Droplet microfluidic sequencing of HIV proviruses with their integration sites in patients receiving antiretroviral therapy

The human immunodeficiency virus (HIV) integrates its genome in that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. The resulting HIV DNA is termed a provirus. Sequencing individual proviruses with the adjacent human cellular junctions may elucidate mechanisms of infected cell persistence in humans. Here, we introduce a high throughput microfluidic assay where droplet-based whole genome amplification of the HIV DNA in its native context is followed by a polymerase chain reaction to tag droplets containing proviruses for sequencing, resulting in the assembly of full-length viral genomes connected to their contiguous HIV-human DNA junctions, regardless of the 150 million-fold higher amount of human DNA present in the background. We analyzed infected cells from patients with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which weren’t recovered by commonly used nested PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.