A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers.

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer's disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap® DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.

Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using Digital PCR.

The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology "toolkit" of methods and materials to support the current standardization efforts.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes.

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.

An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.

Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

Control Materials and Digital PCR Methods for Evaluation of Circulating Cell-Free DNA Extractions from Plasma.

Cell-free DNA is an accessible source of genetic material found naturally in plasma that could be used in many diagnostic applications. Translation of cfDNA analysis methods from research laboratories into the clinic would benefit from controls for monitoring the efficiency of patient sample purification and for quality control of the whole workflow from extraction through to analysis. Here we describe two types of control materials that can be "spiked" into plasma samples to monitor and evaluate different aspects of the workflow. The first control material is an internal control that enables evaluation of extraction efficiency, fragment size bias, and sample inhibition. The second control material serves as a parallel quality control material for measurement of specific genetic targets such as tumor mutations.

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR.

ABTRACT: Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides 'absolute' quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

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.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Fundamentals of multiplexing with digital PCR.

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.

Digital PCR dynamic range is approaching that of real-time quantitative PCR.

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.

The variability and reproducibility of whole genome sequencing technology for detecting resistance to anti-tuberculous drugs.

BACKGROUND: The emergence of resistance to anti-tuberculosis drugs is a serious and growing threat to public health. Next-generation sequencing is rapidly gaining traction as a diagnostic tool for investigating drug resistance in Mycobacterium tuberculosis to aid treatment decisions. However, there are few little data regarding the precision of such sequencing for assigning resistance profiles. METHODS: We investigated two sequencing platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (TBProfiler, Mykrobe predictor) using a well characterised reference strain (H37Rv) and clinical isolates from patients with tuberculosis resistant to up to 13 drugs. Results were compared to phenotypic drug susceptibility testing. To assess analytical robustness individual DNA samples were subjected to repeated sequencing. RESULTS: The MiSeq and Ion PGM systems accurately predicted drug-resistance profiles and there was high reproducibility between biological and technical sample replicates. Estimated variant error rates were low (MiSeq 1 per 77 kbp, Ion PGM 1 per 41 kbp) and genomic coverage high (MiSeq 51-fold, Ion PGM 53-fold). MiSeq provided superior coverage in GC-rich regions, which translated into incremental detection of putative genotypic drug-specific resistance, including for resistance to para-aminosalicylic acid and pyrazinamide. The TBProfiler bioinformatics pipeline was concordant with reported phenotypic susceptibility for all drugs tested except pyrazinamide and para-aminosalicylic acid, with an overall concordance of 95.3%. When using the Mykrobe predictor concordance with phenotypic testing was 73.6%. CONCLUSIONS: We have demonstrated high comparative reproducibility of two sequencing platforms, and high predictive ability of the TBProfiler mutation library and analytical pipeline, when profiling resistance to first- and second-line anti-tuberculosis drugs. However, platform-specific variability in coverage of some genome regions may have implications for predicting resistance to specific drugs. These findings may have implications for future clinical practice and thus deserve further scrutiny, set within larger studies and using updated mutation libraries.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients.

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR.

Digital PCR (dPCR) offers absolute quantification through the limiting dilution of template nucleic acid molecules and has the potential to offer high reproducibility. However, the robustness of dPCR has yet to be evaluated using complex genomes to compare different dPCR methods and platforms. We used DNA templates from the pathogen Mycobacterium tuberculosis to evaluate the impact of template type, master mixes, primer pairs and, crucially, extraction methods on dPCR performance. Performance was compared between the chip (BioMark) and droplet (QX100) formats. In the absence of any external calibration, dPCR measurements were generally consistent within ∼2-fold between different master mixes and primers. Template DNA integrity could influence dPCR performance: high molecular weight gDNA resulted in underperformance of one master mix, while restriction digestion of a low molecular weight sample also caused underestimation. Good concordance (≤1.5-fold difference) was observed between chip and droplet formats. Platform precision was in agreement with predicted Poisson error based on partition number, but this was a minor component (<10%) of the total variance when extraction was included. dPCR offers a robust reproducible method for DNA measurement; however, as a predominant source of error, the process of DNA extraction will need to be controlled with suitable calibrators to maximize agreement between laboratories.