(abbreviations:   digital PCR  -  DigitalPCR  -  dPCR  -  dePCR)

SPECIAL REPORT  --  highly cited -- HOT PAPER
Guidelines for Minimum Information for Publication of Quantitative Digital PCR Experiments.

Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T,
Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA.

Clinical Chemistry 2013 59(6): 892-902

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

The Digital MIQE Guidelines Update:
Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Jim F Huggett and dMIQE Group:
Alexandra S Whale, Ward De Spiegelaere, Wim Trypsteen, Afif Abdel Nour, Young-Kyung Bae, Vladimir Benes, Daniel Burke, Megan Cleveland, Philippe Corbisier, Alison S Devonshire, Lianhua Dong, Daniela Drandi, Carole A Foy, Jeremy A Garson, Hua-Jun He, Jan Hellemans, Mikael Kubista, Antoon Lievens, Mike G Makrigiorgos, Mojca Milavec, Reinhold D Mueller, Tania Nolan, Denise M O'Sullivan, Michael W Pfaffl, Stefan Rödiger, Erica L Romsos, Gregory L Shipley, Valerie Taly, Andreas Untergasser, Carl T Wittwer, Stephen A Bustin, Jo Vandesompele
Clinical Chemistry 2020 66(8): 1012-1029

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.



Digital PCR (dPCR) is a refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA, or RNA). The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants, point mutations, and it is routinely used for clonal amplification of samples for "next-generation sequencing."

PCR Basics:
The PCR method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample. However, many factors complicate this calculation, creating uncertainties and inaccuracies.
These factors include the following:
  • Initial amplification cycles may not be exponential
  • PCR amplification eventually plateaus after an uncertain number of cycles
  • Low initial concentrations of target nucleic acid molecules may not amplify to detectable levels
  • Variations in PCR amplification efficiency in various biological samples
dPCR Working Principle:

Digital PCR overcomes the difficulties of conventional PCR. With dPCR, a sample is partitionedso that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. The partitioning of the sample allows one to count the molecules by estimating according to Poisson. As a result, each part will contain "0" or "1" molecules, or a negative or positive reaction, respectively. After PCR amplification, nucleic acids may be quantified by counting the regions that contain PCR end-product, positive reactions.
In conventional PCR, starting copy number is proportional to the number of PCR amplification cycles. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification.

The dPCR concept was conceived in 1992 by Sykes et al. using nested PCR. An important development occurred in 1995 with co-inventions by Brown at Cytonix and Silver at the National Institutes of Health of single-step quantitization and sequencing methods employing nano-scale arrays and localized clonal colonies using capillaries, gels, affinity surfaces/particles and immiscible fluid containments, resulting in a 1997 U. S. Patent (U. S. Patent 6,143,496) and subsequent divisional and continuation patents. Vogelstein and Kinzler further developed the concept by quantifying KRAS mutations in stool DNA from colorectal cancer patients. Digital PCR has been shown to be a promising surveillance tool for illnesses such as cancer. Significant additional developments have included using emulsion beads for digital PCR by Dressman and colleagues. Digital PCR has many other applications, including detection and quantitization of low-level pathogens, rare genetic sequences, gene expression in single cells, and the clonal amplification of nucleic acids (cPCR or clonal PCR) for the identification and sequencing of mixed nucleic acids samples or fragments. It has also proved useful for the analysis of heterogeneous methylation.

In 2006 Fluidigm introduced the first commercial system for digital PCR based on integrated fluidic circuits (chips) having integrated chambers and valves for partitioning samples. In March 2010, a patent was published for digital PCR based on emulsions.

Digital PCR has many potential applications, including the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, and relative gene expression in single cells. Clonal amplification enabled by single-step digital PCR is a key factor in reducing the time and cost of many of the "next-generation sequencing" methods and hence enabling personal genomics.

