Population-based nanopore sequencing of the HIV-1 pangenome to identify drug resistance mutations
Hirotaka Ode, Masakazu Matsuda, Urara Shigemi, Mikiko Mori, Yoshimi Yamamura, Yoshihiro Nakata, Reiko Okazaki, Mai Kubota, Yuka Setoyama, Mayumi Imahashi, Yoshiyuki Yokomaku, Yasumasa Iwatani
Abstract
HIV-1 drug resistance genotypic tests have primarily been performed by Sanger sequencing of gene segments encoding different drug target proteins. Since the number of targets has increased with the addition of a new class of antiretroviral drugs, a simple high-throughput system for assessing nucleotide sequences throughout the HIV-1 genome is required. Here, we developed a new solution using nanopore sequencing of viral pangenomes amplified by PCR. Benchmark tests using HIV-1 molecular clones demonstrated an accuracy of up to 99.9%. In addition, validation tests of our protocol in 106 clinical samples demonstrated high concordance of drug resistance and tropism genotypes (92.5% and 98.1%, respectively) between the nanopore sequencing-based results and archived clinical determinations made based on Sanger sequencing data. These results suggest that our new approach will be a powerful solution for the comprehensive survey of HIV-1 drug resistance mutations in clinical settings.