Twenty-one independent laboratories quantified a rare single nucleotide variant using digital PCR (dPCR) with high reproducibility between labs, demonstrating the method’s potential for routine clinical testing, according to a study published in Analytical Chemistry.
The study is the first to examine the reproducibility of digital PCR, an approach to quantifying nucleic acids that partitions samples into thousands of smaller reactions, between such a large number of laboratories at different geographic locations. The findings illustrate the inherent potential advantages of dPCR in measuring clinically important mutations, including greater reproducibility and less variability than real-time quantitative PCR (qPCR). In addition, the findings highlight the ability of dPCR to detect relevant rare sequence variants, according to the authors.
“Digital PCR lends itself to clinical testing where precise and reproducible results are critical not only for interlab data comparability, but also for dynamic testing in the same lab to track disease progression,” said George Karlin-Neumann, co-author and Director of Scientific Affairs at Bio-Rad’s Digital Biology Group.
The method permits precise and accurate quantification of nucleic acid concentration by minimizing the variability from common sources of error that can influence qPCR results. These sources include PCR inhibitors that can alter assay efficiency and skew standard curves. Digital PCR also provides absolute quantification of target nucleic acids without the need to establish standard curves, a major source of variability in qPCR.
The blinded study was part of the BioSITrace project, coordinated by the Laboratory of the Government Chemist (LGC), a private life sciences measurement and testing company and the UK’s designated National Measurement Laboratory for chemical and biomeasurement. Researchers in 21 laboratories across North America and Europe were asked to use dPCR to quantify copy number concentrations and fractional abundance of a KRAS mutant DNA with a single nucleotide variation (G12D) in the presence of excess wild-type DNA (down to ~0.2% minor allele frequency). The laboratories performed the experiments using Bio-Rad’s Droplet Digital™ PCR (ddPCR™) technology, on either a QX100™ or a QX200™ Droplet Digital PCR System from Bio-Rad. Droplet Digital PCR is Bio-Rad’s proprietary method for performing digital PCR in which a sample is divided into 20,000 water-oil emulsion droplets.
Cancer-derived mutations that differ only slightly from a wild-type sequence present in a sample in large excess are particularly difficult to detect and quantify using qPCR, especially when fractional abundance of the mutant is less than 5%.
The results of all 21 laboratories agreed with each other within a rigorously defined margin of error. Though three laboratories initially reported differing results, further analysis revealed methodological error attributed to misclassification of positive and negative droplets rather than measurement error. When the data were reanalyzed according to the recommended guidelines, they generated results consistent with the other 18 labs, validating the potential of dPCR for clinical testing applications.
“The ability to detect and quantify rare single nucleotide variants is another strength of dPCR and a measurement that is difficult to make at all with conventional qPCR,” said Karlin-Neumann. “So, similar to the Olympics, the inter-lab study attempted a very difficult ‘routine’ that, when done well, scores higher marks than a simpler, safer routine.”
Toward Clinical Adoption of dPCR
The study supports a new and more robust approach to nucleic acid analysis to address the reproducibility challenges that have plagued the clinical adoption of other PCR-based platforms.
“In the short term, I would anticipate digital PCR will be increasingly used as a reference method to improve the harmonization across laboratories,” said Jim Huggett, corresponding author of the article and a Principal Scientist at LGC. “As digital PCR is increasingly used for clinical diagnostics and preclinical research, people will find that they are more easily able to obtain comparable results.”