dPCR  -  digital Polymerase Chain Reaction (3)
Digital Polymerase-Chain-Reaction (1)
Digital Polymerase-Chain-Reaction (2)

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We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.

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Editorial - Special issue on dPCR
Digital PCR, a technique for the future.

Background - The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection.
Methods - We prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions.
Results - The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA.
Conclusions - PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.

Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe ‘higher order multiplexing’ that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

We explain how to design classic digital assays, comprising identical partitions, in order to obtain the required precision of the estimate within a defined range of concentrations. The design, including the number and volume of partitions, depends significantly on whether the assay is to assess the concentration of the target analyte in the sample or in the source of the sample (e.g. a patient body) with a given precision. We also show how to translate the result referring to the concentration in the sample into the concentration in the source of the sample, including the significant change in the breath of the confidence intervals.

Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.

Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard. Due to these properties, this technique has the potential to not only improve routine quantitative nucleic acid analysis, but also to be used as a reference method for certification of nucleic acid RM. The article focuses on the use and application of both dPCR and RMs for accurate quantification.

Digital PCR as a novel technology and its potential implications for molecular diagnostics.
Huggett JF and Whale A.
Clin Chem. 2013 59(12): 1691-1693

Comment on -- Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury.

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Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.
Pavšič J, Devonshire A, Blejec A, Foy CA, Van Heuverswyn F, Jones GM, Schimmel H, Žel J, Huggett JF, Redshaw N, Karczmarczyk M, Mozioğlu E, Akyürek S, Akgöz M, Milavec M.
Anal Bioanal Chem. 2017 409(10): 2601-2614

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

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Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.
Belmonte FR, Martin JL, Frescura K, Damas J, Pereira F, Tarnopolsky MA, Kaufman BA
Sci Rep. 2016 28;6: 25186

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

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Evaluating Droplet Digital Polymerase Chain Reaction for the Quantification of Human Genomic DNA: Lifting the Traceability Fog.
Kline MC and Duewer DL
Anal Chem. 2017 89(8): 4648-4654

Digital polymerase chain reaction (dPCR) end point platforms directly estimate the number of DNA target copies per reaction partition, λ, where the partitions are fixed-location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both λ and the partition volume, V, must be metrologically traceable to some accessible reference system, ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the critical dPCR assumptions that are essential for establishing the SI traceability of λ. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable partition volume specifications. Our colleagues at the National Institute of Standards and Technology (NIST) have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus, neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split samples with both cdPCR and ddPCR platforms can transfer the λ traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR estimates of CRM target concentration.

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Model-Based Classification for Digital PCR: Your Umbrella for Rain.
Jacobs BKM, Goetghebeur E, Vandesompele J, De Ganck A, Nijs N, Beckers A, Papazova N, Roosens NH, Clement L
Anal Chem. 2017 89(8): 4461-4467

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

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Use of droplet digital PCR for quantitative and automatic analysis of the HER2 status in breast cancer patients.
Otsuji K, Sasaki T, Tanaka A, Kunita A, Ikemura M, Matsusaka K, Tada K, Fukayama M, Seto Y.
Breast Cancer Res Treat. 2017 162(1): 11-18

PURPOSE: Digital polymerase chain reaction (dPCR) has been used to yield an absolute measure of nucleic acid concentrations. Recently, a new method referred to as droplet digital PCR (ddPCR) has gained attention as a more precise and less subjective assay to quantify DNA amplification. We demonstrated the usefulness of ddPCR to determine HER2 gene amplification of breast cancer.
METHODS: In this study, we used ddPCR to measure the HER2 gene copy number in clinical formalin-fixed paraffin-embedded samples of 41 primary breast cancer patients. To improve the accuracy of ddPCR analysis, we also estimated the tumor content ratio (TCR) for each sample.
RESULTS: Our determination method for HER2 gene amplification using the ddPCR ratio (ERBB2:ch17cent copy number ratio) combined with the TCR showed high consistency with the conventionally defined HER2 gene status according to ASCO-CAP (American Society of Clinical Oncology/College of American Pathologists) guidelines (P<0.0001, Fisher's exact test). The equivocal area was established by adopting 99% confidence intervals obtained by cell line assays, which made it possible to identify all conventionally HER2-positive cases with our method. In addition, we succeeded in automating a major part of the process from DNA extraction to determination of HER2 gene status.
CONCLUSIONS: The introduction of ddPCR to determine the HER2 gene status in breast cancer is feasible for use in clinical practice and might complement or even replace conventional methods of examination in the future.

