Fig. 3: HIV sequence-specific optical bleed-through prohibits tight thresholding on negative populations.

a 2D and env 1D plots of OM5346 virus 3 Ψ region sequence when tested as a synthetic DNA gene fragment (Virus 3 Ψ gBlock) without corresponding env template. Minimal Ψ- to env- channel spillover occurs and the positive droplet threshold can be drawn tightly to the double-negative population without consequence (note the presence of one false double-positive droplet). b 2D and env 1D plots of OM5346 virus 4 Ψ region sequence, tested as a synthetic DNA gene fragment (Virus 4 Ψ gBlock), without corresponding env template. Drawing a tight threshold causes optical bleed-through of Ψ (FAM) fluorescence into the env (VIC) channel to yield a false-positive env (and by extension, false-positive intact) signal. c 2D and env 1D plots of OM5346 Virus 4 Ψ region sequence, tested as a synthetic DNA gene fragment (Virus 4 Ψ gBlock) without corresponding env template, with a threshold drawn at an appropriate distance from the double-negative population. This threshold accommodates the fluorescence shift and thus avoids the creation of false-positive intact or env-positive droplet population (note the presence of a single false double-positive droplet). Representative plots from one of three technical replicates from one experiment are shown.