Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer

Fred Russell Kramer 1,
1Rutgers University, USA; 


PCR assays are the most rapid, most sensitive, and least expen- sive way to assess the abundance of mutant DNA fragments present in liquid biopsies. “SuperSelective” PCR primers, due to their unique design, are extraordinarily specific, able to selectively initiate the synthesis of amplicons on ten mutant DNA fragments in the presence of 1,000,000 wild-type DNA fragments. Sets of SuperSe- lective primers, each possessing unique 5′-tag sequences, enable the amplicons generated from each mutant to be distinguished by differently colored molecular beacon probes. And the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant DNA fragment to be assessed by determining the difference between its threshold value and the threshold value of the reference gene.

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A statistical contribution to the uncertainty of concentration measurements using digital PCR

Andreas Kummrow, 1, Annabell Plauth 1,*, Martin Hussels 1
1 Physikalisch-Technische Bundesanstalt, Germany; 

Measurement of the concentration of biological entities using quantitative PCR (qPCR) usually results in a large spread of results obtained in interlaboratory comparisons. As such, deviations in the range of ±0.6 on a logarithmic scale (base 10) are currently considered acceptable in the external quality assurance of quan- tification of viruses like HBV, HCV, and HIV-1 in Germany. Notably, a considerable contribution to this variance may originate from pre-analytical steps and variable efficacy of the assays utilized. Technical limitations of the instrument remain as a source of uncertainty of measurement, even if a perfect extraction and amplification is assumed. Digital PCR (dPCR) does neither require reference genes nor calibration material to quantify the concentra- tion in biological samples and thus might substantially improve the measurement uncertainty in concentration measurements com- pared to qPCR. An apparent physical limitation for concentration measurements using dPCR is the uncertainty of the reaction vol- ume. Recent studies quote an (unexpanded) uncertainty of around 2% using microscopic measurements of the average size of the respective reaction volume. In this study, we discuss the effect of the limited number of repeat reactions in dPCR on the uncertainty of concentration. It is generally accepted that a Poisson correc- tion must be applied to the concentration determined by dPCR, i.e. for an average of reactive DNA molecules in each reaction chamber, the probability of a positive reaction is p = (1 − exp(− )). Thus, for N reactions one expects A=pN positive reactions and B = (1 − p)N negative reactions. The statistical uncertainty for posi- tive (and negative) reactions does not follow a normal distribution. We show, that the uncertainty of finding positive reactions has to be calculated based on the standard deviation of the binomial distribution, which yields u = (AB/N)1/2 . This result is used to calcu- late the minimum number of reactions required for a given DNA concentration and targeted statistical uncertainty u of the con- centration. Additionally, the implications of this result on setting up dPCR experiments is discussed. We calculated the sweet spot for the fewest number of reactions around = 1.5, which falls to = 1 for large statistical uncertainties (>10%). Even in this case, at least 4000 reactions are required to reach a statistical uncertainty of 2%. To maintain this uncertainty for a dynamic range of 3 orders of mag- nitude requires using more than 300000 reactions. The research receives funding from the EMPIR project HLT-07 AntiMicroResist. The EMPIR programme is co-financed by the Participating States and the European Union’s Horizon 2020 research and innovation programme.

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dpcReport: Web server and software suite for unified analysis of digital PCRs and digital assays

Michał Burdukiewicz 1,*,Jim Hugget2, Alexandra Whale2, Boris Fehse3, Piotr Sobczyk4, Paweł Mackiewicz1, Andrej-Nikolai Spiess5, Peter Schierack 6 , Stefan Rödiger 6
1 Department of Genomics, University of Wrocław, Poland
2 Molecular and Cell Biology Team, LGC, Teddington, United Kingdom
3 Research Department Cell and Gene Therapy, Department of SCT, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
4 Faculty of Pure and Applied Mathematics, Wrocław University of Science and Technology, Wrocław, Poland
5 University Medical Center Hamburg-Eppendorf, Hamburg, Germany
6 Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany