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Calibration-free assays on standard real-time PCR devices.
Debski PR, Gewartowski K, Bajer S, Garstecki P
Sci Rep. 2017 22;7: 44854

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

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Digital PCR analysis of circulating nucleic acids.
Hudecova I
Clin Biochem. 2015 Oct;48(15): 948-956

Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date.

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Quantitative nucleic acid amplification by digital PCR for clinical viral diagnostics.
Zhang K, Lin G, Li J.
Clin Chem Lab Med. 2016 54(9): 1427-1433

In the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications.

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Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision.
Majumdar N, Wessel T, Marks J
PLoS One. 2015 10(3): e0118833

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

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Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.
Zonta E, Garlan F, Pécuchet N, Perez-Toralla K, Caen O, Milbury C, Didelot A, Fabre E, Blons H, Laurent-Puig P, Taly V
PLoS One. 2016 11(7): e0159094

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

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Polymerase chain reaction in microfluidic devices.
Ahrberg CD, Manz A, Chung BG
Lab Chip. 2016 16(20): 3866-3884

The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

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RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.
Alikian M, Whale AS, Akiki S, Piechocki K, Torrado C, Myint T, Cowen S, Griffiths M, Reid AG, Apperley J, White H, Huggett JF, Foroni L
Clin Chem. 2017 63(2): 525-531

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification.
METHODS: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay.
RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range.
CONCLUSIONS: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

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Analysis of extracellular RNA by digital PCR.
Takahashi K, Yan IK, Kim C, Kim J, Patel T
Front Oncol. 2014 4: 129 -- eCollection 2014.

The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

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Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.
Liesbet Deprez, Philippe Corbisier, Anne-Marie Kortekaas, Stéphane Mazoua, Roxana Beaz Hidalgo, Stefanie Trapmann, Hendrik Emons
Biomolecular Detection and Quantification, Vol 9, Pages 29-39

Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use.
Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

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Quantification of cell-free DNA in normal and complicated pregnancies -- overcoming biological and technical issues.
Manokhina I, Singh TK, Peñaherrera MS, Robinson WP.
PLoS One. 2014 9(7): e101500 -- eCollection 2014

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal 'fetal' markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2-39.5 weeks). Two fetal- or placental-specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

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Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.
Taylor SC, Carbonneau J, Shelton DN, Boivin G
J Virol Methods. 2015 Nov; 224: 58-66

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.

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Flexible analysis of digital PCR experiments using generalized linear mixed models.
Matthijs Vynck, Jo Vandesompele, Nele Nijs, Björn Menten, Ariane De Ganck, Olivier Thas
Biomolecular Detection and Quantification, Vol 9, Pages 1-13

The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability. We propose the generalized linear mixed models framework as a flexible tool for analyzing a wide range of experiments. We also introduce a method for estimating reference gene stability to improve accuracy and precision of copy number and relative expression estimates. We demonstrate the usefulness of the methodology on a complex experimental setup.

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Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.
Liao GJ, Gronowski AM, Zhao Z.
Clin Chim Acta. 2014 428: 44-50

The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made non-invasive prenatal testing (NIPT) possible. Maternal plasma cell free DNA is a mixture of maternal and fetal DNA, of which, fetal DNA represents a minor population in maternal plasma. Therefore, methods with high sensitivity and precision are required to detect and differentiate fetal DNA from the large background of maternal DNA. In recent years, technical advances in the molecular analysis of fetal DNA (e.g., digital PCR and massively parallel sequencing (MPS)) has enabled the successful implementation of noninvasive testing into clinical practice, such as fetal sex assessment, RhD genotyping, and fetal chromosomal aneuploidy detection.With the ability to decipher the entire fetal genome from maternal plasma DNA, we foresee that an increased number of non-invasive prenatal tests will be available for detecting many single-gene disorders in the near future. This review briefly summarizes the technical aspects of the NIPT and application of NIPT in clinical practice.