Application of digital PCR for Absolute Quantitation
Digital PCR is quantitative PCR method that can be used to measure absolute quantitation. In this technique, the number of positive and negative amplification reactions is used to the determine precise measurement of target concentration.

dPCR Applications:
  • Absolute Quantification of Viral Load
  • Absolute Quantification of Nucleic Acid Standards
  • Absolute Quantification of Next-Gen Sequencing Libraries
  • Rare Allele Detection
  • Low-Fold Copy Number Discrimination
  • Enrichment and Separation of Mixtures
Summary Advantages of Digital PCR:
  • No need to rely on references or standards
  • Desired precision can be achieved by increasing total number of PCR replicates
  • Highly tolerant to inhibitors
  • Capable of analyzing complex mixtures
  • Unlike traditional qPCR, digital PCR provides a linear response to the number of copies present to allow for small fold change differences to be detected


Absolute Quantification:   digtal-PCR  vs.  classical standard curve based 'absolute' Quantification
by Life Technologies

When calculating the "absolute" results of your real-time PCR (qPCR) experiment, you can use either digital PCR method or classical standard curve based "absolute quantification".  More info at Life Technologies

Absolute Quantification at a Glance

Absolute Quantification
(Digital PCR Method)
Absolute Quantification
(Standard Curve Method)
Overview In absolute quantification using Digital PCR, no known standards are needed.  The target of interest can be directly quantified with precision determined by number of digital PCR replicates.  In absolute quantification using the Standard Curve Method, you quantitate unknowns based on a known quantity. First you create a standard curve; then you compare unknowns to the standard curve and extrapolate a value.
Example Quantify copies of rare allele present in heterogenous mixtures.

Count the number of cell equivalents in sample by targeting genomic DNA.

Determine absolute number of viral copies present in a given sample without reference to a standard.
Correlating viral copy number with a disease state.

Absolute Quantification Using the Digital PCR Method

Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.

Absolute Quantification Using the
                              Digital PCR Method
Figure 1: Digital PCR uses the ratio of positive (White) to negative (Black) PCR reactions
to count the number of target molecules.

Absolute Quantification Using the Standard Curve Method

The standard curve method for absolute quantification is similar to the standard curve method for relative quantification, except the absolute quantities of the standards must first be known by some independent means.

Amplification Plot and Standard Curve
                              for Absolute Quantification
Figure 2: Amplification Plot and Standard Curve for Absolute Quantification
Critical Guidelines
The guidelines below are critical for proper use of the standard curve method for absolute quantification:
  • It is important that the DNA or RNA be a single, pure species. For example, plasmid DNA prepared from E. coli often is contaminated with RNA, which increases the A260 measurement and inflates the copy number determined for the plasmid.
  • Accurate pipetting is required because the standards must be diluted over several orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A260 value. This concentrated DNA or RNA must then be diluted 106–1012 -fold to be at a concentration similar to the target in biological samples.
  • The stability of the diluted standards must be considered, especially for RNA. Divide diluted standards into small aliquots, store at –80 °C, and thaw only once before use.

It is generally not possible to use DNA as a standard for absolute quantification of RNA because there is no control for the efficiency of the reverse transcription step.

The absolute quantities of the standards must first be known by some independent means. Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute standards. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA.

Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets -- from variable nonsense to publication quality data
Sean C. Taylor, Genevieve Laperriere, Hugo Germain
Scientific Reports 7, Article number: 2409 (2017)

Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment

Digital PCR Using the OpenArray® Real-Time PCR System
More info at Life Technologies

Digital PCR is a new approach to nucleic acid detection and quantification, which is a different method of absolute quantification and rare allele detection relative to conventional qPCR.

Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.

The OpenArray® Real-Time PCR System enables digital PCR experiments at a scale previously unattainable—in a single day, one user can generate >36,000 digital PCR data points on the OpenArray® Real-Time PCR System, without the use of robotics. Other features of the system include:
  • Accurate and sensitive—detect and count individual molecules to quantify viral load, gDNA, cDNA, plasmids, or next-generation sequencing libraries
  • Fast—produce up to 144 digital answers per 3 hour run
  • Flexible—enables use of your existing assays and has the capacity to test from one to 48 assay/sample dilutions per plate
  • Wide dynamic range—with as few as 64 data points per replicate group, a dilution series can easily be loaded into the TaqMan® OpenArray® Digital PCR Plate, expanding the range of sample concentrations which can be analyzed to produce a digital answer.
  • Intuitive and economical—software includes a Poisson calculator to design your individual digital PCR experiment, eliminating optimization time and minimizing sample usage

button Stilla Technologies
The Naica™ System: Discover the Simplicity of Crystal™Digital PCR
by Stilla Technologies

