Comparison of short‐read and long‐read next‐generation sequencing technologies for determining HIV‐1 drug resistance

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.