Statistical Modeling of Targeted Resequencing Using Microdroplet-Based PCR

RP-22 RainDance Technologies has developed a Targeted Sequencing platform that leverages the sensitivity and the specificity of the Polymerase Chain Reaction (PCR) to target genomic regions from either thousands of exonsor large contiguous loci. The Sequence Enrichment application utilizes a novel microdroplet-based format to rapidly and reproducibly generate over 1 million independent PCR reactions. The process control achieved with this approach enables statistical modeling to tune the performance of the assay. Researchers are able to design experiments using pooled genomic DNA samples and have the confidence that they have the statistical power to detect variants at the frequency necessary for their biological models. Microdroplet based PCR generates stable picoliter-volume PCR reactions in aqueous droplets. Each PCR droplet is produced by combining a primer droplet containing a single primer pair and a genomic DNA template droplet containing all of the PCR reaction components (minus the forward and reverse primers). The primer droplet represents a single element from a Primer Library that represents thousands of different primer pairs that are designed to represent the researchers' targets of interest. Each Library has an equal representation of each unique primer droplet. The primer and genomic DNA template droplets are introduced into a microfluidic chip environment and merged together to form a single PCR droplet. Each element of the Primer Library creates multiple PCR reactions which powers the statistical modeling. The exquisite control and reproducibility of the droplet is the foundation for the statistical modeling. We will present targeted resequencing data to support the statistical models that can be generated using the RainDance Platform. By adjusting the amount of genomic DNA and the complexity of the Primer Library the researcher can tune the assay to the appropriate confidence interval for detecting sequence variants critical for the elucidation of the molecular basis of complex diseases.