Background: HIV drug resistance (HIVDR) testing remains essential for optimizing antiretroviral therapy (ART), yet access remains constrained by high cost, low throughput, and limited availability of Sanger sequencing in resource-limited settings (RLS). Recent improvements in Oxford Nanopore Technologies (ONT) sequencing offer a potential alternative, especially following the expansion of genomic surveillance infrastructure established during the COVID-19 pandemic.
Methods: A cross-sectional laboratory validation study was conducted comparing ONT MinION sequencing with Sanger sequencing for HIV-1 protease/reverse transcriptase (PR/RT) and integrase (IN) genotyping. Sixty-four stored amplicons from patients with virological failure on second-line protease inhibitor (PI)- or integrase strand transfer inhibitor (INSTI)-based regimens were sequenced on the MinION Mk1C (R10.4.1). ONT-derived consensus sequences were generated using a custom pipeline and compared to Sanger reference sequences to assess nucleotide identity and drug resistance mutation (DRM) concordance using the Stanford HIVdb v9.8. We also evaluated the cost and scalability of ONT to determine its feasibility for broader implementation.
Results: ONT MinION sequencing produced high-quality consensus sequences with a median pairwise identity of 99.4% (IQR: 99.2-99.7). After resolving initial discrepancies through chromatogram review and high-depth Nanopore data, high concordance for major DRMs was observed across PR, RT, and IN coding regions. Using a 48-sample multiplexing configuration, the estimated reagent and consumable cost for ONT sequencing was approximately US$23.47 per sample. Published Sanger-based HIV drug resistance assays report per-sample costs in the range of approximately US$43 to US$100, although costs vary depending on laboratory workflow and testing volume. Moreover, ONT provided a substantially shorter turnaround time (~ 5 h from library prep to sequence data), offering a more efficient and cost-effective workflow overall.
Conclusions: ONT MinION sequencing provides an accurate, rapid, and cost-effective alternative to Sanger sequencing for HIVDR genotyping in RLS. Its scalability, ability to detect minority variants, and compatibility with infrastructure setup up in response to the COVID-19 pandemic, make it a viable platform for both national HIVDR surveillance and decentralized clinical testing. Integrating ONT workflows into routine HIVDR monitoring could expand diagnostic access and enable more timely ART optimization in high-burden settings.
Keywords: Drug resistance; Genotyping; HIV-1; Oxford Nanopore Technologies.
© 2026. The Author(s).