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

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
DOI: 10.3389/fgene.2023.1213457 Publication Date: 2023-06-24T14:06:23Z
ABSTRACT
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.
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