User manual

52 | Droplet Digital
™
PCR Applications Guide
Rare Mutation and Sequence Detection
Additional wells may be screened to ensure detection of more than one positive droplet.
For ddPCR, the ability to merge multiple wells into a meta-well provides more experimental
flexibility to tune the experiment to the required LoD. The number of cells or amount of
DNA needed to screen a given number of background molecules is described in Table 5.1.
The number of wells needed is a conservative estimate. The row in bold reflects the best
performance in one well. Researchers may choose to screen additional wells to ensure
detection of more than one positive droplet.
Table 5.1. Requirements for different LoDs for RMD, assuming an ideal assay with a droplet
false-positive rate of zero.
LoD
Total Copies
to Screen Diploid Cells Amount of DNA Number of Wells
1 in 1,000 3,000 1,500 0.010 µg 1
1 in 10,000 30,000 15,000 0.10 µg 1
1 in 25,000 75,000 37,500 0.25 µg 1
1 in 100,000 300,000 150,000 1.0 µg 4
1 in 1,00,000 3,000,000 1,500,000 10.0 µg 40
Required Starting Material (human)
Recommended Controls
If a single wild-type or negative sample control well is run and the observed positive droplets
are zero, then it is good practice to require at least three positive droplets in order to call a
sample positive. The three positive droplets can be in either a single well or across merged
wells. On the other hand, if you run a full plate of negative sample control wells and observe
zero positive droplets, consider making a positive call based on a single positive droplet.
However, we don’t usually recommend this because it may be difficult to control for handling
or contamination issues. If the same sample is spread across multiple wells, then the
number of positive droplets should be pooled across all the wells and a similar number
of negative control wells should be run.
Our guidelines assume that the number of positives in no template control (NTC) wells
is also zero, which indicates good handling practices. If these numbers are not zero,
the rate of potential false positives must be accounted for in calling a positive sample.
Control wells may not be zero for the rare sequence. This could mean that contaminating
template DNA is in the reaction wells. If positive droplets in the NTC wells occur, make
sure that good laboratory practices for PCR are being followed in the laboratory (see Kwok
and Higuchi 1989).
To limit workspace contamination, wipe down your pipets, tip boxes, and benchtops with
5–10% bleach, prepare master mixes in a template-free environment, add samples and
generate droplets in an amplicon-free environment, read droplets in a room separate from
the sample preparations, do not reuse DG8
™
droplet generator cartridges, oils, gaskets,
plates, or pipet tips, and wear appropriate personal protective equipment that is discarded
or confined to appropriate locations.
In addition to workspace contamination, consider careful experimental setup procedures
before droplet generation. Be careful about anything that might produce aerosolized DNA.
We recommend mixing your sample by pipetting up and down the full volume 10–12 times
rather than vortexing or centrifuging your plate because removing the seal after these
processes can spray contaminating DNA into other wells.