User manual

Droplet Digital
™
PCR Applications Guide | 49
Rare Mutation and Sequence Detection
Once the best melting temperatures have been identified using 1-D plots, use 2-D amplitude
plots to assess assay sensitivity. Look for no false positives or “spray” of droplets into the
double-positive region in the 2-D amplitude plot of the wild-type–only samples.
The 2-D amplitude plot for the SNP assay should have no positive droplets in the double-
positive region in the wild-type–only well. Wild-type–only control wells should have the same
concentration of DNA as the highest concentration experimental well. Figure 5.4 shows
an example of false positives or spray into the double-positive region. For more sensitive
mutant detection, assay redesign may be recommended to resolve this.
Fig. 5.4. 2-D amplitude plot of a wild-type control well containing 2,000 copies/µl of an SNP assay
with spray of false positives into the double-positive region of the plot.
Fig. 5.3. Two examples of SNP assay temperature gradients. A, 1-D amplitude plot of the FAM-labeled
mutant assay results of PI3Kca_E545K; B, 1-D amplitude plot of the FAM-labeled mutant assay results of
PI3Kca_H1047R.
0 50,000 100,000 150,000
Event number
0 50,000 100,000 150,000 200,000
Event number
Channel 1 amplitude
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
50% mutant/wild-type DNA
50% mutant/wild-type DNA
A03 B03 C03 D03 E03 F03 G03 H03 A04 B04 C04 D04 E04 F04 G04 H04
A
Channel 1 amplitude
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
A06 B06 C06 D06 E06 F06 G06 H06 A07 B07 C07 D07 E07 F07 G07 H07
B
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000
Channel 2 amplitude
Channel 1 amplitude
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
100% wild-type DNA
100% wild-type DNA