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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.

Current commercially available digital PCR (dPCR) systems and assays are capable of detecting individual target molecules with considerable reliability. As tests are developed and validated for use on clinical samples, the need to understand and develop robust statistical analysis routines increases. This chapter covers the fundamental processes and limitations of detecting and reporting on single molecule detection. We cover the basics of quantification of targets and sources of imprecision. We describe the basic test concepts: sensitivity, specificity, limit of blank, limit of detection, and limit of quantification in the context of dPCR. We provide basic guidelines how to determine those, how to choose and interpret the operating point, and what factors may influence overall test performance in practice.

Current commercially available digital PCR (dPCR) systems and assays are capable of detecting individual target molecules with considerable reliability. As tests are developed and validated for use on clinical samples, the need to understand and develop robust statistical analysis routines increases. This chapter covers the fundamental processes and limitations of detecting and reporting on single molecule detection. We cover the basics of quantification of targets and sources of imprecision. We describe the basic test concepts: sensitivity, specificity, limit of blank, limit of detection, and limit of quantification in the context of dPCR. We provide basic guidelines how to determine those, how to choose and interpret the operating point, and what factors may influence overall test performance in practice.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

A central theme in neural coding concerns the role of response variability and noise in determining the information transmission of neurons. This issue was investigated in single cells of the lateral geniculate nucleus of barbiturate-anesthetized cats by quantifying the degree of precision in and the information transmission properties of individual spike train responses to full field, binary (bright or dark), flashing stimuli. We found that neuronal responses could be highly reproducible in their spike timing (approximately 1-2 ms standard deviation) and spike count (approximately 0.3 ratio of variance/mean, compared with 1.0 expected for a Poisson process). This degree of precision only became apparent when an adequate length of the stimulus sequence was specified to determine the neural response, emphasizing that the variables relevant to a cell's response must be controlled to observe the cell's intrinsic response precision. Responses could carry as much as 3.5 bits/spike of information about the stimulus, a rate that was within a factor of two of the limit the spike train could transmit. Moreover, there appeared to be little sign of redundancy in coding: on average, longer response sequences carried at least as much information about the stimulus as would be obtained by adding together the information carried by shorter response sequences considered independently. There also was no direct evidence found for synergy between response sequences. These results could largely, but not entirely, be explained by a simple model of the response in which one filters the stimulus by the cell's impulse response kernel, thresholds the result at a fairly high level, and incorporates a postspike refractory period.

Targeted therapies in many cancers have allowed unprecedented progress in the treatment of disease. However, routine implementation of genomic testing is constrained due to: 1) limited amounts of sample (pg–ng range) per biological specimen, 2) diagnostic turnaround time and workflow, 3) cost, and 4) difficulties in detection of mutational loads below 5%. KRAS is mutated in approximately 40% of colorectal cancers (CRCs). The majority of mutations affect codons 12, 13, and 61 and indicate a negative response to anti–epidermal growth factor receptor (EGFR) therapy. To optimize therapy strategies for personalized care, it is critical to rapidly screen patient samples for the presence of multiple KRAS mutations.

