Population-based nanopore sequencing of the HIV-1 pangenome to identify drug resistance mutations

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.

HIV-1 diversity and pre-treatment drug resistance in the era of integrase inhibitor among newly diagnosed ART-naïve adult patients in Luanda, Angola

“…To date, Sanger sequencing capable of identifying mutations with a prevalence of 15–25% or more has been used to analyse specific viral protein-coding sequences encoding ARVs drug targets 9 . In contrast, deep sequencing approaches based on nanopore technology (Oxford Nanopore Technologies [ONT]), have been developed for DR testing as they are capable of detecting less abundant mutations (< 15%), although the clinical impact of detecting such Low-abundance mutations remain controversial and merit further investigation 10 . The next-generation sequencing (NGS) approach based on ONT technology is cost-effective, portable/accessible and applied in genome assembly, full-length transcript detection and base modification detection and in more specialised areas, such as rapid clinical diagnoses and outbreak surveillance for countries where resources are limited, but the burden of infectious diseases such as the HIV is high 11 .…”