New HIV-1 infections are genotyped as part of standard of care testing to ensure that antiretroviral treatment will be efficacious against the virus. Historically this has been performed by sequencing the pol region of the HIV-1 genome only. The popularity of next-generation sequencing (NGS) methods during the SARS-CoV-2 pandemic has resulted in a shift towards using NGS in diagnostic sequencing, but there remain limited methodologies utilising the strengths of NGS for robust diagnostic sequencing of longer regions of the HIV-1 genome. Given the acceptance and success of tiling PCR methodologies during the SARS-CoV-2 pandemic, we aimed to design and verify a novel tiling PCR method for routine HIV-1 sequencing. A set of tiling PCR primers was designed to amplify the 5' half of HIV-1 in six overlapping segments of 1,000 bp in only two PCR reactions. The assay can move from sample to sequencer in under a day. The tiling PCR was able to generate HIV-1 sequences from 90 (100%) samples in a comparison panel, and complete protease-reverse transcriptase and integrase regions were amplified in > 90% of samples with a viral load > 5000 copies/mL. Seven additional drug resistance mutations were identified when using this novel method. As such, this novel designer tiling PCR is a promising method for the routine NGS-based diagnostic sequencing of HIV-1.
Keywords: Assay verification; Clinical sequencing; HIV-1; Next-generation sequencing; Tiling PCR.
© 2025. The Author(s).