Single genome amplification (SGA)-derived envelope sequencing

SGA sequencing was performed at viral sequencing core in Walter Reed Army Institute of Research (WRAIR) as described previously (Trinh et al., 2019; Keele et al., 2008). Briefly, viral RNA was extracted from the plasma of the infected RV217 participants using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) and complementary DNA (cDNA) was synthesized using the SuperScript III RT kit (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. cDNA was amplified as a full genome or 2 half genomes overlapping by 1.5 kb as previously described using SGA strategy, which was then end-point diluted in 96-well plate, such that to yield less than 30% amplification product. Env specific primers were used to amplify env gene from the HIV genome.