Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

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.

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

“…Molecular dropout has been demonstrated by observing when partitions containing 2 linked assays amplify only 1 target using duplex dPCR (31 ). The prevalence of single assay dropout provides an estimate of the templates that have not amplified, which in turn can be used to better estimate the true amount of template (32 ). Substantial molecular dropout requires a calibration control when absolute counts are required (see Calibrators below).…”

“…If results from different assays are in good agreement, this provides another level of confidence that the assays are likely to be optimal. However, if the intention is to perform multiplex analysis, the efficacy of multiplex formats should be compared to singleplex reactions (32 ).…”

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

“…A negative partition is routinely assumed to contain no DNA; however, it is known that when using methods like digital LAMP (31 ) and when detecting RNA by reverse transcriptase dPCR (30 ) it is possible for a large number of partitions to contain template that is not amplified. This "molecular dropout" is likely to be low in a dPCR reaction with a pure DNA template (36 ). However, template must be amplifiable and factors that damage DNA or that make DNA less accessible, like plasmid supercoiling (23 ), are likely to also lead to molecular dropout.…”

“…We used this in combination with multiplex analysis to estimate molecular dropout and investigate the impact of different template types on error (36 ). This application also has the potential to offer clinically valuable tools to investigate haplotypes in prenatal screening for diseases like ␤-thalassemia (43 ) and polymorphic gene complex arrangements encoding immunological cell receptors (21 ).…”

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…96) Digital PCR is a new technique for direct counting of the copy number of mRNA in samples without using calibration curves. 97) Transcriptomics research has been driven by the development of new technologies. On the other hand, versatile LC-MS instruments have been developed recently, mainly for the pharmacokinetics research, since high throughput analysis of basic drugs has been attained by the development of high-pressure binary gradient pumps with stainless steel tubing for reverse-phase chromatography using a column of silica gel with chemically bound ODS groups and electrospray ionization (ESI).…”