The origin of the rain is not clear. Rain often is attributed to delayed PCR onset [3] or partial PCR inhibition in individual droplets [4]. However, it could also be a consequence of damaged positive droplets with corresponding reduced fluorescence, or damaged negative droplets with increased background fluorescence, or a mixture of both [5].

we found that the clouds can become more compact and produce substantially less rain upon: (i) reducing the ramp rate to 1 °C/sec in every PCR step (Fig. 10a), (ii) increasing the annealing/extension time to 2 minutes (up from 1 minute; Fig. 10b), and (iii) increasing the number of cycles to 50 (Fig. 10c)28

Do not exceed 5,000 copies of target/μl of the final ddPCR reaction mix

Restriction Digestion

DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well

Digestion is recommended whenever DNA input is greater than 75 ng

For those shorter than 25 bases, a standard (dual labeled) BHQ™ Probe may be perfectly satisfactory.

process of dPCR is, for the most part, the same as that of qPCR; the key difference is that the nucleic acid sample is diluted and separated into numerous individual partitions that are independently amplified

The copy number of nucleic acids is then calculated using Poisson statistics on the number of positive versus negative partitions22.

the concentration is calculated based on the fraction of droplets that is empty (that is, the fraction that does not contain any target DNA).

QXDeveloperusestheformulaandvariablesshownbelow

Table10.Pre-RunErrorIndicators

We recommend the following changes to the default settings when designing ddPCR assays:

Strive for a Tm between 50 and 65°C. One way to calculate Tm values is by using the nearest-neighbor method. Use the Tm calculator at http://www.basic.northwestern.edu/biotools/oligocalc.html, with values of 50 mM for salt concentration and 300 nM for oligonucleotide concentration

To detect foreign DNA in 5 ml of lake water, 15 ml of lake water must be screened.

average of 5 copies/droplet (the upper end of the recommended loading range), we expect to see about 134 empty droplets in a total of 20,000 droplets

At the extreme ends of the concentration range (for example, fewer than ten copies of target in a well or more than 120,000 copies of target in a well), fewer droplets in a well will lead to slightly larger error bars.

Repeated freezing and thawing of the supermix is not recommended. DTT should be aliquoted to multiple tubes and stored at –20°C to minimize freezing and thawing

LOD was defined as <x>bi + ksbi, where <x>bi equals the mean of the no-template controls, sbi is s.d. of no-template controls and k = 2.479 (99% confidence interval)

Designing Probes for a ddPCR Assay

There is a simple mathematical relationship between the fraction of droplets that are unoccupied (black bar) and the concentration of target molecules.

Some droplets are lost in transfer steps and others are eliminated by the stringent metrics applied by QuantaSoft Software as the droplets pass through the Droplet Reader, resulting ultimately in data from 12,000–16,000 droplets being used in subsequent concentration calculations

Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device.

An estimate of par-titioning error can be found based on the analysis of Dube et al.,39 which models the partitioning as a binomial process.

Digital assays have two sources of error: subsampling error and partitioning error

Partitioning the sample into small containers results in a statistical distribu-tion of targets

because it measures indi-vidual molecules rather than an ensemble concentration

purifying the target of interest from interfering compounds.

Subsampling error sets the lower detection limit at low concentrations and is independent of the instrument, while partitioning error domi-nates at high concentrations and may depend on the sampling and partitioning instrument

The average number of droplets read for each ddPCR was 13,825 with a standard deviation of 1,892 droplets

NTCs showed a low-level background signal of approximately three positive droplets per NTC assay, which could possibly be attributed to low-level template contamination during the preparation of the reaction mixture

simple calibration procedure generated an instrument-specific color compensation matrix that was subsequently stored on the droplet reader and automatically applied to data to eliminate cross talk between FAM and VIC labeled probes.

Poisson distribution can be used in place of other standard curves to determine template concentration, which means that data can be reliably obtained from the same samples across multiple instruments and laboratories.