Abstract
Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.
Highlights
The high precision offered by digital PCR has the potential for measuring smaller fold changes than established techniques like quantitative real-time PCR
We have evaluated the performance characteristics of the Adh assays in both uniplex and duplex formats with quantitative real-time PCR (qPCR) followed by investigation of the effects of the assay format on the accuracy and precision of the ratio measurement between two Adh assays using digital PCR (dPCR)
We demonstrated that performing duplex dPCR can be more precise than uniplex and, when template is limiting, performing pre-amplification may not be necessary as it does not improve the measurement of the low concentration sample
Summary
The high precision offered by digital PCR (dPCR) has the potential for measuring smaller fold changes than established techniques like quantitative real-time PCR (qPCR). This potentially offers a new tool for clinical measurement applicable to approaches such as assessment of DNA copy number variation (CNV) [1]. For maximum precision, the DNA sample must be at an optimum concentration [2] and this can be challenging due to the fact that clinical samples are frequently of limited size and concentration. Duplex PCR, where two targets are analysed per reaction, and sample pre-amplification offer methods that would allow an increase in the number of tests that can be performed on a sample, while reducing the required sample size needed. Aspects of duplex PCR that could introduce bias include a preferential amplification of one target over the other or inhibitors presence in the sample that affect one assay more than the other [3]
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