Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods
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.