Methods for comparing multiple digital PCR experiments

Abstract

The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series.Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment.

Accurate design of translational output by a neural network model of ribosome distribution

“…For each circularization reaction, two qPCR reactions were performed: one where the formation of a product was dependent upon oligo circularization, and one where it was not (oligos defined in Supplementary Table 3). qPCR data was analyzed using custom R scripts whose core functionality is based on the packages qpcR 39 & dpcR 40 (qpcr_functions.R, available on github). The signal from the circularization dependent amplicon was normalized to that from the circularization independent amplicon, and then expressed as a fold-change compared to the mean of the oligonucleotide with the most favored sequence under the model.…”

“…For each reverse transcription reaction, two qPCR reactions were performed: one with primers specific to the mCherry ORF, and one with primers specific to the eCitrine variant ORF in question (oligos defined in Supplementary Table 3). qPCR data was analyzed using custom R scripts whose core functionality is based on the packages qpcR 39,46 & dpcR 40 (qpcr_functions.R, available on github). The signal from each eCitrine variant ORF was normalized to that from the mCherry ORF in the same sample, and then expressed as a fold-change compared to the median of these values for the MIN (fastest predicted sequence) eCitrine variant.…”

High dimensional surrogacy: computational aspects of an upscaled analysis

“…When compared to conventional statistical tests, visual inference showed promising results in experiments, for example, for testing linear model coefficients using boxplots and scatterplots (Majumder, Hofmann, and Cook 2013). With the package nullabor (Wickham, Chowdhury, and Cook 2014) helpful general purpose functions for visual inference are available within R. Due to the often uncertain or even misleading nature of funnel plot based conclusions, we identified funnel plots as a prime candidate field for the application of visual inference. For this purpose, we developed the function funnelinf which is available within the R package metaviz.…”

Accurate design of translational output by a neural network model of ribosome distribution

“…Table 2). qPCR data was analyzed using custom R scripts whose core functionality is based on the packages qpcR 33 & dpcR 34 (qpcr_functions.R, available on github). The signal from the circularization dependent amplicon was normalized to that from the circularization independent amplicon, and then expressed as a fold-change compared to the mean of the oligonucleotide with the most favored sequence under the model.…”

“…Table 2). qPCR data was analyzed using custom R scripts whose core functionality is based on the packages qpcR 33,41 & dpcR 34 (qpcr_functions.R, available on github). Allowing for the measured differences in PCR efficiency between the eCitrine variant specific primer pairs, the signal from the eCitrine variant ORF was normalized to that from the mCherry ORF, and then expressed as a fold-change compared to the median of these values for the parental eCitrine variant.…”