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Droplet Digital™ PCR (ddPCR™)
ddPCR technology enables highly sensitive detection and absolute quantification of gene expression. While qPCR can satisfy several parameters for scientists, ddPCR technology offers other avenues, particularly research questions that require absolute quantification of rare transcripts or low-abundance targets. Improvements in ddPCR instrumentation have also increased throughput.
Building on the ability of Droplet Digital PCR to make many thousands of discrete measurements in a single sample (compared to one measurement in a tube or well), improvements in instrumentation amplifies throughput. For instance, the Bio-Rad QX600™ Droplet Digital PCR System features multiplexing capabilities, including quantification of up to 12 targets per well, six-color detection, and one to eight samples per cartridge with a reader capacity of 1–96 samples.
Obtaining Quantification
As researchers build genome-and transcript-wide profiles with NGS, accurate and precise quantification is required to deepen their understanding of libraries and panels obtained from NGS.
qPCR
As qPCR is the prevailing standard for gene expression quantification, it is often used in tandem with NGS. In addition, its ease of use, a wealth of existing literature, and technical support make qPCR an attractive means of quantifying gene expression.
- When performed appropriately, qPCR is excellent for amplifying and quantifying DNA libraries obtained from NGS. qPCR is particularly effective in the quantification of gene expression when the number of target regions is low (less than 5), and the study aims to screen or identify known variants
- Though qPCR has associated challenges, such as its reliance on reference standards, well-to-well variability, and susceptibility to contamination, optimization in protocols have made data more consistent. As discussed in the Real-Time PCR (qPCR) section, current instruments include automation and streamlined data analysis pipelines, significantly reducing variability and improving data quality
The Pros and Cons of RT-qPCR and Digital PCR across Therapeutic Development
ddPCR Technology
Droplet Digital PCR is the preferred method for absolute quantification for low-abundance targets in rare samples. Like qPCR, Droplet Digital PCR can also qualify and quantify NGS libraries. Moreover, its high tolerance to PCR inhibitors results in robust quantification, thus making ddPCR technology highly attractive for researchers studying rare targets in complex backgrounds.
- One excellent example of the precise and accurate quantification of Droplet Digital PCR was carried out by Campomenosi et al.1 The authors compared the quantitative capabilities of qPCR versus Droplet Digital PCR in measuring microRNA (miRNA), a selection of which has been proposed as a biomarker for lung cancer. Interestingly, they found that while both qPCR and Droplet Digital PCR can detect and quantify miRNA with high concordance, the coefficient of variation for Droplet Digital PCR was significantly smaller than qPCR and included higher throughput
- While qPCR results are highly susceptible to contamination, Droplet Digital PCR can overcome these challenging situations when measuring samples with high background. 1 study utilized Droplet Digital PCR and demonstrated that while qPCR measurements of cDNA in contaminated samples or low levels of nucleic acids (quantification cycle [Cq]>29) resulted in uninterpretable data, Droplet Digital PCR can measure the same samples with precision and accuracy2
Developing Sensitivity for Rare Transcripts or Subtle Changes in Expression
NGS
Generation of genome wide libraries can be difficult in poor-quality samples or degraded RNA specimens. However, improvements in technology have greatly increased NGS sensitivity.
- For example, the Bio-Rad SEQuoia RiboDepletion Kit increases assay sensitivity by removing irrelevant rRNA fragments and retains rare transcripts that are often lost from a pre-library RNA depletion strategy
- The Bio-Rad ddSEQ Single-Cell Library Preparation Kits for 3’ RNA-seq and ATAC-Seq allow for reliable high-throughput profiling of single cells with high capture efficiency and sensitivity
qPCR
For many researchers, detecting twofold or greater changes is sufficient; qPCR is adequate for these research questions. However, as a researcher studies more subtle changes or detection of rare variants, qPCR becomes a relatively difficult technique to adopt.
- Since the sensitivity of qPCR relies on the linearity of the standard curve (R2>0.98) that is assay specific and dependent on the sample type and quality, researchers should take care of their sensitivity needs when considering qPCR. Detection of rare variants in qPCR requires increased cycles, thus leading to potential distortions in library heterogeneity that prevent the detection of rare variants.
qPCR Technology
ddPCR technology, on the other hand, has risen to prominence due to its excellent capabilities in detecting and quantifying rare variants or subtle changes in gene expression.
- Droplet Digital PCR has seen significant utility in oncology research and includes the measurement of structural variant analysis, copy number variation, methylation detection, and loss of heterozygosity
- Indeed, while sample heterogeneity can impact the accurate measurement of copy number variations, the ability of Droplet Digital PCR to process a high number of independent amplifications in a single sample allows for resolution and accurate measurement of copy number differences often seen in a variety of cancer
- Optimized qPCR is capable of detecting as little as 1% mutant fraction. For researchers requiring more sensitive assays, Droplet Digital PCR has been shown to detect 0.001% mutant fraction (that is, detection of mutant DNA in a 1,000-fold excess of wild-type background)3
Ensuring Reproducibility
Reproducibility is a key need across scientific fields, particularly for those studying human disease. Indeed, replicating datasets is critical for advancing research and allows scientists to address future directions posed by existing literature.