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Direct elicitation of template concentration from quantification cycle (Cq) distributions in digital PCR.
Mojtahedi M, Fouquier d'Hérouël A, Huang S.
Nucleic Acids Res. 2014 42(16): e126

Digital PCR (dPCR) exploits limiting dilution of a template into an array of PCR reactions. From this array the number of reactions that contain at least one (as opposed to zero) initial template is determined, allowing inferring the original template concentration. Here we present a novel protocol to efficiently infer the concentration of a sample and its optimal dilution for dPCR from few targeted qPCR assays. By taking advantage of the real-time amplification feature of qPCR as opposed to relying on endpoint PCR assessment as in standard dPCR prior knowledge of template concentration is not necessary. This eliminates the need for serial dilutions in a separate titration and reduces the number of necessary reactions. We describe the theory underlying our approach and discuss experimental moments that contribute to uncertainty. We present data from a controlled experiment where the initial template concentration is known as proof of principle and apply our method on directly monitoring transcript level change during cell differentiation as well as gauging amplicon numbers in cDNA samples after pre-amplification.

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Methods for comparing multiple digital PCR experiments.
Burdukiewicz M, Rödiger S, Sobczyk P, Menschikowski M, Schierack P, Mackiewicz P
Biomol Detect Quantif. 2016 Aug 10;9: 14-19 -- eCollection 2016.

The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series. Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment.

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Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.
Dreo T, Pirc M, Ramšak Z, Pavšič J, Milavec M, Zel J, Gruden K.
Anal Bioanal Chem. 2014 406(26): 6513-6528

Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

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A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples.
Sedlak RH, Kuypers J, Jerome KR.
Diagn Microbiol Infect Dis. 2014 S0732-8893 (14) 00366-6

We demonstrate the development of a multiplex droplet digital PCR assay for human cytomegalovirus (CMV), human adenovirus species F, and an internal plasmid control that may be useful for PCR inhibition-prone clinical samples. This assay performs better on inhibition-prone stool samples than a quantitative PCR assay for CMV and is the first published clinical virology droplet digital PCR assay to incorporate an internal control.

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Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery.
Christopher J. Hayes, Tara M. Dalton
Biomol Detect Quantif. 2015(4): 22-32

PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR) has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA) in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits.

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Impact of variance components on reliability of absolute quantification using digital PCR.
Jacobs BK, Goetghebeur E, Clement L.
BMC Bioinformatics. 2014 Aug 22;15: 283

BACKGROUND:  Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.
RESULTS:  We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.
CONCLUSIONS:  Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

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One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses.
Rački N, Morisset D, Gutierrez-Aguirre I, Ravnikar M.
Anal Bioanal Chem. 2014 406(3): 661-667

Water contamination by viruses has an increasing worldwide impact on human health, and has led to requirements for accurate and quantitative molecular tools. Here, we report the first one-step reverse-transcription droplet digital PCR-based absolute quantification of a RNA virus (rotavirus) in different types of surface water samples. This quantification method proved to be more precise and more tolerant to inhibitory substances than the benchmarking reverse-transcription real-time PCR (RT-qPCR), and needs no standard curve. This new tool is fully amenable for the quantification of viruses in the particularly low concentrations usually found in water samples.

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Assessing the accuracy of quantitative molecular microbial profiling.
O'Sullivan DM, Laver T, Temisak S, Redshaw N, Harris KA, Foy CA, Studholme DJ, Huggett JF
Int J Mol Sci. 2014 Nov 21;15(11): 21476-21491

The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

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Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer.
Li N, Ma J, Guarnera MA, Fang H, Cai L, Jiang F.
J Cancer Res Clin Oncol. 2014 Jan;140(1): 145-150

PURPOSE: MicroRNAs (miRNAs) play important roles in the initiation and progression of lung cancer. Measuring miRNA expression levels in sputum could provide a potential approach for the diagnosis of lung cancer. The emerging digital PCR is a straightforward technique for precise, direct, and absolute quantification of nucleic acids. The objective of the study was to investigate whether digital PCR could be used to quantify miRNAs in sputum for lung cancer diagnosis.
METHODS: We first determined and compared dynamic ranges of digital PCR and conventional quantitative reverse transcriptase PCR (qRT-PCR) for miRNA quantification using RNA isolated from sputum of five healthy individuals. We then used digital PCR to quantify copy number of two lung cancer-associated miRNAs (miR-31 and miR-210) in 35 lung cancer patients and 40 cancer-free controls.
RESULTS: Copy number of the miRNAs measured by digital PCR displayed a linear response to input cDNA amount in a twofold dilution series over seven orders of magnitude. miRNA quantification determined by digital PCR assay was in good agreement with that obtained from qRT-PCR analysis in sputum. Furthermore, combined quantification of miR-31 and miR-210 copy number by using digital PCR in sputum of the cases and controls provided 65.71 % sensitivity and 85.00 % specificity for lung cancer diagnosis.
CONCLUSION: As digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number in sputum for lung cancer diagnosis.

