title: Impact of variance components on reliability of absolute quantification using digital PCR.
abstract: BACKGROUND
  Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.
  
  
  RESULTS
  We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.
  
  
  CONCLUSIONS
  Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.
authors.affiliation: Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Krijgslaan 281, S9, 9000 Ghent, Belgium. BartKM.Jacobs@UGent.be.
authors.forename: Bart K M
authors.initials: BK
authors.lastname: Jacobs
authors.forename: Els
authors.initials: E
authors.lastname: Goetghebeur
authors.forename: Lieven
authors.initials: L
authors.lastname: Clement
mesh_terms: D000704:Analysis of Variance; D018628:Gene Dosage; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D035501:Uncertainty