HIV-1 infection requires life-long therapy with anti-retroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end there are numerous ongoing studies to evaluate immunologic approaches including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here we describe a relatively high throughput combined quantitative polymerase chain reaction (qPCR) and next generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any 2 of the 4 probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV- 1 latent reservoir. Clinical Trial Registration ID #[NCT02825797][1]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02825797&atom=%2Fbiorxiv%2Fearly%2F2019%2F05%2F31%2F641951.atom

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