Digital PCR (dPCR) is a variant of PCR, where the PCR ampli- fication is conducted in multiple small volume reactions (termed partitions) instead of a bulk. The dichotomous status of each par- tition (positive or negative amplification) is used for absolute quantification of the template molecules by Poisson transforma- tion of the proportion of positive partitions. The vast expansion of dPCR technology and its applications has been followed by the development of statistical data analysis methods. Yet, the software landscape is scattered, consisting of scripts in various programming languages, web servers with narrow scopes or closed source vendor software packages, that are usually tightly tied to their platform. This leads to unfavourable environments, as results from differ- ent platforms, or even from different laboratories using the same platform, cannot be easily compared with one another.
To address these problems, we developed the dpcReport web server that provides an open-source tool for the analysis of dPCR data. dpcReport provides a streamlined analysis framework to the dPCR community that is compatible with the data output (e.g., CSV, XLSX) from different dPCR platforms (e.g., Bio-Rad QX100/200, Biomark). This goes beyond the basic dPCR data analysis with vendor-supplied softwares, which is often limited to the compu- tation of the mean template copy number per partition and its uncertainty. dpcReport gives users more control over their data analysis and they benefit from standardization and reproducible analysis.
Our web server analyses data regardless of the platform ven- dor or type (droplet or chamber dPCR). It is not limited to the commercially available platforms and can also be used with exper- imental systems by importing data through the universal REDF format, which follows the IETF® RFC 4180 standard. dpcReport provides users with advanced tools for data quality control and it incorporates statistical tests for comparing multiple reactions in an experiment, currently absent in many dPCR-related soft- ware tools. dpcReport provides users with advanced tools for data quality control. The conducted analyses are fully integrated within extensive and customizable interactive HTML reports including figures, tables and calculations. To improve reproducibility and transparency, a report may include snippets in the programming language R enabling an exact reproduction of the analysis per- formed by dpcReport through functions from the dpcR package. Our software follows the standardized dPCR nomenclature of the dMIQE guidelines. Since the vast functionality offered by dpcRe- port may be overwhelming at first, our web server is extensively documented.
The server is freely accessible at: http://www.smorfland.uni.

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Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices

Maja Sidstedt 1,2,*,Erica L. Romsos3, Ronny Hedell2,4, Carolyn R. Steffen3, Peter M. Vallone3, Peter Rådström1, Johannes Hedman 1,2
1Applied Microbiology, Department of Chemistry, Lund University, PO Box 124, 221 00 Lund, Sweden
2 Swedish National Forensic Centre, 581 94 Linköping, Sweden
3 Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899-8314, United States
4 Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 41296 Gothenburg, Sweden; 

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions [1]. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA con- centration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds [2,3]. In this study, we present how certain inhibitors disturb dPCR quantification and sug- gest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to eluci- date the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance dif- fers greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory- substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of posi- tive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By under- standing more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.
1 M. Baker, Digital PCR hits its stride, Nat. Methods 9 (2012) 541–544.
2 C. Coudray-Meunier, A. Fraisse, S. Martin-Latil, L. Guillier, S. Delannoy, P. Fach,
S. Perelle, A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples, Int. J. Food Microbiol. 201 (2015) 17–26.
3 T. Hoshino, F. Inagaki, Molecular quantification of environmental DNA using microfluidics and digital PCR, Syst. Appl. Microbiol. 35 (2012) 390–395.

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Automated cell picking and single cell digital PCR focusing on mitochondrial transfer

David Svec 1,2,
1Institute of Biotechnology AS CR, Prague
2 TATAA Biocenter, Czech Republic; 

Mitochondria are unique organelles comprising their own genetic information in the form of mitochondrial DNA (mtDNA). Cell type mtDNA heteroplasmy was reported before as a common event observed not only in patients with mitochondrial diseases but also in healthy individuals. In parallel reports show that mitochon- dria move between mammalian cells. We performed single cell expression profiling focusing on horizontal mitochondrial transfer. Using automated cell picking and single cell digital PCR, we showed that generation of tumors in syngeneic mice by cells devoid of mito- chondrial (mt) DNA ( 0 cells) is linked to acquisition of the host mtDNA, leading to the normalization of mitochondrial respiration.

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Partition volume in dPCR – Monodispersity not really important

Joel Tellinghuisen 1,
1Vanderbilt University, USA; 

The role of partition volume variability, or polydispersity, in digital polymerase chain reaction methods is examined through formal considerations and Monte Carlo simulations. Contrary to intuition, polydispersity causes little precision loss for low average copy number per partition and can actually improve precision when exceeds ∼4. It does this by negatively biasing the esti- mates of , thus increasing the number of negative (null) partitions N0. In keeping with binomial statistics, this increases the relative precision of N0 and hence of the biased estimate m of . Below = 1, the precision loss and the bias are both small enough to be negligible for many applications. For higher the bias becomes more important than the imprecision, making accuracy depend- ent on knowledge of the partition volume distribution function. This information can be gained with optical microscopy or through calibration with reference materials.

