Droplet Digital PCR Provides Accurate Quantification of NGS Libraries

Here, we caught up with Dr George Karlin-Neumann of Bio-Rad Laboratories, and Jason Bielas of the Fred Hutchinson Cancer Research Center to find out how this technique has been utilized to its full potential to accurately quantify next-generation sequencing libraries for scientific research. 

Dr. George Karlin-Neumann, Director of Scientific Affairs, The Digital Biology Center, Bio-Rad Laboratories (in Pleasanton, CA) 

TN: Can you tell me a little more about QX100 Droplet Digital PCR (ddPCR) system? 

Dr. Karlin-Neumann: Bio-Rad's QX100 Droplet Digital PCRTM system is most simply put a DNA molecule counting technology for quantifying specific target sequences in a sample. It counts (or “digitizes”) the starting number of copies of a DNA molecule by subdividing each PCR reaction mix into ten’s of thousands of partitions (uniformly sized droplets) in the Droplet Generator, thermocycling these to endpoint in a 96-well plate in a standard thermocycler, and counting the target-containing fraction of droplets by way of their elevated fluorescence (i.e. positive droplets) in the Droplet Reader (a 2-color droplet flow cytometer). At low ddPCR concentrations of the specific target -- where positive droplets rarely contain more than one target molecule -- the number of positives per unit volume closely approximates the molecule’s concentration in the reaction; however, at high ddPCR concentrations, droplets may have up to an average of 5 target copies per droplet, and it is important to apply Poisson statistics to correctly determine the reaction’s true target concentration. The digitized measurement does not, however, require a standard curve for absolute quantification, in contrast to analogue real-time PCR measurements which do. 

TN: What are the key features of ddPCR that allowed researchers to accurately detect mitochondrial DNA? 

DKN: Droplet Digital PCR is designed to precisely, sensitively and reproducibly quantify DNA targets regardless of the source of that DNA, whether it is from human genomic or mitochondrial DNA, bacterial genomic or plasmid DNA, or even cDNA that has been derived from RNA by reverse transcription either in the droplets or prior to droplet formation. 

Once an assay has been validated to show that it is specific and quantitative for the target of interest (under the run conditions used), ddPCR can properly be used to accurately quantify the target as well as precisely quantifying it. The key features of Bio-Rad’s ddPCR technology that enable these properties lie in its robust chemistry, instrumentation and signal analysis. The chemistry and microfluidics together produce highly uniform and stable droplets that are robust to pipetting, thermocycling and reading. These properties enable high sensitivity (down to several molecules per reaction well) and high precision across a large dynamic range (~5 logs), and coupled with endpoint thermocycling, this allows detection and accurate quantitation of diverse-sized (deletion target) amplicons as demonstrated in the Bielas lab study. Importantly, this combined system robustness is reflected in consistent results from day to day and lab to lab, resulting in high reproducibility and ease of use. 

TN: This is one application for ddPCR, what other applications can this be used in? 

DKN: In a number of published studies, one of the common uses of the QX100 is for rare sequence and rare mutation detection or “needle in a haystack” problems, such as: ultra-sensitive measurements of latent proviral reservoirs in HIV eradication efforts; assessing contamination of foodstuffs by GMO products; liquid biopsy studies (e.g. monitoring transplant rejection by abundance of graft DNA in the blood or monitoring cancer patient response to therapy through oncogene markers in cell-free DNA); and detection of somatic CNV mosaicism in human skin cells. Additionally, searches are under way for CSF biomarkers to identify pre-symptomatic individuals at risk for Alzheimer’s Disease and for miRNA cancer biomarkers in patient serum. A further area that is showing great benefit from droplet digital PCR is the measurement of copy number variation (CNV), both in revealing the multi-allelic CNV landscape of the human genome and its potential involvement in inherited disease, and for identifying oncogenic CNV’s for personalized treatment of cancer patients. It is also commonly used for making absolute gene expression measurements without the need for a standard curve. Other established and developing uses are for NGS library quantification and QC, validation of NGS results (e.g. for CNV’s, RNA editing or differential heteroallelic expression) and for phasing of markers through their physical linkage in droplets. 

Jason Bielas, representing Fred Hutchinson Cancer Research Center 

TN: You have developed a new tool to accurately quantify and characterize de novo detection events, Digital Deletion Detection (3D). Can you tell me more about this technique? 

Jason Bielas: Mitochondrial DNA (mtDNA) deletions are known to contribute to a number of neuromuscular diseases, cancer and aging. It is therefore of great importance to study their genesis, and mechanisms of clonal expansion to phenotypic expression. Furthermore, these variants may serve as valuable biomarkers for early diagnosis of disease. However, there were no assays available, which are sensitive enough to accurately analyze these rare events. 

As a result, we established a novel assay, termed Digital Deletion Detection (3D), which permits the measure of DNA deletion mutations with unprecedented sensitivity. 3D is a three-step process that includes enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. 

We demonstrate the unparalleled throughput, sensitivity, and technical advancement of 3D by (1) interrogating over 8 billion mitochondrial genomes, (2) reporting the first ever, unbiased absolute measure of spontaneous mitochondrial DNA deletions, and (3) establishing that the expansion of pre-existing deletion mutations is the primary factor underlying the age-related accumulation of mtDNA deletions in human brain. 

TN: What benefits does 3D have over current techniques? 

JB: The work presented in our manuscript represents significant advancements in the ability to detect, quantify, and characterize rare allelic variants or polymorphisms of any nature. While the initial utility and motivation presented in our manuscript is the detection of rare deletions within mtDNA, the assay is by no means limited to this application. Given that our assay provides the first true measure of these mutations, their true utility can only now begin to be determined. The same principles used for the detection of rare deletion events also apply to the detection of rare nuclear variants or the detection of disease biomarkers. The means to sensitively detect such events have been the subject of intense research across a wide range of disciplines and applications. 

TN: Your research supports the hypothesis that pre-existing mutations are the primary factor contributing to age-related accumulation of mitochondrial DNA detection. What are the wider implications of this finding? 

JB: If aging and disease are driven by the accumulation of mitochondrial DNA deletions, then research focused on extending human life should focus on preventing the expansion of these events. However, this also implies that level of deletions events acquired in early life, might influence/set the length of ones lifespan (without intervention). Hence, also preventing the onset of mutation events early in life becomes increasing important in extending lifespan. 

TN: How do you see the 3D assay helping future research? 

JB: The primary benefit of the 3D assay is that it provides a powerful means to detect extremely rare mutations. Mitochondria are critical for proper cellular function, and maintaining the integrity of mitochondrial DNA (mtDNA) is key to ensuring functional mitochondria. However, the mechanisms of mtDNA maintenance are not well understood, nor is it known how or when the large deletions that are associated with aging and disease are acquired. Drs. Sean Taylor and I are currently using 3D technology to help us trace the origins of these mutations and to identify the critical time period when the adult pattern is set. Then we can also begin to address the really critical question of whether the expansion of these deletions actually drives aging and disease and whether or not that process can be controlled. 

Technology Networks Editorial Team