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Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.
Fu GK, Wilhelmy J, Stern D, Fan HC, Fodor SP.
Anal Chem. 2014 Mar 18;86(6): 2867-2870

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

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Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.
Yang R, Paparini A, Monis P, Ryan U.
Int J Parasitol. 2014 S0020-7519 (14) 00226-4

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R2⩾0.999) and faecal samples (R2⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies.

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Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.
Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW, Wright WE.
Nucleic Acids Res. 2014; 42(13): e104

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼ 2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

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GMO quantification: valuable experience and insights for the future.
Milavec M, Dobnik D, Yang L, Zhang D, Gruden K, Zel J.
Anal Bioanal Chem. 2014 Oct;406(26): 6485-6497

Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.

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A decade with nucleic acid-based microbiological methods in safety control of foods.
Kuchta T, Knutsson R, Fiore A, Kudirkiene E, Höhl A, Horvatek Tomic D, Gotcheva V, Pöpping B, Scaramagli S, To Kim A, Wagner M, De Medici D.
Lett Appl Microbiol. 2014 59(3): 263-271

In the last decade, nucleic acid-based methods gradually started to replace or complement the culture-based methods and immunochemical assays in routine laboratories involved in food control. In particular, real-time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry-over contamination. Basic advantages provided by nucleic acid-based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid-based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.

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Digital PCR hits its stride
by Monya Baker
Nature Methods 9, 541–544 (2012)

A few years ago, Ramesh Ramakrishnan had to spend so much time explaining what digital PCR was that he had to rush through his explanations of applications when he gave talks at meetings. Now, he says, most audiences are at least familiar with the term, even if they have not performed the technique themselves. “It's no longer an exotic thing,” says Ramakrishnan, director of R&D at Fluidigm Corporation.
The strategy for digital PCR (dPCR) has been summarized as 'divide and conquer': a sample is diluted and partitioned into hundreds or even millions of separate reaction chambers so that each contains one or no copies of the sequence of interest. By counting the number of 'positive' partitions (in which the sequence is detected) versus 'negative' partitions (in which it is not), scientists can determine exactly how many copies of a DNA molecule were in the original sample. Among other applications, researchers have used digital PCR to distinguish differential expression of alleles1, to track which viruses infect individual bacterial cells2, to quantify cancer genes in patient specimens3 and to detect fetal DNA in circulating blood4.
The concept behind digital PCR was first described in 1992 (ref. 5). A few years later, Bert Vogelstein and Ken Kinzler at Johns Hopkins University named the technique and showed that it could be used to quantify disease-associated mutations in stool from patients with colorectal cancer. But although the theory was simple, its implementation was not. Initial demonstrations were performed in commercially available 384-well plates with 5 microliters per partition, requiring volumes of reagents that would daunt most researchers6.
Advances in nanofabrication and microfluidics have now led to systems that produce hundreds to millions of nanoliter- or even picoliter-scale partitions. Academic technology developers have described several implementations, but so far only a handful of companies have commercialized products or announced plans to do so (Table 1). Fluidigm and Life Technologies create reaction chambers within specially designed chips or plates. Bio-Rad and RainDance sequester reagents into individual droplets ... ... ... ... ... ... ...

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Viral diagnostics in the era of digital polymerase chain reaction.
Sedlak RH, Jerome KR.
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
Diagn Microbiol Infect Dis. 2013 75(1): 1-4

Unlike quantitative polymerase chain reaction (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR. Here we review current literature that demonstrates dPCR's potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection.

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Analytical detection techniques for droplet microfluidics - a review.
Zhu Y, Fang Q.
Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, China.
Anal Chim Acta. 2013 (787): 24-35

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.

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Absolute quantification by droplet digital PCR versus analog real-time PCR.
Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M.
Digital Biology Center, Bio-Rad Laboratories, Inc., Pleasanton, California, USA.
Nat Methods. 2013 Sep 1.

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

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Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR to Inhibitory Substances.
Dingle TC, Sedlak RH, Cook L, Jerome KR.
Clin Chem. 2013 Sep 3.

To the Editor:  Real-time quantitative PCR (qPCR)1 is a rapid and sensitive method that forms the foundation for many clinical diagnostic tests. Droplet digital PCR (ddPCR) shares these qualities with qPCR, but owing to reaction partitioning, ddPCR is proposed to exhibit increased tolerance to interfering substances, making it an attractive alternative to qPCR for diagnostic applications (1, 2). The data to support this phenomenon and its mechanism, however, are currently lacking in the literature. Herein, we describe a series of experiments to compare the inhibition tolerance of laboratorydeveloped CMV qPCR and ddPCR (Bio-Rad Laboratories, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA,and heparin) directly into the PCR reactions (4 ). Differences in the resulting inhibition curves and the half-maximal inhibitory concentrations (IC50) were then assessed.

