Statistical Analysis of Nonuniform Volume Distributions for Droplet-Based Digital PCR Assays

We present a method to determine the concentration of nucleic acids in a sample by partitioning it into droplets with a nonuniform volume distribution. This digital PCR method requires no special equipment for partitioning, unlike other methods that require nearly identical volumes. Droplets are generated by vortexing a sample in an immiscible oil to create an emulsion. PCR is performed, and droplets in the emulsion are imaged. Droplets with one or more copies of a nucleic acid are identified, and the nucleic acid concentration of the sample is determined. Numerical simulations of droplet distributions were used to estimate measurement error and dynamic range and to examine the effects of the total volume of droplets imaged and the shape of the droplet size distribution on measurement accuracy. The ability of the method to resolve 1.5- and 3-fold differences in concentration was assessed by using simulations of statistical power. The method was validated experimentally; droplet shrinkage and fusion during amplification were also assessed experimentally and showed negligible effects on measured concentration.