Fig 1.
Use of the IPDA to evaluate PCR efficiency in nFGS methods.
(A-D) IPDA analysis of PCR products from the outer PCR used in all nFGS methods (S1 Table). Template was DNA from a treated PLWH diluted to limit dilution with respect to proviruses. Each IPDA dot plot is shown below a diagram illustrating a possible proviral structure that would give rise to the observed plot. (A) Exponential amplification of an intact provirus. Each of the ~20,000 droplets in the reaction is positive for both the ψ and env amplicons. As a result, negative droplets which normally appear in the lower left quadrant are absent. (B) Exponential amplification of a provirus lacking the targeted psi sequence, for example due to a deletion such as that shown in the white box. (C) Representative well in which there was exponential amplification of a provirus lacking the targeted env sequence. (D) Representative negative well. (E-H) Use of the IPDA to quantitate molecular yields in nFGS methods. Positive PCRs from outer or nested reactions in nFGS methods are serially diluted until most droplets are negative. In this example, DNA from the J-Lat 6.3 clone was plated at limit dilution with respect to proviruses and amplified with the outer and nested PCRs of Method 4. An aliquot from a positive well was serially diluted and reanalyzed by IPDA. With more dilution, negative wells become dominant, allowing digital counting of PCR products as double positive droplets. Some shearing between IPDA amplicons is also evident in the form of single positive droplets at higher dilutions.
Fig 2.
Quantitative analysis of nFGS methods.
(A) Proviral constructs used to evaluate efficiency of nFGS methods. NL4-3-derived proviral constructs are shown in relation to the HIV-1 proviral map. Numbers in italics indicate the size of the amplicons obtained with the nFGS outer primers (black arrows). Deleted regions are shown in white. Positions of the IPDA ψ (blue arrows) and env (green arrows) amplicons are indicated. (B) Synthetic double stranded DNA templates used to evaluate efficiency of nFGS methods. (C,D) Experimental protocol. Published methods (red arrows) and our quantitative analysis of those methods (black arrows) are summarized in the flow diagram. Numbers in red circles refer to individual methods described in S1 Table. Analysis of the long-distance outer PCR step common to all nFGS methods is described in Panel C. Methods 1–6 all use the same outer PCR primers to generate a 9064 bp amplicon. Outer PCR wells were screened by IPDA analysis of a 1/10 dilution of the PCR products. The fraction of wells giving exponential amplification (see Fig 1) was determined, and then a large dilution of each positive well was analyzed by IPDA to count individual product molecules. The nested inner PCRs used in each nFGS method were analyzed as described in Panel D. For Method 6, a 9 kb inner PCR is run only for wells that have more than one positive nested subgenomic qPCR. Methods 3 and 5 are very similar to Method 4 but use a different polymerase and/or cycle number (see S1 Table for details) and were not tested here.
Fig 3.
Proviral amplicon length affects both the fraction of limiting dilution PCRs with amplification and the yield of product molecules in positive reactions.
(A) Effect of sequence length on the fraction of positive wells. Carefully quantitated NL4-3-derived proviral constructs of different lengths (Fig 2A and 2B) diluted into DNA from HIV-negative donors were plated at 30 proviruses per 96 well plate. A full length hypermutated (FLHM) provirus [p2g10, reference [29]] was also analyzed. After amplification by the outer PCR common to most nFGS methods, the fraction of positive wells was determined by IPDA analysis of each well. (B) Number of product molecules per reaction in the positive wells from A was determined by IPDA analysis of highly diluted aliquots from positive wells. Black lines show the geometric mean values.
Fig 4.
Amplification of full length and deleted proviruses by different nFGS methods.
(A) Number of limiting dilution outer PCRs that are positive for amplification of full length and deleted proviruses by different nFGS methods. Carefully quantitated proviral constructs diluted into HIV-1 negative DNA were plated at 30 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. The expected number of positive wells (dashed line) was observed for the proviral construct with a large internal deletion representing 70% of the genome (green bars) but not for the full-length provirus (pink bars). (B) Number of product molecules generated in the positive PCRs from A. Aliquots from positive wells from a representative plate were diluted extensively and analyzed by IPDA for digital counting of product molecules as described in Fig 1E–1H. Black lines show the geometric mean values. (C) Number of limiting dilution nested PCRs that are positive for full length and deleted proviral constructs amplified by different nFGS methods. After outer and nested amplification for 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. 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 colors). (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. Black lines show the geometric mean values.
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.
Fig 6.
Efficiency of the nested qPCRs of Method 6.
(A) Analysis with J-Lat cells. Carefully quantitated genomic DNA from J-Lat 6.3 cells was plated at limit dilution with respect to proviruses (30 proviruses/96 well plate). After the initial 9 kb outer PCR, wells were screened by IPDA. A total of 47 positive wells were identified in 3 independent experiments. Aliquots from these wells were subjected to 4 qPCR reactions used in Method 6. For each amplicon, successful qPCR amplification is indicated by a blue rectangle. (B) Analysis of qPCR efficiency with NL4-3. Carefully quantitated amounts of NL4-3 plasmid were diluted into HIV-negative donor DNA, plated at the indicated number of copies/well, and amplified using the four qPCRs of Method 6. For each amplicon, successful qPCR amplification is indicated by a blue rectangle.