Human genomic DNA NA10860 (left 5 panels) and the RPP30 synthetic construct (right 5 panels) were quantitated using the RPP30 (FAM) assay on this digital array.

<p>The two bottom panels are NTC (no template control). Digital PCR Analysis software can count the number of positive chambers in each panel. When two assays with two fluorescent dyes are used in a multiplex digital PCR reaction, two genes can be independently quantitated. This is the basis of the CNV study using the digital array.</p

A digital array has 12 panels of 765 reaction chambers each.

<p>PCR mixes are loaded into each panel and single DNA molecules are randomly partitioned into the chambers. The digital array can be thermocycled, imaged on a BioMark instrument, and the data analyzed using the Digital PCR Analysis software.</p

Illustration of a numerical projection algorithm to compute the sampling distribution of ratio of two random variables with arbitrary probability distributions by slicing the 2-D space into thin wedges and accumulating the joint probabilities in the wedges.

<p>Most of the contribution would come from the confidence ellipse region.</p

Given number of chambers <i>C</i> and counts <i>H</i>₁ and <i>H</i>₂ of the positive chambers in a digital array for the target gene and the reference gene, respectively, list of formulas needed to analyze copy number variation.

<p>See the details in the paper for assumptions made so that these equations are close approximations to actual values.</p

Consider an infinite universe of chambers.

<p>A digital array panel is a finite sampling of this universe. The goal is to determine <i>λ</i>, the mean number of the target molecules per chamber in the DNA sample. The number of positive chambers, which have hits of one or more molecules, shown as filled green squares in the panel with <i>C</i>( = 765) chambers is <i>H</i>.</p

Histogram of number of positive chambers <i>H</i> = <i>P</i>×<i>C</i> obtained by choosing <i>M</i> = 400 as the mean number of molecules per panel over 70 thousand panels and running a simulation using a random number generator.

<p>The green curve is the sampling distribution predicted by the theory.</p

Comparison of the metrics of histogram, shown in Figure 5, of number of positive chambers obtained in simulation with those predicted by the theory.

<p>Comparison of the metrics of histogram, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002876#pone-0002876-g005" target="_blank">Figure 5</a>, of number of positive chambers obtained in simulation with those predicted by the theory.</p

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics