Portable Nanopore sequencing solution for next-generation HIV drug resistance testing

Background: Tackling HIV drug resistance is one of major challenges for ending AIDS epidemic, but the elevated expense of cutting-edge genomics hampers the advancement of HIV genotype testing for clinical care. Methods: We developed a HIV genotype testing pipeline that centers on a cost-efficient portable Nanopore sequencer. Accuracy verification was conducted through comparison with parallel data obtained via fixed-site Pacbio sequencing. Our complete pol-gene sequencing strategy coupled with portable high-throughput sequencing was applied to identify drug resistance mutations across 58 samples sourced from the ART-treated Los Angeles General Medical Center Rand Schrader Clinic (LARSC) cohort (7 samples from 7 individuals) and the ART-naive Center for HIV/AIDS Vaccine Immunology (CHAVI) cohort (51 samples from 38 individuals). Results: A total of 472 HIV consensus sequences, each tagged with a unique molecular identifier, were produced from over 1.4 million bases acquired through portable Nanopore sequencing, which matched those obtained independently via Pacbio sequencing. With this desirable accuracy, we first documented the linkage of multidrug cross-resistance mutations across Integrase Strand Transfer inhibitors (INSTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) from an individual failing a second-generation INSTI regimen. By producing more than 500 full-length HIV pol gene sequences in a single portable sequencing run, we detected Protease Inhibitor (PI), Nucleoside Reverse Transcriptase Inhibitor (NRTI), NNRTI and INSTI resistance mutations. All drug resistance mutations identified through portable sequencing were cross-validated using fixed-site Pacbio sequencing. Conclusions: Our accurate and affordable HIV drug resistance testing solution is adaptable for both individual patient care and large-scale surveillance initiatives.