A simple and fast workflow: 2h30 time-to-result

Combining two digital PCR technology, Crystal Digital™ PCR is Stilla Technologies next generation solution that delivers results in 2h30 with less than 5 minutes hands-on time. The heart of Stilla’s Naica System™ is the microfluidic Sapphire Chip, fully integrating the 3 steps of Digital PCR (droplet formation, amplification and readout) in a single consumable. Using the Sapphire Chips, the sample is partitioned into a droplet crystal, i.e. a 2D monolayer of 30 000 droplets. The partitioning and amplification are both performed by the Naica Geode™ thermocycler while the Naica Prism3™ instrument offers high multiplexing capability by visualizing targets through 3 fluorescent channels (blue, red and green). Once the image acquisition has been performed, Stilla’s Crystal Miner™ analysis software enables the automatic identification of positive and negative droplets with an intuitive visual inspection and analysis of the Digital PCR experiment. From the number of droplets in each population, it gives the absolute concentration of the target sequences.

Stilla’s Crystal Digital PCR is compatible with both TaqMan probes and Eva Green chemistry.

In brief, The Naica System is:

Publications & Posters:
Application Notes:

Digital PCR - A breakthrough in quantitative PCR
by Bio-Rad

The QX200 Droplet Digital PCR system is Bio-Rad's second generation of PCR technology. Droplet Digital™ PCR (ddPCR™) provides an absolute measure of target DNA molecules with unrivaled accuracy, precision, and sensitivity. Applications include copy number variation, rare sequence detection, mutation detection, and gene expression analysis.

The QX200 ddPCR system lets you:

  • Enrich for rare target sequences
  • Detect small fold target differences
  • Determine copy number without a standard curve
Unparalleled performance:

=> Bio-Rad`s ddPCR Application Guide

=> Bio-Rad's ddPCR publication list

RainDrop Digital PCR from RainDance Technologies

RainDance Technologies RainDrop Digital PCR System shifts the PCR paradigm from a single color per marker approach to a more scalable and precise multi-color and intensity-per-marker method. This novel approach increases sensitivity by generating between 1 million and 10 million pico-liter sized droplets per lane, which is a 500 – 10,000x improvement over existing PCR methods. Since each droplet encapsulates a single molecule, researchers can quickly determine the absolute number of droplets containing specific target DNA and compare that to the amount of droplets with normal, background wild-type DNA. The RainDrop System is the third generation of RainDance’s droplet-based PCR instrumentation and uses the fourth generation of the company’s microfluidic chips.

The RainDrop System supports a number of important applications including low-frequency tumor allele detection, gene expression, copy number variation, and SNP measurement.

  • Superior sensitivity: Detect 1 mutant amongst 250,000 wild-type molecules with a lower limit of detection of 1 in more than 1,000,000.
  • Unprecedented multiplexing: Conduct up to 10 tests or more on the same sample using the single molecule multi-color detection technique.
  • Greater study design flexibility: Optimize number of PCR reactions based on your sensitivity AND multiplex requirements.
  • Closed-tube design: Ensure highest quality by eliminating contamination or carryover.
  • Proven picodroplet technology platform: Leverage a decade of experience with the same core technology found in RainDance’s Targeted Sequencing Systems.
  • Lowest cost per data point: Generate true digital answers with orders of magnitude more data per dollar.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR
Q. Zhong, S. Bhattacharya, S. Kotsopoulos, J. Olson, V. Taly, A.D. Griffiths, D.R. Link and J.W. Larson, Lab on a Chip 2011, DOI: 10.1039/C1LC20126C.

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics
D. Pekin, Y. Skhiri, J. Baret, D. Le Corre, L. Mazutis, C. Ben Salem, F. Millot, A. El Harrak, J. B. Hutchison, J.W. Larson, D.R. Link, P. Laurent-Puig, A.D. Griffiths and V. Taly, Lab on a Chip 2011, DOI: 10.1039/C1LC20128J.


Quantitative detection of circulating tumor DNA by droplet-based digital PCR
Poster presented at the American Association for Cancer Research (AACR) 2012:

Single molecule droplet-based digital PCR for high sensitivity detection of cancer biomarkers
Poster presented at the Advances in Genome Biology and Technology (AGBT) 2012

Single molecule digital PCR approach offers fundamental breakthroughs in multiplexing and sensitivity
Poster presented at the International Congress of Human Genetics (ICHG) 2011

New digital PCR reporting guidelines for molecular diagnostics.
National Measurement System
LGC scientists have collaborated on the development of best practice guidelines for the reporting of digital PCR data. Digital PCR is an emerging tool for DNA analysis showing great promise in new challenging areas of clinical diagnostics. These reporting guidelines provide a gold standard checklist of experimental information that should be included in all digital PCR publications to enable the research community to review and compare.

