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P193 – A High-resolution HLA-A Genotyping Workflow Based on Nanopore Sequencing and its Forensic Applicability
The human leukocyte antigen (HLA) gene complex is one of the most polymorphic regions in the human genome, and HLA-A, a core member of the HLA class Ⅰ gene family, holds crucial application value in forensic individual identification. Traditional HLA-A genotyping techniques (e.g., PCR-SSP and PCR-SBT) suffer from low resolution, while next-generation sequencing (NGS) is limited by short read lengths. As a third-generation sequencing (TGS) technology, nanopore sequencing offers a novel technical strategy for high-resolution HLA-A genotyping owing to its ultra-long read length advantage. In this study, we established a comprehensive HLA-A genotyping workflow based on nanopore sequencing. Six standard reference materials with known HLA-A genotypes were used to validate the genotyping accuracy of this workflow, and 50 unrelated individuals from the Chengdu Han population were recruited to evaluate its sequencing performance and forensic applicability. The results demonstrated stable sequencing data quality, with an average of 330,634 reads generated per sample and a mean read length exceeding 3.3 kb. This ultra-long read length enabled full allele resolution covering exons, introns, and untranslated regions, thus achieving high-resolution eight-digit genotyping. The genotyping results of the six standard materials were 100% consistent with their reference genotypes. For the individual samples, the genotyping success rate reached 100%, with a total of 16 HLA-A alleles identified; the dominant alleles (A02:07:01:01 and A24:02:01:01) were consistent with the findings of previous population studies. In conclusion, the developed nanopore sequencing-based HLA-A genotyping workflow exhibits high accuracy, universality, robustness, and practical feasibility for forensic applications. Through subsequent systematic optimization and improvement, this workflow is expected to integrate with other HLA genes (e.g., HLA-B, -C, and -DRB1) to establish a multi-marker combined typing system. This system will serve as a valuable supplementary tool for conventional STR genotyping and provide more comprehensive technical support for the investigation of complex criminal cases.
Authors
- Jing Liu (Sichuan University, China)
- Minxiao Hui (Sichuan University, China)
- Shuyuan Zhang (Sichuan University, China)
- Linchuan Liao (Sichuan University, China)
- Zheng Wang (Sichuan University, China)
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