Abstract
The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.
Highlights
The digital method distributes target molecules into a large number of partitions such that each partition gets a number of molecules (0, 1, 2, etc.) theoretically following a Poisson distribution
Digital PCR results are compared against analogous quantitative PCR results running the same original samples
Each sample A through E is interrogated on QS3D digital PCR system (Thermo Fisher Scientific) in the following way: Two chips are employed per sample: One at the original concentration, One diluted 1: 50
Summary
The digital method distributes target molecules into a large number of partitions such that each partition gets a number of molecules (0, 1, 2, etc.) theoretically following a Poisson distribution. Performing PCR on these partitions results in amplification being detected (positives) in those partitions containing one or more target molecules and no amplification being detected (negatives) in those partitions containing zero molecules. Digital PCR experiments resulting in the production of all positive or all negative results do not typically provide sufficient information regarding the investigated samples in question The focus of this article is on theoretical results that are fully applicable to digital PCR on any platform. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors
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