Evaluation of digital PCR for clinical diagnostics

Polymerase chain reaction (PCR), the method that amplifies DNA sequences by multiple rounds of thermal cycling, has been used extensively throughout molecular research over the last 25 years. It has many different applications including gene expression analysis, DNA mutation detection, cloning and sequencing and, as a result, has been widely used in clinical medicine, forensics and biological research. In 1999, cancer research pioneers Kenneth Kinzler and Bert Vogelstein of Johns Hopkins University (Baltimore), modified the standard PCR method in order to improve its sensitivity for cancer diagnostics. They named this method digital PCR.

The main principle of digital PCR is that a single sample is split into many fractions, all of which are subsequently analysed by a standard PCR method (Figure 1). The sample is fractionated by the simple process of dilution so that each fraction contains approximately one copy of DNA template or less. By isolating individual DNA templates this process effectively enriches DNA molecules that were present at very low levels in the original sample. Therefore, this method has applications for the detection of DNA mutations (or any other DNA/RNA targets) that are present at very low levels relative to a high background of normal DNA, for example the early stages of cancer development. The other major feature of digital PCR is that data are recorded as positive or negative (i.e. generation of an amplification product or not). Hence, the individual readout signals are qualitative or ‘digital’ in nature.

Figure 1 – The principle of digital PCR. a) Conventional PCR: The sample contains a heterogeneous mixture of DNA molecules where the target molecule (e.g. DNA mutation) is present at very low levels relative to a large background of normal DNA. Therefore, an average signal is acquired. b) Digital PCR: The sample is split into many fractions by dilution (approximately 1 copy of DNA or less per fraction) prior to PCR, thereby, enriching minority targets within individual reactions
Digital PCR can be performed manually, but it is labour-intensive, prone to pipetting errors and replication levels are limited by the format of plate used (i.e. 96 or 384 well). Alternative approaches to the multi-well plate method are now emerging. One of these is the use of a microfluidic sample handling system to split one sample into hundreds of individual reactions chambers which reside on an ‘integrated fluidic circuit’ or chip (Figure 2). This method also involves miniaturisation of the PCR whereby reactions are performed in nl volumes on the chip compared with ml volumes typically used in conventional PCR. This miniaturised process allows performance of close to 10000 individual PCR reactions per chip.

Figure 2 – Fluidigm BioMark digital chip. Microfluidic channels split each sample into 765 reaction chambers (inset) prior to standard thermal cycling and real time PCR analysis. Image courtesy of Fluidigm.
The purpose of this Chemical and Biological Metrology Programme project (DX3 - Performance standards for early diagnostic test methods) is to evaluate the application of sensitive emerging nucleic acid detection methods, such as digital PCR, for clinical diagnostics by investigating the reproducibility and robustness of measurements with regard to detection and quantitation of low copy number/minority biomarkers compared with the current gold standard method (i.e. conventional real time PCR).

Leukemia represents a relevant case study as it provides a difficult diagnostic challenge that requires detection of low numbers of leukemic cells amongst a majority of normal blood cells. Proof-of-principle experiments at LGC have measured the expression levels of the Bcr-Abl fusion transcript in a leukemia cell line. Figure 3 illustrates data from the BioMark digital chip. Panels A to E show decreasing expression levels (i.e. fewer positive samples) from a 2-fold serially diluted sample. Mixed leukemia/normal samples at different ratios are now being analysed to mimic the in vivo situation prior to the analysis of real clinical samples.

Figure 3 – An example of mRNA expression data generated by the Fluidigm BioMark digital PCR. mRNA expression of Bcr-Abl was determined using a Taqman ™ real time PCR assay and cDNA from the K562 leukemia cell line. Panel A-E represents a 2-fold dilution series of a cDNA sample. Panel F represents a negative control. Each panel consists of 765 individual PCR reactions which were analytical replicates of the same original sample. Red dots represent individual reactions where PCR products were detected. Red dots are simply counted per panel to give the total copy number of template in the sample (note: a correction factor is applied to account for occasional wells that may contain >1 copy/well). Numbers down the right-hand side represent estimated RNA copy number previously determined by conventional real time PCR using Bcr-Abl standards as well as the experimental copy number determined by digital PCR.