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Droplet digital PCR for MYD88L265P mutation detection in Waldenström Macroglobulinemia: Minimal residual disease monitoring and characterization on circulating free DNA

Daniela Drandi 1,*, Elisa Genuardi1,
Irene Dogliotti1, Martina Ferrante1, Cristina Jimenez2, Francesca Guerrini3, Mariella Lo Schirico1, Vittorio Muccio1, Barbara Mantoan1, Milena Gilestro4, Paola Omedè4, Sara Galimberti3,
Lorella Orsucci4, Federica Cavallo1, Ramon Garcia Sanz2, Mario Bocca 1,4,Marco Ladetto5, Simone Ferrero1
1 Department of Molecular Biotechnologies and Health Sciences, Hematology Division, University of Torino, Italy
2 Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain
3 Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
4 Division of Hematology, ASO S.Giovanni Battista, Torino, Italy
5 Division of Hematology, Az Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy; 

Background: MYD88L265P mutation might represent an ideal marker for minimal residual disease (MRD) monitoring in Walden- ström Macroglobulinemia (WM). However, the conventional allele-specific quantitative PCR (ASqPCR) is not sensitive enough for MRD monitoring on peripheral blood (PB), harboring low concentrations of tumor cells. Besides, cell-free DNA (cfDNA) is increasingly used for mutational studies. We set up a new, highly sensitive, droplet digital PCR (ddPCR) assay for MYD88L265P detec- tion and described: 1) its feasibility for mutation screening and MRD monitoring in bone marrow (BM) and PB; 2) its application for mutational studies on cfDNA.
Methods: BM, PB and plasma from local series of WM, IgG- lymphoplasmacytic lymphoma (LPL) and IgM-MGUS patients (pts) were collected at baseline and during follow-up (FU). 40 healthy subjects were used as negative controls. Genomic DNA (gDNA) and plasmatic cfDNA were extracted by Maxwell RSC system (Promega). MYD88L265P was assessed on gDNA (100 ng) and cfDNA (from 1 ml of plasma) by a custom ddPCR assay on a QX100 System (Bio-Rad). For comparison ASqPCR was assessed on gDNA (100 ng), as described [Xu L, 2013]. MYD88L265P cut-off was settled based on the healthy samples background level. IGH-based MRD analysis was performed as described [Drandi D, 2015].
Results: Sensitivity of ddPCR versus ASqPCR was assessed on a ten-fold serial dilution standard curve. Whereas ASqPCR con- firmed the sensitivity of 1.00E−03, ddPCR reached a sensitivity up to 5.00E−05. Overall, 291 samples from 148 pts, 194 base- line (128 BM, 66 PB) and 97 follow-up (43 BM and 54 PB), were analyzed. 123/128 (96.1%) diagnostic BM and 47/66 (71.2%) PBsamples scored positive for MYD88L265P (BM median 3.6%, range: 0.02-72.6%: PB median 0.3%, range: 0.01-27.8%). 11/46 (24%) pts with both BM/PB collected at diagnosis showed a positive/negative match. Concordances between ddPCR and qPCR methods were investigate on 100 samples (60 BM, 40 PB) and overall a good concordance was observed (p=0.0005). Of note the majority of discordances were observed in the follow-up samples. Moreover, to investigate whether MYD88L265P ddPCR could be used for MRD monitoring we compared it to the gold standard IGH-based MRD assay in baseline and FU samples (23 BM, 15 PB) from 10 pts. The comparison showed highly superimposable results between meth- ods. Finally, ddPCR performed on cfDNA from 60 plasma samples showed 1 log higher median levels of MYD88L265P mutation by mutation in plasma (1.4%, range 0–72.2%) compared to PB (0.1%, range: 0.01–27.8%).
Conclusion: ddPCR is a feasible and highly sensitive assay for mutational screening and MRD monitoring in WM, particularly in samples harboring low concentrations of circulating tumor cells. Moreover, plasmatic cfDNA represents a promising tissue source and might be an attractive, less invasive alternative to PB or BM for MYD88L265P detection.

