(736a) Single Proviral Sequencing of Latent HIV-1 Proviruses Using Droplet Microfluidics
AIChE Annual Meeting
2019 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Omics and High-Throughput Technologies
Thursday, November 14, 2019 - 3:30pm to 3:48pm
Here, we develop a droplet microfluidic based single provirus sequencing (SiP-seq) method to sequence HIV genome together with itâs adjacent human genome from latently infected T cells. Genomic DNA fragments are compartmentalized and amplified in isolated pico-liter water-in-oil emulsions, followed by PCR-activated sorting of them based on the presence of HIV sequence. The amplification of genome in individual pico-liter droplets also enables maximum amplification uniformity and minimal contamination. We have validated the method using J-Lat cell line (a Jurkat based cell line, with each cell containing a full-length integrated HIV-1 genome with a frameshift in Envand GFP in place of the Nef). We have then sorted and sequenced single HIV genomes as well as its integration sites in latently HIV-infected CD4 T cells from ART-suppressed patients. We showed the first true full-length sequencing of HIV provirus as well as aberrant HIV-1 integrations from ART-suppressed patients. Our approach allows isolation and sequencing large number of inserted single provirus from latently infected T cells, which will provide novel insights to the genetic mechanisms regulating HIV persistence.