Ultrarapid High-Resolution HLA Class I Typing Using Transposase-Based Nanopore Sequencing for Pharmacogenetic Testing in Standard Samples

Nanopore sequencing has been increasingly examined as a method for rapid, high-resolution HLA typing in recent years. In this study, we aimed to apply ultrarapid nanopore-based HLA typing to HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01, using standard DNA samples. Most previous studies have used the Oxford Nanopore Ligation Sequencing Kit for HLA typing, which requires multiple enzymatic steps and remains relatively expensive, even when samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding Kit, a transposase-based approach, which required less than 1 hour of hands-on library preparation time and only minimal reagents. Twenty standard DNA samples were genotyped for HLA-A, -B, and -C, including 11 samples from individuals of diverse ethnic backgrounds and 9 samples from Thai individuals. Two primer sets, one commercial and one previously published, were used to amplify the HLA-A, -B, and -C genes. HLA typing tools based on different algorithms were applied and compared. We found that, without the need for several third-party reagents, the transposase-based workflow reduced hands-on time from approximately 9 hours to 4 hours, making it a practical approach for achieving same-day results for 2-24 samples. However, imbalance in PCR amplification between haplotypes could affect typing accuracy. This work demonstrates that transposase-based nanopore sequencing can accurately report 6-digit HLA alleles from standard samples and highlights its potential for race- and population-independent pharmacogenetic testing with substantially reduced time and cost.