Ultrarapid and high-resolution HLA class I typing using transposase-based nanopore sequencing applied in pharmacogenetic testing

• DOI: 10.3389/fgene.2023.1213457
• Publication Date: 2023-06-24T14:06:23Z
• Authors: Nampeung Anukul; Piroon Jenjaroenpun; Chonticha Sirikul; Natnicha Wankaew; Pattaraporn Nimsamer; Ekkapong Roothumnong; Manop Pithukpakorn; Nipapan Leetrakool; Thidathip Wongsur...
• Keywords: Regulatory T Cell Development and Function; Radiology, Nuclear Medicine and Imaging; Genotyping; Therapeutic Antibodies: Development, Engineering, and Applications; Hypersensitivity Reactions to Drugs and Medications; Nanopore sequencing; Genotype; HLA-B*5701; Immunology; Population; QH426-470; Gene; HLA Genotyping; Drug Hypersensitivity; Computational biology; human leukocyte antigen; Health Sciences; Genetics; DNA sequencing; nanopore; Biology; turnaround time; Multiplex; pharmacogenomics; Pharmacology; Immunology and Microbiology; Allele; Human leukocyte antigen; FOS: Clinical medicine; Life Sciences; Typing; Environmental health; FOS: Biological sciences; Antigen; long-read sequencing; Medicine; Thai

Nanopore sequencing has been examined as a method for rapid and high-resolution human leukocyte antigen (HLA) typing in recent years. We aimed to apply ultrarapid nanopore-based HLA typing for HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01. Most studies have used the Oxford Nanopore Ligation Sequencing kit for HLA typing, which requires several enzymatic reactions and remains relatively expensive, even when the samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding kit, which is transposase-based, with library preparation taking less than 1 h of hands-on time and requiring minimal reagents. Twenty DNA samples were genotyped for HLA-A, -B, and -C; 11 samples were from individuals of different ethnicity and nine were from Thai individuals. Two primer sets, a commercial set and a published set, were used to amplify the HLA-A, -B, and -C genes. HLA-typing tools that used different algorithms were applied and compared. We found that without using several third-party reagents, the transposase-based method reduced the hands-on time from approximately 9 h to 4 h, making this a viable approach for obtaining same-day results from 2 to 24 samples. However, an imbalance in the PCR amplification of different haplotypes could affect the accuracy of typing results. This work demonstrates the ability of transposase-based sequencing to report 3-field HLA alleles and its potential for race- and population-independent testing at considerably decreased time and cost.