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Droplet Digital
™
PCR Applications Guide | 51
Rare Mutation and Sequence Detection
Fig. 5.7. 2-D plot of BRAF V600E assay.
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Channel 2 amplitude
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In Figure 5.7, the four clusters represent the droplets as follows:
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Blue cluster (top left), FAM-positive droplets with only mutant template
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Brown cluster (top right), double-positive droplets with both templates inside
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Gray cluster (bottom left), negative droplets with no template
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Green cluster (bottom right), VIC-positive droplets with only wild-type template
The orthogonality of the 2-D pattern means the four clusters are located spatially at right
angles to each other. In an RMD assay, clusters are commonly deviated from the corner
(right angles). For example, the blue cluster is shifted to right relative to the negative cluster
in Figure 5.7, a result of wild-type probe (labeled with VIC) cross-reacting with mutant
amplicon. Also, the double-positive cluster in an RMD assay is usually located between the
two single-positive clusters rather than at the upper-right corner, due to cross-reactivity.
Statistical Considerations for Rare Detection Experimental Design
The limit of detection (LoD) is the minimum concentration of the mutant (rare) sequence
that can be reliably differentiated from a negative control (100% wild type). In RMD,
LoD is typically quoted as a ratio or a percentage: for example, 1 in 10,000, or 0.01%.
In quantitative PCR, the LoD is largely a function of the cross-reactivity of the probes.
In contrast, in ddPCR, the LoD is determined primarily by the number of wild-type molecules
that are screened. The LoD can be adjusted to the precise requirement of any application
by adjusting the number of wells, and therefore molecules, screened.
Consider a sample at a mutant or rare sequence concentration of 1 mutant or sequence in
10,000 wild type. To guarantee with 95% confidence that at least 1 mutant molecule will be
screened in this sample, statistics dictates that at least 30,000 wild-type molecules must be
screened, or three times the number of expected wild-type target molecules. This is about
100 ng/well of human DNA where the target has a copy of 1/haploid genome (Table 5.1).
# of molecules to screen = 3 × background molecules