The advent of new commercial systems that facilitate the ease of use of digital PCR will further promote the application of this powerful method in other clinical and experimental research applications such as the detection of cancer biomarkers in body fluids such as blood, urine and sputum, as well as pre-natal screening biomarkers in maternal blood. Such methods hold promise towards non-invasive diagnostics.

Reference:  http://www.nmschembio.org.uk

Digital PCR:  a powerful new tool for noninvasive prenatal diagnosis?
Zimmermann BG, Grill S, Holzgreve W, Zhong XY, Jackson LG, Hahn S.
Prenat Diagn. 2008 28(12): 1087-1093.
Fluidigm Corporation, South San Francisco, USA.

Recent reports have indicated that digital PCR may be useful for the noninvasive detection of fetal aneuploidies by the analysis of cell-free DNA and RNA in maternal plasma or serum. In this review we provide an insight into the underlying technology and its previous application in the determination of the allelic frequencies of oncogenic alterations in cancer specimens. We also provide an indication of how this new technology may prove useful for the detection of fetal aneuploidies and single gene Mendelian disorders.

Non-invasive prenatal diagnosis by single molecule counting technologies
Chiu RW, Cantor CR, Lo YM.
Trends Genet. 2009 Jul;25(7): 324-331
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR, China.

Non-invasive prenatal diagnosis of fetal chromosomal aneuploidies and monogenic diseases by analysing fetal DNA present in maternal plasma poses a challenging goal. In particular, the presence of background maternal DNA interferes with the analysis of fetal DNA. Using single molecule counting methods, including digital PCR and massively parallel sequencing, many of the former problems have been solved. Digital mutation dosage assessment can detect the number of mutant alleles a fetus has inherited from its parents for fetal monogenic disease diagnosis, and massively parallel plasma DNA sequencing enables the direct detection of fetal chromosomal aneuploidies from maternal plasma. The analytical power of these methods, namely sensitivity, specificity, accuracy and precision, should catalyse the eventual clinical use of non-invasive prenatal diagnosis.

Digital polymerase chain reaction;  new diagnostic opportunities
European Pharmaceutical Review  - Genomics page 7-9;   published 22 February 2010
Jim Huggett 1 &  Daniel J Scott 2
1 Molecular and Cell Biology, LGC;  2 Project Manager, Research and Technology Division, LGC

LGC is an international science-based company located in South West London. A progressive and innovative enterprise, LGC operatesin socially responsible fields underpinning the health, safety and security of the public, and regulation and enforcement for UKgovernment departments and blue chip clients. Our products and services enable our customers to have a sound basis on whichto base their scientific and commercial decisions or conformity to international statutory and regulatory standards.

DNA diagnostics gets digitized
by Mikael Kubista and Anders Stahlberg
Drug Discovery Wold - Fall 2011

Quantitative real-time PCR (qPCR) has during the last two decades emerged as the preferred technology for nucleic acid analysis in routine as well as in research. qPCR has the sensitivity to detect a single molecule, the specificity to differentiate targets by a single nucleotide, and, because of its exponential nature, virtually unlimited dynamic range.

PCR’s next frontier
Nathan Blow reports.
PCR - the workhorse of modern molecular biology - is charging forward using both conventional and digital methods to explore single cells and even single molecules. Follow this report!

Emerging real-timePCR applications
Mikael Kubista
Drug Discovery World Summer 2008

Since its introduction on the commercial market little more than 10 years ago,real-time PCR has become the main technical platform for nucleic aciddetection in research and development, as well as in routine diagnostics. In2007 the real-time PCR market revenue in the US was estimated at $740million with annual growth of more than 10%. In this article latestdevelopments and future expectations are presented.

Video on Digital PCR by TATAA Biocenter
The video on Digital PCR describing latest platform at TATAA Biocenter. http://www.youtube.com/watch?v=qdFOpRbrYE0&noredirect=1
For more information how to do dPCR at TATAA Biocenter see http://www.tataa.com/Services/Projects.html#Digital

Concept of the limiting dilution assay

Limiting dilution analysis:  from frequencies to cellular interactions

Dozmorov I, Eisenbraun MD, Lefkovits I.
Immunol Today. 2000 Jan;21(1):15-8.
Dept of Pathology, University of Michigan, Ann Arbor, MI 48109, USA.

