyping in Chinese Populations: Resolving Ambiguities at High Resolution

Haider Cuello Garcia¹Yinfeng Wang¹Zhoufan Zhang¹Changling Cao²Yiying Zhu¹Abdelhak Ouzaouit¹Huiqiang Huang²Haitao Liu²Ting-Ya Jiang²Xueping Dong³Yang Zhou^1*Yu Wu^3*

• ¹Jiangsu University, Zhenjiang, China
• ²AlloDx (Shanghai) Biotech Co, Ltd, Shanghai, China
• ³The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, China

Accurate human leukocyte antigen (HLA) genotyping is critical for organ transplantation to ensure donor-recipient compatibility. Conventional methods, such as sequence-based typing (SBT), often face challenges in resolving allelic ambiguities, particularly in highly polymorphic regions of HLA loci. Therefore, this study aimed to develop 6 locus multiplex primers combined with high-resolution sequencing, focusing on improving sequencing depth and reducing costs. Multiplex PCR primers targeting HLA-A, -B, -C, -DPB1, -DQB1, -DRB1 loci were designed using high-frequency alleles from public databases. Primers were optimized using as reference the sequencing depth across loci. The method was validated using SBT and probe capture-based targeted next-generation sequencing to evaluate its approach accuracy. Moreover, 770 samples from Chinese population were further studied to verify the allele frequency adding information about HLA types of this population. The optimized multiplex PCR-NGS sequencing showed depths within a target range of 100-1000 with high accuracy determined in the two-digit, four-digit and six-digit, with a reliability of ≥ 98%, ≥ 95% and ≥ 95% respectively in both methods.The developed multiplex PCR-NGS method offers a reliable, cost-effective approach for high-resolution HLA genotyping, and may be particularly suitable for clinical studies, especially in donor-recipient matching during organ transplantation.