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Highly precise measurement of HIV DNA by droplet digital PCR.
Strain MC, Lada SM, Luong T, Rought SE, Gianella S, Terry VH, Spina CA, Woelk CH, Richman DD.
University of California San Diego, La Jolla, California, United States of America.
PLoS One. 2013;8(4): e55943

Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.

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Methods for applying accurate digital PCR analysis on low copy DNA samples.
Whale AS, Cowen S, Foy CA, Huggett JF.
Molecular and Cell Biology Team, LGC Ltd, Teddington, United Kingdom.
PLoS One. 2013;8(3): e58177

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.

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Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.
Shuga J, Zeng Y, Novak R, Lan Q, Tang X, Rothman N, Vermeulen R, Li L, Hubbard A, Zhang L, Mathies RA, Smith MT.
School of Public Health, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA, UC San Francisco/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, MD 20852, USA, Guangdong Poison Control Center, Guangzhou 510300, China and Environmental Epidemiology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, NL-3508, The Netherlands.
Nucleic Acids Res. 2013 41(16): e159

Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10-6) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1 × 10-7 or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.

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Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine.
Day E, Dear PH, McCaughan F.
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Methods. 2013 59(1): 101-107

The efficient delivery of personalized medicine is a key goal of healthcare over the next decade. It is likely that PCR strategies will play an important role in the delivery of this goal. Digital PCR has certain advantages over more traditional PCR protocols. In this article we will discuss the current status of digital PCR, highlighting its advantages and focusing on how it can be utilized in biomarker development and analysis, including the use of individualized biomarkers. We will explore recent developments in this field including examples of how digital PCR may integrate with next generation sequencing to deliver truly personalized medicine.

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Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis.
Devonshire AS, Sanders R, Wilkes TM, Taylor MS, Foy CA, Huggett JF.
Molecular and Cell Biology, LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.
Methods. 2013 Jan;59(1): 89-100

Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed.

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Limitations and possibilities of small RNA digital gene expression profiling.
Linsen SE, de Wit E, Janssens G, Heater S, Chapman L, Parkin RK, Fritz B, Wyman SK, de Bruijn E, Voest EE, Kuersten S, Tewari M, Cuppen E.
Nat Methods. 2009 Jul;6(7): 474-476

To the Editor:  High-throughput sequencing (HTS) has proven to be an invaluable tool for the discovery of thousands of microRNA genes across multiple species1,2. At present, the throughput of HTS platforms is sufficient to combine discovery with quantitative expression analysis allowing for digital gene expression (DGE) profiling3. We observed that methods for small RNA DGE profiling are strongly biased toward certain small RNAs, preventing the accurate determination of absolute numbers of small RNAs. The observed bias is largely independent of the sequencing platform but strongly determined by the method used for small RNA library preparation. However, as the biases are systematic and highly reproducible, DGE profiling is suited for determining relative expression differences between samples.

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Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients.
Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Le Corre D, Li X, Atochin I, Link DR, Griffiths AD, Pallier K, Blons H, Bouché O, Landi B, Hutchison JB, Laurent-Puig P.
Université Paris Sorbonne Cité, INSERM UMR-S775, Centre Universitaire des Saints-Pères, Paris, France;
Clin Chem. 2013 Aug 12.

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumors samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected.CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

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High-throughput microfluidic single-cell digital polymerase chain reaction.
White AK, Heyries KA, Doolin C, Vaninsberghe M, Hansen CL.
Centre for High Throughput Biology and ‡Department of Physics and Astronomy, University of British Columbia , 307-2125 East Mall, Vancouver, British Columbia, Canada V6T 1Z4.
Anal Chem. 2013 Aug 6;85(15): 7182-7190

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118 900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204 000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

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Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.
Didelot A, Kotsopoulos SK, Lupo A, Pekin D, Li X, Atochin I, Srinivasan P, Zhong Q, Olson J, Link DR, Laurent-Puig P, Blons H, Hutchison JB, Taly V.
Université Paris Sorbonne Cité, INSERM UMR-S775, Paris, France.
Clin Chem. 2013 May;59(5): 815-823

BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.
METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.
RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.
CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.