Limiting dilution analysis (LDA)1has gained widespread accept-ance as a tool for quantifyingcells that possess observablefunctional activities. Thoroughly plannedtitration experiments can produce straight-forward and interpretable single-hit kinetics,whereas analyses of unfractionated cellpopulations over a broader dilution rangeresult in data that deviate from linearity anddo not adhere to all-or-none functionality(e.g. virgin and memory CD4T cells2andothers3–10). However, by studying the factorsthat cause the deviation from linearity, theinteractions between different cell types inthe population can be identified and charac-terized. As a corollary, it follows that, alongwith quantification of desired cells, LDA al-lows an analysis of the regulatory processesthat underlie an observed activity.

End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: performance under optimal and suboptimal conditions
Ramachandran S, Xia GL, Ganova-Raeva LM, Nainan OV, Khudyakov Y.
J Virol Methods. 2008 Aug;151(2): 217-224
Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.

An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.

Digital PCR

Digital PCR
Vogelstein B, Kinzler KW.
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41.
The Howard Hughes Medical Institute and the Johns Hopkins Oncology Center, Baltimore, MD 21231, USA

The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant ras oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

Nanoliter scale PCR with TaqMan detection
Kalinina O, Lebedeva I, Brown J, Silver J.
Nucleic Acids Res. 1997 May 15;25(10):1999-2004.
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.

We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.

Digital polymerase chain reaction for characterisation of DNA reference materials
Somanath Bhat, , Kerry R. Emslie
Biomolecular Detection and Quantification; Available online 3 May 2016
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.

Principle and applications of digital PCR
Pohl G, Shih IeM.
Expert Rev Mol Diagn. 2004 Jan;4(1): 41-47
Department of Pathology, 418 North Bond Street, B-315, Baltimore, MD 21231, USA.

Digital PCR represents an example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis in cancer. The technique is to amplify a single DNA template from minimally diluted samples, therefore generating amplicons that are exclusively derived from one template and can be detected with different fluorophores or sequencing to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles). Thus, digital PCR transforms the exponential, analog signals obtained from conventional PCR to linear, digital signals, allowing statistical analysis of the PCR product. Digital PCR has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens, providing a promising molecular diagnostic tool for cancer detection. The scope of this article is to review the principles of digital PCR and its practical applications in cancer research and in the molecular diagnosis of cancer.

Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma
Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM.
Clin Chem. 2008 Oct;54(10): 1664-1672.
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

BACKGROUND: The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA.
METHODS: We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma.
RESULTS: Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was > or =2 times higher for all 3 trimesters of pregnancy than previously reported.
CONCLUSIONS: Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.

Digital PCR for the molecular detection of fetal chromosomal aneuploidy
Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK, Leung TY, Zee BC, Cantor CR, Chiu RW.
Proc Natl Acad Sci U S A. 2007 7;104(32): 13116-13121
Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, School of Public Health, The Chinese University of Hong Kong, Sha Tin, New Territories, Hong Kong Special Administrative Region, People's Republic of China.

Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis
Lo YM, Chiu RW.
Clin Chem. 2008 54(3): 461-466
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.

BACKGROUND: The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. The potential application of this technology for the noninvasive prenatal detection of fetal chromosomal aneuploidies is an aspect of this field that is being actively investigated. The main challenge of work in this area is the fact that cell-free fetal nucleic acids represent only a minor fraction of the total nucleic acids in maternal plasma. Methods and
RESULTS: We performed a review of the literature, which revealed that investigators have applied methods based on the physical and molecular enrichment of fetal nucleic acid targets from maternal plasma. The former includes the use of size fractionation of plasma DNA and the use of the controversial formaldehyde treatment method. The latter has been achieved through the development of fetal epigenetic and fetal RNA markers. The aneuploidy status of the fetus has been explored through the use of allelic ratio analysis of plasma fetal epigenetic and RNA markers. Digital PCR has been shown to offer high precision for allelic ratio and relative chromosome dosage analyses.
CONCLUSIONS: After a decade of work, the theoretical and practical feasibility of prenatal fetal chromosomal aneuploidy detection by plasma nucleic acid analysis has been demonstrated in studies using small sample sets. Larger scale independent studies will be needed to validate these initial observations. If these larger scale studies prove successful, it is expected that with further development of new fetal DNA/RNA markers and new analytical methods, molecular noninvasive prenatal diagnosis of the major chromosomal aneuploidies could become a routine practice in the near future.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy
Fan HC, Blumenfeld YJ, El-Sayed YY, Chueh J, Quake SR.
Am J Obstet Gynecol. 2009 200(5): 543.e1-7
Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, CA, USA.

