HIV SMRTcap, a novel single molecule, long-read sequencing assay to characterize the HIV-1 reservoir in cells and tissues across multiple subtypes


BACKGROUND: HIV reservoir characterization has focused on classifying integration sites and examining the integrity of proviral genomes, many of which contain defects rendering them replication incompetent. Current methods mainly examine either integration sites or proviral integrity, and perform robustly in cell-based assays, with limited application to tissue-specific reservoirs. There has also been scant effort to expand these assays to non-subtype B samples. To address these limitations, we developed a novel single molecule assay, HIV SMRTcap, that provides simultaneous resolution of the 'HIV integron' (proviral genome and matched integration sites) in cells and tissues across all major HIV-1 subtypes.
METHODS: HIV+ genomic DNA is extracted from cells or tissues and sheared into 11-15kb fragments. Oligo-based enrichment is performed using a custom panel of biotinylated 120-mers specific for HIV genomes representing all major subtypes. Oligo-bound fragments are enriched with streptavidin beads and used for sequencing library preparation. Sequencing is performed on the Sequel IIe system to obtain highly accurate (>99.99%) long reads, which allow for simultaneous characterization of the integration site, proviral intactness, and clonality.
RESULTS: HIV SMRTcap successfully enriched HIV integron events by >2400-fold from multiple sources: in vitro infection, primary PBMC, multiple autopsy- and biopsy-derived tissues (including spleen, kidney, liver, lymph node, basal ganglia), and across samples infected with HIV subtypes A, B, C, and D. HIV SMRTcap resolved both intact and defective proviral genomes, demonstrating that partial tiling of oligo baits was sufficient for capture. Intriguingly, analysis of samples using HIV SMRTcap revealed integration into repeat elements (i.e., LINEs, SINEs) absent from matched data obtained with linker-mediated PCR, likely due to limitations in mapping short-read data.
CONCLUSIONS: Standard short-read HIV reservoir measurement methods do not provide direct simultaneous evaluation of the complete HIV integron, limiting investigations of the impact of integration site on clonal expansion and retention of intact proviral genomes in patient samples. Moreover, specialized methods that can link this information (i.e., MIP-seq) are often prohibitively expensive and labor intensive. HIV SMRTcap utilizes long-read sequencing to characterize complete HIV integrons in a cost-effective, scalable manner, and has tremendous potential to accurately investigate HIV reservoir dynamics during evaluation of HIV cure strategies.