NGS
In recent years advances in NGS technologies have been exciting but not without challenges in reproducibility. Of note, several studies have been conducted to assess reproducibility standards across several NGS platforms.
- One of the largest projects investigating the reproducibility of RNA-Seq is the Sequencing Quality Control (SEQC) project.4 A multilaboratory endeavor, SEQC found that RNA-Seq platforms are capable of accurate and reproducible measurements of relative expression across sites and platforms if specific filters are used
- However, others are discovering reproducibility issues in RNA-Seq, particularly in using graphical tools and statistical models — though reproducibility across sample replicates and FlowCells are acceptable5
- In general, scientists should take care when employing NGS and utilize multiple methods to validate and replicate their results
qPCR
Though a standard in the industry, qPCR has faced several reproducibility challenges across its lifetime. These include random errors from biological variability and the more alarming systemic errors arising from improper equipment usage and analyses.6
- Fortunately, advances in data standards, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines and real-time PCR data markup language (RDML), and streamlined protocols and instrumentation (for example, CFX Opus System connections to wireless network [WiFi] or Ethernet) are improving reproducibility standards when using qPCR
qPCR Technology
ddPCR technology involves two critical advantages that significantly impact accuracy and reproducibility: 1) partitioning of a sample into thousands of individual PCR reactions and 2) acquiring data at the reaction endpoint. These factors allow for direct quantification of DNA without dependence on standard curves. This results in more precise and reproducible data, especially in the presence of sample contaminants. These capabilities significantly improve reproducibility2
- For example, 1 group of researchers studying Streptococcus agalactiae, the causative pathogen of puerperal sepsis and pneumonia, achieved excellent reproducibility with Droplet Digital PCR and obtained a coefficient of variation (CV) of 4.5% across their experiments7
- Others have also been successful in replicating results using Droplet Digital PCR. For example, one group aimed to develop and characterize DNA reference material with Droplet Digital PCR across 7 independent laboratories and obtained a CV of less than 4.5%8
Protecting Limited Sample
Especially in fields where the sample is precious, making the most of a sample is critical.Fortunately, advances in most gene expression platforms now allow researchers to maximize the output of their samples, limited or otherwise.
NGS
Most NGS platforms require as little as 10 ng of DNA, and improvements in instrumentation have made progress in ensuring researchers can make the most of their samples.
- RNA-Seq platforms can now capture all RNA biotypes from poor-quality RNAs, such as formalin-fixed paraffin-embedded (FFPE) samples. The Bio-Rad SEQuoia workflow, for example, also includes the SEQuoia RiboDepletion Kit, which allows for the efficient filtering and removal of irrelevant rRNA
- The Bio-Rad ddSEQ Single-Cell Library Preparation Kits for 3’ RNA-seq and ATAC-Seq can process from 500 to 5,000 cell output per sample with high cell utilization, providing flexibility depending on sample requirements. The ddSEQ Single-Cell Isolator is capable of processing hundreds to tens of thousands of cells per experiment.
ddPCR Technology
Droplet Digital PCR is preferred in situations requiring a limited sample or low-abundance targets. The unique sample partitioning step of Droplet Digital PCR, paired with Poisson statistical data analysis, allows higher precision than traditional PCR and qPCR methods.The ddPCR Systems are designed to quantify multiple targets with high accuracy and sensitivity, and are capable of quantifying up to 12 targets in a single well.
- Droplet Digital PCR can be applied to several sample types, from low-abundance circulating tumor DNA (ctDNA) in blood, urine, and cerebrospinal fluid, to FFPE tissue. Among the other advantages of ddPCR technology outlined here for researchers requiring absolute quantification, this gene expression analysis platform is also excellent when samples are limited
Conclusions
Across the multitude of parameters and needs outlined in this article, it is important to remember that one technique is not necessarily better than the other and is highly dependent on the research question. However, combining multiple methods with orthogonal applications will maximize your research output once you determine your research needs.
References
- Campomenosi P et al. (2016). A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer. BMC Biotechnol 16, 60.
- Taylor SC et al. (2017). Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: From variable nonsense to publication quality data. Sci Rep 7, 2,409.
- Hindson BJ et al. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83, 8,604–8,610.
- SEQC/MAQC-III Consortium (2014). A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium. Nat Biotechnol 32, 903–914.
- Yu L (2020). RNA-seq reproducibility assessment of the Sequencing Quality Control Project. Cancer Inform 19, 1176935120922498.
- Taylor SC et al. (2019). The ultimate qPCR experiment: Producing publication quality, reproducible data the first time. Trends Biotechnol 37, 761–774.
- Zeng YF et al. (2020). Development of a droplet digital PCR method for detection of Streptococcus agalactiae. BMC Microbiol 20, 179.
- Pinheiro LB et al. (2017). Interlaboratory reproducibility of droplet digital polymerase chain reaction using a new DNA reference material format. Anal Chem 89, 11,243–11,251.
- Saxena D et al. (2019). Rapid and ultrasensitive digital PCR (dPCR) profiling of EGFRvIII in tumor cells and tissues. Neurooncol Adv 1, vdz030.
- Kibschull M et al. (2016). Quantitative large scale gene expression profiling from human stem cell culture micro samples using multiplex pre-amplification. Syst Biol Reprod Med 62, 84–91.
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