Extended Data Fig. 5: Plasmid controls confirm specificity of the IPDA. | Nature

Extended Data Fig. 5: Plasmid controls confirm specificity of the IPDA.

From: A quantitative approach for measuring the reservoir of latent HIV-1 proviruses

Extended Data Fig. 5

a, Map of the plasmid pNL4-3 carrying an intact HIV-1 provirus. Positions of the Ψ (blue) and env (green) IPDA amplicons and of a distinct set of plasmid shearing control (PSC, magenta boxes) amplicons are indicated. Spacing between PSC amplicons is equal to spacing between Ψ and env amplicons. Dotted lines indicate positions of deletions in plasmids carrying previously described12 defective proviruses E44E11 and 4F12 with 5′ and 3′ deletions, respectively. b, IPDA analysis of the indicated ZraI-cut plasmids representing intact, 5′-deleted and 3′-deleted proviruses. c, Summary of droplet counts for the experiment shown in b. E44E11 and 4F12 give positive droplets only in quadrants 4 and 1 (Q4 and Q1), respectively. For pNL4-3, more than 95% of droplets are in Q2, with the remainder attributable to shearing between the Ψ and env amplicons. d, Analysis of shearing. For IPDA analysis of patient samples, shearing was measured using amplicons in the RPP30 gene (Fig. 3a, d, e). For plasmid control experiments, shearing of ZraI-cut plasmids was analysed using two sets of amplicons, the Ψ and env IPDA amplicons and the equally spaced PSC amplicons shown in a. ddPCR analysis was done on fresh (D0) maxipreps of pNL4-3 linearized with ZraI at a concentration mimicking patient samples. To assess the effects of higher levels of DNA fragmentation, IPDA analysis was also done on pNL4-3 DNA that had been incubated at 4 °C for 5 days (D5), and on pNL4-3 DNA cut with both ZraI and EcoRI (two cuts). The mean and range of duplicate determinations of the DSI is shown for each set of amplicons. The DSI was the same for the IPDA and PSC amplicons at three different levels of shearing. The DSI was used to correct the IPDA droplet counts in e. Negative values were set to 0. e, Uncorrected and DSI-corrected IPDA analysis of the intact proviral construct pNL4-3 at different levels of fragmentation. After correction, positive droplets were almost exclusively in Q2 even at higher levels of fragmentation.

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