MrHAMER2: high-accuracy long-read RNA sequencing to decode isoform-specific variation in viral transcripts during latency.

Alternative splicing (AS) greatly expands the repertoire of proteins encoded by the human genome. Viruses have been shown to hijack AS cellular pathways to sustain replication or lead to latency. In HIV-1 infection, the virus integrates into the host genome, becoming a transcriptional unit that directly engages in AS to regulate its gene expression. Sequencing advances have enabled insights into HIV-1 gene expression dynamics during productive replication. However, viral isoform dynamics during latency remain largely uncharacterized due to the low abundance of spliced viral transcripts in associated CD4+ T cell subsets, making their accurate detection and quantification challenging. MrHAMER2 is a high-accuracy long-read RNA sequencing method that leverages dual Unique Molecular Identifier (UMI) tagging of cDNA to accurately capture and quantify full-length isoforms with high dynamic range and 99.968% single-nucleotide accuracy. We used MrHAMER2 to decode the spliced HIV-1 transcriptome in a primary CD4+ T cell model of latency and showed substantial changes in viral isoforms bearing intron retentions accompanied by changes in their potential to generate translatable protein.