Values measured by digital PCR do not vary with cycle number.

<p>Plasmid templates were emulsified into droplets and thermally cycled for 30 to 50 cycles before analysis. No significant differences in measured <i>pol</i> (<b>A</b>) or 2-LTR (<b>B</b>) copy numbers were observed over this range of cycling times. No positive events were observed after 20 cycles (not shown). Similar results were obtained using dilutions of infected CD4+ T cells into uninfected PBMC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055943#pone.0055943.s001" target="_blank">Fig. S.1</a>). Error bars indicate the observed standard deviation between wells.</p

Assay limit of detection and quantification.

<p>CD4 cells infected in vitro were serially diluted into a background of PBMC DNA. The number of replicate wells was increased from 3 to 36 in proportion to the dilution, allowing accurate measurement below one copy per 10⁶ cells. The shaded area indicates concentrations below the theoretical limit of detection for a single well.</p

Effect of sequence variation.

<p>Patient isolates with previously determined <i>pol</i> sequences that differ from the consensus primer/probe set in at least two positions were analyzed by ddPCR and by qPCR. Both assays were conducted in parallel using the mismatched consensus primer/probe set and a patient-specific matched primer/probe set. Use of consensus primers and probe resulted in an underestimate of copy number by one to two log₁₀ by qPCR, with complete loss of detection in the extreme case of 5 total mismatched bases. The underestimate was largely mitigated (mean 57% reduction in log₁₀ copy number change) by ddPCR in all cases. These 4 cases reflect the most extreme mismatches observed in 84 patients, suggesting that sequence variation is unlikely to significantly impact ddPCR assay results in clinical studies. All samples analyzed were HIV-1 subtype B.</p

Correlation of ddPCR and qPCR measurements.

<p>(<b>a</b>) Pol copy numbers measured by ddPCR and qPCR were significantly correlated (Pearson R² = 0.64, slope = 0.98±0.08). The correlation weakened at low copy numbers, primarily due to a rapid increase in the variance of the qPCR assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055943#pone.0055943.s003" target="_blank">Fig. S3</a> (a)). For copy numbers measured in the bottom tertile (<230 HIV DNA copies/10⁶ cells) by ddPCR, the correlation was not significant (R² = 0.08, P = 0.12). In the central tertile, the correlation was weak but significant (R² = 0.15, P = 0.03). In the top tertile, the correlation was strong (R² = 0.53, P<0.0001). (<b>b</b>) 2-LTR copy numbers measured by both methods were significantly but weakly (R² = 0.14, P = 0.002) correlated.</p