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96 | Droplet Digital
™
PCR Applications Guide
Appendix B: Technical Error Bars in Droplet Digital
™
PCR
Partitioning
The second contribution to the technical error bars comes from partitioning of the DNA
targets into droplets. This is the error that dominates at high concentration.
The illustration in Figure 2 shows 288 target molecules partitioned into 144 droplets.
These grids are snapshots in time: imagine molecules bouncing around in the sample,
then at any given instant divide the sample into 288 partitions. It’s evident that most of
the partitions do not contain exactly two molecules.
Fig. 2. Target molecule partitioning. CPD, copies per droplet.
Empty droplets: 22 (19 expected)
Calculated concentration: 1.88 CPD
Empty droplet
Occupied droplets
Statistics tells us that we should on average see 19 (or more precisely, 19.5) empty
partitions or droplets in this experiment. In a particular instant in time, we see 22 empty
droplets and we estimate a concentration of 1.88 based on this number. The partitioning
in Figure 3 shows different instants in time, and correspondingly different numbers of empty
droplets (note that the empty “droplets” are highlighted by a thicker black line). This type of
error is governed by Poisson statistics, just like the subsampling error, and is referred to as
the “partitioning error.”
Fig. 3. Target molecule partitioning repeated three times. CPD, copies per droplet.
Empty droplets: 20
Estimated concentration: 1.97 CPD
Empty droplets: 17
Estimated concentration: 2.14 CPD
Empty droplets: 19
Estimated concentration: 2.03 CPD