User manual

Droplet Digital
™
PCR Applications Guide | 33
High Concentration Example
Consider the case in which 50,000 target molecules are present in 20 µl (50,000 targets
in 20 µl = 2,500 targets/µl = 2.5 CPD). With an average of 2.5 copies of target/droplet,
it’s not immediately obvious that there will be any empty droplets. But Poisson statistics
predicts that there will be some empty droplets (1,642 empty droplets) and gives a precise
relationship between the average number of copies/droplet and the expected fraction of
empty droplets. Table 3.2 shows how many droplets we expect to see with 0, 1, 2, 3, 4,
or more copies/droplet when there are on average 2.5 copies of target/droplet, or 50,000
copies in a 20 µl reaction volume.
Table 3.2. Expected percentage of droplets containing the target at high concentration.
Number of Target Molecules Count of Droplets Total Droplets, %
0 1,642 8.21
1 4,101 20.5
2 5,130 25.7
3 4,275 21.4
4 2,672 13.4
5 1,336 6.68
6 557 2.78
7 199 0.99
8 62 0.31
9 17 0.086
10 4 0.02
11 1 0.0049
Absolute Quantification and the Statistics of Droplet Digital
™
PCR
Intermediate Concentration Example
Consider the case in which there are 5,000 target molecules in 20,000 droplets (5,000
targets in 20 µl = 250 copies/µl = 0.25 CPD). Random partitioning of target molecules into
droplets will lead to some droplets with 2, 3, or even 4 copies, and correspondingly more
than 75% of the droplets will have zero copies. Poisson statistics tells us exactly how many
droplets to expect in each category. Table 3.1 shows the expected percentage of droplets
in each category. Note that in ddPCR, each droplet is distinguished as either empty/negative
(zero targets) or occupied/positive (one or more targets). For the 0.25 CPD case, 78%
(not 75%) of the droplets will be positive and 22% (not 25%) will be negative on average.
Since Poisson statistics arise in multiple different contexts in ddPCR, we describe this
phenomenon as “partitioning statistics.”
Table 3.1. Expected percentage of droplets
containing target DNA copies.
Targets Droplets, %
0 78
1 19.5
2 2.4
3 0.2
4 0.01