OBJECTIVE: The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy.
STUDY DESIGN: Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes.
RESULTS: Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested.
CONCLUSION: Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

Digital PCR provides sensitive and absolute calibration for high throughput sequencing
White RA 3rd, Blainey PC, Fan HC, Quake SR.
BMC Genomics. 2009 Mar 19;10:116.
Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA.

BACKGROUND: Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
RESULTS: We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
CONCLUSION: The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays
Wang J, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, Jones RC, Ma PC.
Clin Chem. 2010 Apr;56(4):623-32. Epub 2010 Mar 5.
Fluidigm Corporation, South San Francisco, CA, USA.

BACKGROUND: The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable.
METHODS: We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.
RESULTS: The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors.
CONCLUSIONS: The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.

Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR
McCaughan F, Darai-Ramqvist E, Bankier AT, Konfortov BA, Foster N, George PJ, Rabbitts TH, Kost-Alimova M, Rabbitts PH, Dear PH.
J Pathol. 2008 216(3): 307-316
Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, The Rayne Institute, London WC1E 6JJ, UK.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Single-molecule genomics
McCaughan F, Dear PH.
J Pathol. 2010 Jan;220(2):297-306.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

The term 'single-molecule genomics' (SMG) describes a group of molecular methods in which single molecules are detected or sequenced. The focus on the analysis of individual molecules distinguishes these techniques from more traditional methods, in which template DNA is cloned or PCR-amplified prior to analysis. Although technically challenging, the analysis of single molecules has the potential to play a major role in the delivery of truly personalized medicine. The two main subgroups of SMG methods are single-molecule digital PCR and single-molecule sequencing. Single-molecule PCR has a number of advantages over competing technologies, including improved detection of rare genetic variants and more precise analysis of copy-number variation, and is more easily adapted to the often small amount of material that is available in clinical samples. Single-molecule sequencing refers to a number of different methods that are mainly still in development but have the potential to make a huge impact on personalized medicine in the future.

Microfluidic digital PCR enables multigene analysis of individual environmental bacteria
Ottesen EA, Hong JW, Quake SR, Leadbetter JR.
Science. 2006 314(5804): 1464-1467
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs
Pradervand S, Weber J, Lemoine F, Consales F, Paillusson A, Dupasquier M, Thomas J, Richter H, Kaessmann H, Beaudoing E, Hagenbüchle O, Harshman K.
Biotechniques. 2010 48(3): 219-222
Genomic Technologies Facility, Center for Integrative Genomics, University of Lausanne, Genopode Building, Lausanne, Switzerland

Profiling microRNA (miRNA) expression is of widespread interest given the critical role of miRNAs in many cellular functions. Profiling can be achieved via hybridization-based (microarrays), sequencing-based, or amplification-based (quantitative reverse transcription-PCR, qPCR) technologies. Among these, microarrays face the significant challenge of accurately distinguishing between mature and immature miRNA forms, and different vendors have developed different methods to meet this challenge. Here we measure differential miRNA expression using the Affymetrix, Agilent, and Illumina microarray platforms, as well as qPCR (Applied Biosystems) and ultra high-throughput sequencing (Illumina). We show that the differential expression measurements are more divergent when the three types of microarrays are compared than when the Agilent microarray, qPCR, and sequencing technology measurements are compared, which exhibit a good overall concordance.

Amplification-free digital gene expression profiling from minute cell quantities
Ozsolak F, Ting DT, Wittner BS, Brannigan BW, Paul S, Bardeesy N, Ramaswamy S, Milos PM, Haber DA.
Nat Methods. 2010 7(8):619-21. Epub 2010 Jul 18.
Helicos BioSciences Corporation, Cambridge, Massachusetts, USA

Generating reliable expression profiles from minute cell quantities is critical for scientific discovery and potential clinical applications. Here we present low-quantity digital gene expression (LQ-DGE), an amplification-free approach involving capture of poly(A)(+) RNAs from cellular lysates onto poly(dT)-coated sequencing surfaces, followed by on-surface reverse transcription and sequencing. We applied LQ-DGE to profile malignant and nonmalignant mouse and human cells, demonstrating its quantitative power and potential applicability to archival specimens. Amplification-free digital gene expression profiling from minute cell quantities.

