User manual

Droplet Digital
™
PCR Applications Guide | 53
Rare Mutation and Sequence Detection
We recommend always running negative controls to monitor for all possible sources of
false-positive droplets, given the risk of laboratory contamination when working with
templates and amplicons. With an extremely sensitive technology such as ddPCR,
we recommend running enough negative sample controls as part of every experiment so
that you can demonstrate that the probability of calling a true negative sample incorrectly
is below a certain threshold.
Experimental Strategies for RMD
Once the number of target molecules to be screened has been determined for a given LoD,
the number of wells required to screen that number of targets must be determined. In the
QX100
™
or QX200
™
Droplet Digital PCR system, up to 1 µg of DNA can be loaded into a
single well with a recommended limit of 5,000–6,000 copies/µl per well. To achieve a lower
LoD than 1 in 25,000 using human DNA (with a copy number of one per haploid), more
wild-type molecules can be screened by running the same sample in multiple wells.
Factors that Impact RMD Calculations
Table 5.1 outlines RMD experimental design based on statistical requirements. Additional
real-world factors will impact these numbers in an assay-specific and sample-specific way
and should be considered when planning an RMD experiment.
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Sample availability — depending on the source (formalin-fixed, paraffin-embedded
[FFPE] or fresh frozen), a typical tissue biopsy will provide <250 ng of sample, meaning
<75,000 genome copies to screen and at best a 1 in 25,000 LoD. With a more typical
100 ng of DNA from a biopsy, an LoD of 1 in 10,000 is more practical
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Sample integrity and preparation — the calculations above assume that every target
present can be successfully assayed and does not account for nonamplifiable DNA.
Samples can have varying degrees of degradation and chemical damage. FFPE
samples, for instance, are routinely used in cancer research, but can provide DNA of
poor quality. Consider this impact when calculating the amount of starting material
needed to effectively screen the desired number of target molecules
Sample Preparation
To achieve optimal accuracy in copy number measurements, restriction digestion of
genomic DNA is required. Restriction digestion separates tandem gene copies, ensuring
proper random partitioning into droplets. Restriction digestion can also reduce sample
viscosity and improve assay performance by improving template accessibility.
Rare Sequence Detection
An RSD application might require one or two assays. If the goal is to quantify the amount of
target sequence in a given volume of starting material, a single assay is used (Figure 5.8).
If the goal is to quantify the amount of target sequence with respect to a given reference
sequence, two independent assays are required (Figure 5.9). In either case, assay design
and optimization for RSD has less risk of cross-reactivity of the target assay with
background DNA.