PoDCall – Positive Droplet Caller

Digital droplet PCR (ddPCR) is a promising method that can provide a solution for absolute DNA quantification with very high sensitivity, but for DNA methylation data there has been a lack of robust and standardized methods for analyzing such data. We therefore developed PoDCall, an R package that aims to provide a robust calling of positive droplets for DNA methylation ddPCR data performed on the Bio-Rad platform. The package provide functions to read raw two-channel amplitude data exported from either QuantaSoft or QX Manager from a run of ddPCR, set thresholds to call positive droplets and calculate concentrations and normalized concentrations for each well, and write results and optional plots to file. All of the above can be performed either using the functions of PoDCall in a regular R session, or by using the accompanying Shiny app included in the package for a more interactive analysis of the data. The latter allows for visual inspection of the data and results, and also the possibility to manually adjust thresholds if desired. The PoDCall package (Brodal et al, 2023) is available from the Bioconductor repository: https://bioconductor.org/packages/release/bioc/html/PoDCall.html

A more detailed description of the algorithm and concepts of PoDCall is discussed in the application note “PoDCall: positive droplet calling and normalization of droplet digital PCR DNA methylation data” (Jeanmougin et al, 2023).

PoDCall has primarily been developed for DNA methylation ddPCR experiments but does also seem to work well for ddPCR experiments looking at DNA mutations (Vynck et al, 2023).

References

  • Marine Jeanmougin, Hans Petter Brodal, Heidi Dietrichson Pharo, Hege Marie Vedeld, Guro Elisabeth Lind, PoDCall: positive droplet calling and normalization of droplet digital PCR DNA methylation data, Bioinformatics, Volume 39, Issue 1, January 2023, btac766, https://doi.org/10.1093/bioinformatics/btac766
  • Brodal H, Jeanmougin M, Lind G (2023). PoDCall: Positive Droplet Calling for DNA Methylation Droplet Digital PCRdoi:10.18129/B9.bioc.PoDCall, R package version 1.10.0, https://bioconductor.org/packages/PoDCall.
  • Matthijs Vynck, Yao Chen, David Gleerup, Jo Vandesompele, Wim Trypsteen, Antoon Lievens, Olivier Thas, Ward De Spiegelaere, Digital PCR Partition Classification, Clinical Chemistry, Volume 69, Issue 9, September 2023, Pages 976–990, https://doi.org/10.1093/clinchem/hvad063
 
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