Background Droplet digital PCR (dPCR) is a potentially appealing technology for quantitative detection of specific mutations with simultaneous measurement of the reference gene and without the need for standard curves. This makes dPCR particularly suitable for minimal residual disease (MRD) diagnostics in chronic myeloid leukaemia. However, a limitation of the dPCR assays compared to standard quantitative PCR (qPCR) is the background (termed lower limit of blank, LoB) of 1 or 2 positive droplets for BCR-ABL (Franke et al, ASH 2015, Cross et al, Leukemia 2016). The resulting false positive rate (FPR) limits the sensitivity and the ability of the test to detect deep molecular remissions. Aims The aim was to establish BCR-ABL (b2/a2 and/or b3/a2 transcript) dPCR assays with a sensitivity at least equivalent to that of qPCR, in which Methods One assay (Assay 1) was developed in Leipzig and another (Assay 2) by Bio-Rad laboratories. Sample preparation was identical for both assays up to the stage of reverse transcription (RT). Assay 1 used routine local RT procedure followed by a duplex BCR-ABL / ABL dPCR reaction modified from the M-BCR-ABL PCR published by Jennings et al. 2014. We tested ABL primer combinations from exons 4, 8, 10, and 11 generating a range of product lengths and optimised quenchers and PCR conditions to maximise the separation between positive and negative droplets. Assay 2 consisted of a reverse transcriptase reaction followed by a duplex PCR specific for ABL and both the b2/a2 and b3/a2 transcripts of BCR-ABL. Digital droplet PCR was performed in both cases using the Bio-Rad QX200 system. Background noise and cross hybridisation were assessed in non-template controls (NTC), BCR-ABL negative cell lines and healthy wild-type donor samples. In addition, assay 2 was tested on 40 CML patient samples with MRD levels ranging from BCR-ABL negative to 1% BCR-ABL/ABL as defined by standard qPCR. Results Assay 1 yielded a FPR of 4% (4/94) in NTC with 1 positive droplet for BCR-ABL and a FPR of 3% for ABL (n = 3/94), resulting in a specificity of >95% and a LOB of 0. The BCR-ABL FPR using wild type target (HEK 293T cell line and healthy donors) was 5% (5/92 and 2/37, respectively). Assay2: The specificity was >95% for both BCR-ABL and ABL in NTC and wild type samples. Extensive NTC analysis yielded no false positives for BCR-ABL PCR (n = 0/176; LoB = 0) and 1% false positives in ABL PCR (n = 2/176; 1-2 positive droplets, LoB = 0). The BCR-ABL negative cell line was confirmed negative for BCR-ABL PCR (n = 44), while the BCR-ABL assays of healthy donors were positive in 2% (n = 5/234) with 1 positive droplet per positive sample. The BCR-ABL copy numbers of the 40 CML samples were lower by dPCR than by RT-PCR (median 3/6) with similar ABL transcripts (median 55500/54949). This resulted in lower ratios by dPCR (mean 0.0313 vs. 0.0461 by qPCR). However, after conversion of the qPCR results with our lab specific conversion factor (CF) Conclusions An assay with a FPR of Disclosures Franke: Novartis: Honoraria, Research Funding. Tzonev:Bio-Rad: Employment, Equity Ownership. Shelton:Bio-Rad: Employment. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau. Lange:Novartis: Research Funding.

Determining the chromosomal phase of pairs of sequence variants – the arrangement of specific alleles as haplotypes – is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11–116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, routine implementation of genomic testing is still limited by: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. In metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor antibodies (αEGFR) are used to target the wild-type EGFR receptor. However, KRAS is mutated in approximately 40% of colorectal cancers and is indicative of a negative response to αEGFR therapy. BRAF and PIK3CA mutations are also associated with poor response in the remaining patients with KRAS wild-type genotype. To optimize therapy strategies for personalized care, it is therefore critical to rapidly screen patient samples during the course of disease for the presence of multiple mutations. The low abundance of mutants and limited amount and quality of available clinical samples (FFPE and cfDNA) render it difficult to reliably detect multiple mutations with current platforms and methods. We have developed a multiplexing strategy for screening clinically-actionable KRAS, BRAF, and PIK3CA mutations in mCRC clinical samples using digital PCR. No pre-amplification step was required. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented in molecular diagnostic laboratories for rapid, routine screening and monitoring of residual disease in cancer patients. Citation Format: Wei Yang, Dawne N. Shelton, Samantha Cooper, Jennifer Berman, Svilen Tzonev, Eli Hefner, John F. Regan. Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2014-LB-115

From simultaneous recording of closely located neurons one can obtain information about local inter-neuronal connectivity and acquire fine-scale neuronal response mappings. A multitrode (by which we mean a tight bundle of traditional extracellular electrodes) (McNaughton et al., 1983) is a natural choice for this kind of recording, because it allows discrimination of multiple neurons at a single recording site. Separation of spikes from different neurons is based on the assumption that spike amplitude decreases with the distance between the neuron and the tip of the electrode. Thus, the ratio of spike amplitudes measured on different wires provides reliable information for spike discrimination. The tetrode, which is made of four very thin wires (typical wire diameter with the insulation is 20 μm) is the minimal set of electrodes that provides “triangulation” in the three- dimensional space. If tetrode tips are not located in a plane, and under the idealization that neurons are point sources in a homogenous medium, the ratios of spike amplitudes will be unique for each neuron. In contrast to the tetrode, a single electrode is “spatially blind”, and its use for multi-neuron recordings can easily lead to mixing of signals from different cells whose spike amplitudes as measured at the electrode site are equal.

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.