User manual

Droplet Digital
™
PCR Applications Guide | 29
Fig. 3.1. Example of a well-performing assay for absolute quantification.
Absolute Quantification and the Statistics of Droplet Digital
™
PCR
Note: Do not exceed 5,000 copies of target/µl of the final ddPCR reaction mix.
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Use a thermal gradient to optimize ddPCR results
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In the Well Editor of QuantaSoft software, designate ABS for the experiment type
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In the Well Editor, indicate which assay is the target and which assay is the reference
when performing multiplex reactions
Absolute Quantification Data Analysis
The ABS experiment is designed to quantify the concentration of the target and give a result
in copies/µl of the final 1x ddPCR reaction.
If the plate was set up for ABS analysis, automatic thresholding determines concentrations
and populates the data tables in the analysis mode of the software. The threshold may
be manually adjusted. Note specifically any wells that are flagged as No Call in the status
column of the data tables.
QuantaSoft software will return a No Call for wells with too many positive droplets
(not enough empty droplets to apply Poisson statistics), wells with Quality Scores below
0.85, and wells with fewer than 10,000 droplets. After visually inspecting the data, you may
set a threshold manually and QuantaSoft software will complete the calculations.
Figure 3.1 shows excellent separation between positive droplets (green) and negative
droplets (black) in the chart on the left, showing droplets (event number) vs. fluorescence
amplitude. The histogram on the right plots amplitude vs. the frequency of the populations
of droplets and can assist in setting the threshold.
QuantaSoft software measures the number of positive and negative droplets for each
fluorophore in each sample. It then fits the fraction of positive droplets to a Poisson
algorithm to determine the starting concentration of the target DNA molecule
in units of copies/µl input from the sample (Figure 3.2).