Svilen Tzonev

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.

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.

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).