Virtual-Partition Digital PCR for High-Precision Chromosomal Counting Applications

Digital PCR (dPCR) is the gold-standard analytical platform for rapid high-precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platform's ability to address some clinical applications that require the simultaneous monitoring of 20-50 analytes per sample. Here, we present virtual-partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescence intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for noninvasive fetal aneuploidy testing. This demonstration assay─tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA─is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers the variance of the chromosomal ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding > 98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultrahigh-precision quantitation.