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Droplet Digital
™
PCR Applications Guide | 55
Rare Mutation and Sequence Detection
Fig. 5.9. Detection of a very low target concentration in a high background (such as a low viral load
in a given number of PBMCs) by analyzing samples at two different concentrations.
DNA from 300,000
human cells
Dilute
100-fold
Quantify rare sequence
Quantify background
Target
Background
The target sequence (the “rare” sequence) might be quantified at either concentration,
depending on the amount present in the starting sample. The background sequence is
quantified in the low concentration well.
In the examples shown in Table 5.2, the LoD is defined with respect to the number of
human cells. The number of wells needed is a conservative estimate that considers that
droplets can be lost due to workflow or quality control specifications.
Table 5.2. Case 2: Experimental setup for different LoD and LoQ in rare sequence detection.
LoD LoQ (<10% CV)
Human
Diploid Cells
Amount
of DNA, µg
High
Concentration
Low
Concentration
1 in 5,000 cells 100 in 15,000 cells 15,000 0.10 1 0
1 in 12,500 cells 100 in 37,500 cells 37,500 0.25 1 0
1 in 100,000 cells 100 in 300,000 cells 300,000 2.0 2 1
1 in 500,000 cells 100 in 1,500,000 cells 1,500,000 10.0 10 1
Number of Wells
Case 2: Quantification with Respect to Second DNA Sequence
To detect a very low target concentration in a high background sample, for example 1 copy
of virus/100,000 peripheral blood mononuclear cells (PBMCs), the sample can be analyzed
at two different concentrations (Figure 5.9). The high-concentration wells provide sensitive
detection of the rare sequence while the low-concentration wells enable quantification of
the background sequence. For example, to reliably detect an average of 1 virus in
100,000 PBMCs, we must screen 300,000 PBMCs, or 2 µg of DNA (Table 5.1).
This requires three wells on the QX100 system: two wells at high concentration to screen
enough PBMCs for the presence of rare virus, and a third well at a lower concentration to
quantify the PBMCs. This experimental setup is shown in Figure 5.9.