User manual

Droplet Digital
™
PCR Applications Guide | 41
Copy Number Variation Analysis
Running a CNV Assay
Select Copy Number Variation (CNV) as the experiment type in QuantaSoft software
when loading wells. Double click on the experiment name in the main software window to
set the ploidy for the reference (Figure 4.3).
Fig. 4.3. Setting the ploidy for the CNV reference.
Note: R2 means 2 copies/genome (diploid) or 1 copy/haploid genome. If CNV experimental
type is not selected for your wells, the CNV tab will not be available. This affects only the
CNV charting calculations and can be applied during setup or any time after data collection.
For the reference assay, select Reference as type. Put this assay’s concentration as the
denominator, for example, “b” for the ratio chart and for the fractional abundance chart,
and assign the CN selected to this assay. If both Ch1 and Ch2 are selected as “unknown,”
Ch1 is automatically “a” and Ch2 is automatically both “b” and the CNV reference.
Restriction Digestion
To achieve optimal accuracy in CN measurements, restriction digestion of genomic DNA
is required. Restriction digestion separates tandem gene copies, ensuring proper random
partitioning into droplets (Figure 4.4). Restriction digestion can also reduce sample viscosity
and improve assay performance by improving template accessibility.
Fig. 4.4. Restriction digestion separates tandem gene copies.
Single copy variant Two tandem copies Two unlinked copies
3 positive droplets 3 positive droplets:
inaccurate CNV estimate
5 positive droplets:
accurate CNV estimate