Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

Abstract

Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

The Future of Digital Polymerase Chain Reaction in Virology

“…They also found a larger partition volume for the Biomark platform than earlier reported by Bhat et al [62]. Furthermore, the data collected by Dong et al [55] and Pinheiro et al [45] indicate that the partition volume differs between samples and/or cartridges, further providing evidence of the need for partition volume verification. The partition volume used by the QX100 and QX200 ddPCR systems (Bio-Rad) has since been updated to 0.85 nL.…”

“…Furthermore, Pinheiro et al [45] discussed the importance of an accurate volume estimation in more detail, and Corbisier et al [57] found that concentrations of reference materials were underestimated due to real droplet volumes 8 % lower than the provided 0.91 nL. This finding was confirmed by Dong et al [55] who found that the droplet volume provided for the Raindrop ddPCR platform was also overestimated (4.39 pL instead of 5 pL, or a 14 % overestimation). They also found a larger partition volume for the Biomark platform than earlier reported by Bhat et al [62].…”

“…In this context, it will be imperative to carefully investigate the impact of different dPCR platforms and the effect of the conformation of the standard DNA, i.e. circular plasmids or linear DNA [55].…”

Characterization of Four Novel Plasmids from Lactobacillus plantarum BM4

“…The principle of ddPCR involves subdividing a single PCR reaction mixture into many small partitions and each undergoing the PCR reaction separately (32). Several studies have reported using ddPCR to determine the relative copy numbers of plasmids (33,34). According to our results, the relative copy numbers of pBM1, pBM2, pBM3, and pBM4 were calculated as 82, 24, 34, and 16 copies per chromosome equivalent using the ddPCR method.…”

Non-invasive diagnostic platforms in management of non-small cell lung cancer: opportunities and challenges

“…There are multiple sequencing platforms that are available for analysis of ctDNA and these platforms all have some various advantages and disadvantages (12). Platforms such has real-time quantitative PCR (qPCR), the Scorpion Amplification-Refractory Mutation System (ARMS) and droplet digital PCR (dPCR) can provide high sensitivity for detection of known genomic alterations and relatively easy to integrate into the clinical workflow with easy bioinformatic burden.…”