User manual

Droplet Digital
™
PCR
Droplet Digital
™
PCR Applications Guide | 9
Figure 1.13 shows the effect of varying the threshold value (Figure 1.13A) on concentration
(Figure 1.13B). Eight ddPCR replicate wells are shown in the plots in Figure 1.13A.
Three scenarios are tested for discriminating positive (high amplitude) from negative
(low amplitude) droplets and the concentration calls associated with each (Figure 1.13B).
A precision of ±5% is maintained regardless of the thresholding method used
(see Fig. 1.13B zoomed-in view).
After optimizing the conditions to conduct a ddPCR experiment, the precision and resolving
power afforded by partitioning are unprecedented. Figure 1.14A provides an example of this,
showing a twofold serial dilution spanning 4 orders of magnitude of Staphylococcus aureus
template concentration (n, FAM channel) in a constant background of human genomic DNA
(gDNA) (n, VIC channel) run as a duplex assay. Figure 1.14B shows a zoomed-in view that
displays the resolution of small copy number differences using ddPCR for a series of 10%
dilutions of the same target and reference assays. The error bars represent Poisson 95%
confidence intervals.
Fig. 1.13. The power of thousands of replicates using ddPCR. A, threshold options;
B, measured concentration for different threshold settings.
A
B
0 2 4 6 8 10
Well location (8 replicates)
0 2 4 6 8 10
Well location (8 replicates)
Measured concentration, copies/µl
1,400
1,20 0
1,000
800
600
400
200
0
1,10 0
1,080
1,060
1,040
1,020
1,000
980
960
940
920
900
0 20,000 40,000 60,000 80,000 100,000 120,000
Event number
A02 B02 C02 D02 E02 F02 G02 H02 A02 B02 C02 D02 E02 F02 G02 H02 A02 B02 C02 D02 E02 F02 G02 H02
0 20,000 40,000 60,000 80,000 100,000 120,000
Event number
0 20,000 40,000 60,000 80,000 100,000 120,000
Event number
Channel 2 amplitude
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
Low threshold
Tight concentrations
Medium threshold
High threshold
High
Medium
Low