Digital transcriptome profiling from attomole-level RNA samples
Ozsolak F, Goren A, Gymrek M, Guttman M, Regev A, Bernstein BE, Milos PM.
Genome Res. 2010 Apr;20(4): 519-525
Helicos BioSciences Corporation, Cambridge, MA 02139, USA

Accurate profiling of minute quantities of RNA in a global manner can enable key advances in many scientific and clinical disciplines. Here, we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput sequencing-based technique allowing whole transcriptome surveys from subnanogram RNA quantities in an amplification/ligation-free manner. LQ-RNAseq involves first-strand cDNA synthesis from RNA templates, followed by 3' polyA tailing of the single-stranded cDNA products and direct single molecule sequencing. We applied LQ-RNAseq to profile S. cerevisiae polyA+ transcripts, demonstrate the reproducibility of the approach across different sample preparations and independent instrument runs, and establish the absolute quantitative power of this method through comparisons with other reported transcript profiling techniques and through utilization of RNA spike-in experiments. We demonstrate the practical application of this approach to define the transcriptional landscape of mouse embryonic and induced pluripotent stem cells, observing transcriptional differences, including over 100 genes exhibiting differential expression between these otherwise very similar stem cell populations. This amplification-independent technology, which utilizes small quantities of nucleic acid and provides quantitative measurements of cellular transcripts, enables global gene expression measurements from minute amounts of materials and offers broad utility in both basic research and translational biology for characterization of rare cells.

Single-molecule sequencing of an individual human genome
Pushkarev D, Neff NF, Quake SR.
Nat Biotechnol. 2009 27(9): 847-852
Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA.

Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.

Digital PCR on a SlipChip
Shen F, Du W, Kreutz JE, Fok A, Ismagilov RF.
Lab Chip. 2010 10(20):2666-72. Epub 2010 Jul 1.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 E 57th St, Chicago, Illinois 60637, USA

This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.

Somatic deletion of the NF1 gene in a neurofibromatosis type 1-associated malignant melanoma demonstrated by digital PCR
Rübben A, Bausch B, Nikkels A.
Mol Cancer. 2006 Sep 10;5:36.
Department of Dermatology, University Hospital RWTH Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany

BACKGROUND: Neurofibromatosis type 1 (NF1) is the most common hereditary neurocutaneous disorder and it is associated with an elevated risk for malignant tumors of tissues derived from neural crest cells. The NF1 gene is considered a tumor suppressor gene and inactivation of both copies can be found in NF1-associated benign and malignant tumors. Melanocytes also derive from neural crest cells but melanoma incidence is not markedly elevated in NF1. In this study we could analyze a typical superficial spreading melanoma of a 15-year-old boy with NF1 for loss of heterozygosity (LOH) within the NF1 gene. Neurofibromatosis in this patient was transmitted by the boy's farther who carried the mutation NF1 c. 5546 G/A.
RESULTS: Melanoma cells were isolated from formalin-fixed tissue by liquid coverslip laser microdissection. In order to obtain statistically significant LOH data, digital PCR was performed at the intragenic microsatellite IVS27AC28 with DNA of approx. 3500 melanoma cells. Digital PCR detected 23 paternal alleles and one maternal allele. Statistical analysis by SPRT confirmed significance of the maternal allele loss.
CONCLUSION: To our knowledge, this is the first molecular evidence of inactivation of both copies of the NF1 gene in a typical superficial spreading melanoma of a patient with NF1. The classical double-hit inactivation of the NF1 gene suggests that the NF1 genetic background promoted melanoma genesis in this patient.

Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution
Suzanne Weaver, Simant Dube, Alain Mir, Jian Qin, Gang Sun, Ramesh Ramakrishnan, Robert C. Jones, Kenneth J. Livak
Methods. 2010 Apr;50(4):271-6. Epub 2010 Jan 15.
Fluidigm Corporation, 7000 Shoreline Court, Suite 100, South San Francisco, CA 94080, USA.

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.

One bacterial cell, one complete genome
Woyke T, Tighe D, Mavromatis K, Clum A, Copeland A, Schackwitz W, Lapidus A, Wu D, McCutcheon JP, McDonald BR, Moran NA, Bristow J, Cheng JF.
PLoS One. 2010 5(4): e10314
Department of Energy Joint Genome Institute, Walnut Creek, California, USA

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

Spinning disk platform for microfluidic digital polymerase chain reaction.
Sundberg SO, Wittwer CT, Gao C, Gale BK.
Anal Chem. 2010 82(4): 1546-1550.
University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA

An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.

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