NCM 2022: Long-read sequencing assay allows accurate characterization of the HIV-1 reservoir

The advent of near full-length (NFL) HIV-1 proviral genome sequencing has expanded our understanding of the reservoir composition, revealing that approximately 2–5% of persistent proviruses in antiretroviral therapy-treated individuals can be considered genome-intact. However, current NFL assays are based on labour-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. We developed a long-read sequencing assay that allows for high-throughput amplicon sequencing of NFL HIV-1 genomes. By tagging individual HIV-1 genomes with two distinct UMIs, the step of limiting dilution can be omitted, enabling the amplification of many NFL genomes in a single reaction. We employed our assay to successfully characterize the viral reservoirs of research samples obtained from a chronic cohort of people living with HIV (n=18). We believe that our long-read assay will have a wide future applicability in the field of HIV-1 research and could help to further increase our understanding of HIV-1 reservoir composition and dynamics.