Background
Understanding and identifying HIV-1 neutralization escape requires isolating envelopes that have undergone antibody selection pressure. However, recombinant artifacts are generated during bulk reverse transcription (RT) and subsequent PCR, which can affect neutralization phenotype. To avoid this, amplification from a single genome is essential. We addressed this by developing a novel droplet-based one step RT-PCR method which can amplify full length envelopes with less recombination and significantly higher throughput than other single genome amplification methods.
Methods
Droplet-based genome amplification (DBGA) partitions starting templates into approximately 20,000 droplets, in which around 30% or less of the droplets contain a single genome. We validated our approach by generating a sequence pool containing either 6 different full-length HIV-1 RNAs or 8 HIV-1 DNA. Envelopes in these sequence pools were amplified using one-step DBGA, bulk RT followed by DBGA (bulk-RT-DBGA), or bulk RT followed by bulk PCR. Envelope sequences were obtained using single molecule PacBio technology. Recombination was quantified using Los Alamos Recombination Identification Program (RIP) and a genetic error quantification (GEQ) method. Differences were compared using Chi-square tests.
Results
Recombinant frequency was significantly lower in the RNA sequence pools amplified with our one-step DBGA (2.6% RIP) as compared to bulk RT followed by bulk PCR (25.5% RIP, p < 0.0001). Recombinant frequency was similar between one-step DBGA and bulk-RT-DBGA (2.6% and 1.5% respectively, RIP). Recombination rates in DBGA scaled with increased genomic input; as expected, because the frequency of droplets containing two or more templates increases with higher inputs amounts.
Conclusions
DGBA reduces recombinant artifacts similarly to established RT-SGA protocols while being high throughput and less laborious compared to SGA. Our approach provides a platform to characterize HIV-1 envelope sequences which have undergone antibody selection pressure with significantly more depth than previous methods.