Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods
Fig 5
Amplification of intact proviruses from the J-Lat 6.3 cell line by different nFGS methods.
(A) Number of limiting dilution outer PCRs that are positive for amplification of full-length proviruses by different nFGS methods. Carefully quantitated amounts of J-Lat DNA were diluted and plated at 30 intact proviruses/96 well plate. After amplification under conditions used by Methods 1 and 6 or Methods 2–5 (see S1 Table), the fraction of wells with amplification was determined by IPDA. Results are expressed as the number of positive reactions per 96 well plate divided by the expected value (30/plate) for each of 5 plates (black circles). The means and standard deviations for the 5 plates tested per condition are shown as bars and black lines, respectively. (B) Number of product molecules generated in the positive PCRs from A. Aliquots from positive wells from a representative plated were diluted extensively and analyzed by IPDA for digital counting of product molecules. Values from individual positive wells are shown as pink circles. (C) Number of limit dilution wells that were positive after nested PCR amplification by different nFGS methods. After outer and nested amplification under indicated conditions (see Fig 2C and 2D and S1 Table), the fraction of wells with amplification was determined by IPDA. Results are expressed as the fraction of positive wells relative to the expected value (30/plate) as described in A (dark pink bars). In addition, 45 ul aliquots of nested PCR wells were analyzed by agarose gel electrophoresis, and the number of wells with visible bands was determined and plotted in the same manner (light pink bars). (D) Number of product molecules generated in the positive PCRs from a representative plate in C. Aliquots from positive wells were diluted extensively and analyzed by IPDA for digital counting of product molecules as described in B. Values from individual positive wells are shown as pink circles.