Chapter 8: Quality Control and Best Practices in ddPCR

While digital PCR (ddPCR) provides highly precise quantification, ensuring data quality requires careful experimental design, appropriate controls, and post-run validation.
This chapter outlines essential quality control (QC) considerations and best practices to ensure reliable and reproducible ddPCR results.

1. Droplet Quality and Thresholding - Minimum Droplet Count: A minimum of 10,000 accepted droplets per well is recommended for statistically robust quantification.
Fewer droplets result in wider confidence intervals and increased error.

  • Rain Detection: “Rain” refers to droplets with intermediate fluorescence that fall between the positive and negative peaks.
    These droplets may result from:

    • Poor probe or primer efficiency
    • Suboptimal thermal cycling
    • Sample degradation or inhibition

Inspection and adjusting thresholds may be necessary.

2. Controls

  • No Template Control (NTC): Included in every run to monitor contamination. Expected result: 0 positive droplets.

  • Positive Control: A sample with a known concentration of target DNA, used to confirm assay performance.

  • Reference Gene Control: In CNV assays, use a stable reference gene (e.g., RPP30 or RNaseP) to normalize copy number.

3. Assessing Reproducibility

Run technical replicates to ensure precision and identify potential outliers.

replicates <- c(10.2, 10.4, 9.9)
mean(replicates) 
## [1] 10.16667
sd(replicates) 
## [1] 0.2516611
cv <- sd(replicates) / mean(replicates) * 100
round(cv, 2) # CV (%) < 10% is ideal
## [1] 2.48

4. False Positives and False Negatives

Error Type Possible Cause Mitigation Strategy
False Positive Contamination, misclassified “rain” droplets Use strict NTC, adjust threshold
False Negative Poor droplet generation, failed amplification Increase input DNA, optimize conditions

For rare mutation detection, even a single false positive can affect interpretation — validation is essential.

5. LoD Confirmation via Dilution Series

To confirm the Limit of Detection (LoD), use a serial dilution of a known template and assess the lowest concentration at which detection is consistent.

  • Run 3 or more replicates per dilution level
  • LoD is typically the lowest level at which all replicates are positive
Practice Recommendation
Droplet Count >10,000 per well
NTC Should have 0 positive droplets
Positive Control Include known copy number sample
Replicates Use technical duplicates or triplicates
Threshold Inspection Adjust for rain manually if needed
LoD Validation Perform dilution series experiments

By incorporating these best practices, we can minimize experimental variability, reduce false results, and ensure that ddPCR data are reliable and publication-ready.