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How to monitor analytical and technical factors influencing qPCR

Jens Bjorkman 1,
1TATAA Biocenter, Sweden

qPCR is today a mature and most established method. Still it is easy to neglect the underlying factors that may compromise the results. With an easy-to-use highly automated qPCR instrument in every lab, scientist can (if they choose) generate data with mini- mum focus on the pre-qPCR parameters that may have significant impact on the data. It is not difficult to produce Cq-values, but how do we know that they truly reflect the amount of target that was actually present in the original sample or even in vivo? As described in the MIQE guidelines proper controls should be included in order to identify and in some cases correct for the bias caused by pre- analytical parameters and technical variation. I will describe quality control measures to test for degradation of RNA, RT and PCR inhibi- tion, genomic DNA background, and provide means to compensate for interplate variation to perform high quality quantitative PCR measurements.

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Influence of PCR consumables on the accuracy of real-time PCR experiments and NGS sample preparation

Gerrit Gutzke, Hanna Oldfield *, Emily Flowers
14titude Ltd, Germany 


Classical PCR and qPCR plates are one-component plates made out of polypropylene (PP). PP is the best plastic material for PCR tubes as it is chemically inert and allows for the production of ultra- thin tube walls which is important for fast temperature transfer. While PP has become the standard material for PCR consumables some of its properties question its suitability for applications like qPCR and NGS:
The material characteristics of PP exhibit a Vicat Softening Tem- perature (VST) of 90◦C and a coefficient of thermal expansion of 180 × 10−6 K−1 which are potential weaknesses for its usage at typical (q)PCR temperatures.
When used for (q)PCR, not only do the plates soften during the denaturation step, but measurements also show that the plates expand by up to 2 mm in the diagonal plane (from room temperature to 95 ◦ C) and they shrink again as the temperature decreases. Therefore, the plate will undergo expansion and contraction in every cycle, placing significant tension on the plate seals. As a result, contact between the seal and plate will be particularly weakened in the corner positions and outer rows leading to evaporation from the plate in these areas, while centre wells will only be affected minimally. This differential evaporation effect is especially eminent when adhesive seals are used (as opposed to heat seals).
Evaporation has a significant effect on the reaction conditions resulting in noticeable effects, especially for qPCR. Identical sam- ples can exhibit significant differences in their Ct values, depending on their position on the plate. This often remains unnoticed as trip- licates are typically placed in neighbouring wells which are affected by similar levels of evaporation.
A solution to the problem of evaporation related qPCR inaccu- racies is the usage of two-component plates. These plates consist of tubes made out of PP but a frame made out of polycarbonate (PC). PC does not show significant temperature-dependant expan- sion and contraction as the VST of this material is 147◦C and the coefficient of thermal expansion is 70 × 10−6 K−1 .
The ongoing trend to continually reduce DNA concentrations can make additional modifications or choices necessary. DNA has been shown to bind to PP tubes in trace amounts, especially in highly ionic conditions. Different PP polymers are used for the pro- duction of PCR consumables as they differ in their characteristics, such as surface charge. As a result, different PCR plates bind DNA in varying amounts. Furthermore, the commercially used term “low- binding” is poorly defined.
DNA binding to PP surfaces has not been reported as an issue for PCR/qPCR as DNA stuck to the walls will be released during denaturation steps. However, NGS protocols contain a number of transfer steps from one tube to another and ultra-low DNA binding characteristics may be required if only trace amounts of nucleic acids are used.

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Validation of a qPCR method – Determining limit of detection, limit of quantification and dynamic range

Mikael Kubista 1, Robert Sjöback1, Jens Björkman1, Björn Sjögreen2, Lucas Luke Linz3, Amin Forootan2

1 TATAA Biocenter AB, Sweden
2 MultiD Analyses AB, Sweden
3 LGC Douglas Scientific, USA 

Quantitative Real-Time Polymerase Chain Reaction (qPCR) is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD), the limit of quantification (LoQ) and the Dynamic Range as appli- cable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies and adapted to the logarithmic response characteristic of qPCR.
The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments
SA Bustin, V Benes, JA Garson, J Hellemans, J Huggett, M Kubista, Clinical chemistry 55 (4), 611-622
Prime time for qPCR–raising the quality bar M Kubista Eur. Pharm. Rev. 19, 60-67

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