Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning

Abstract

Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

Figure 1

Single-molecule partition vs DNA input. Partitions with a single DNA molecule over all DNA-positive partitions are calculated based on Poisson statistics for a partition capacity of 20 K partitions common across commercially available dPCR platforms versus 30 million partitions in UltraPCR’s. Low partition systems fall out of the single-molecule domain rapidly and require Poisson correction to derive quantitative accuracy up to a threshold number of counts.^, In contrast, even at 1 million DNA input copies, UltraPCR still maintains the limited dilution regime, allowing for true single-molecule counting across a 6-log dynamic range.

Figure 2

UltraPCR workflow. UltraPCR uses disposable droplet generators to rapidly partition a PCR mix via centrifugation directly into PCR strip tubes. The same PCR strip tubes are loaded directly into a standard thermal cycler for target amplification and then transferred to the UltraPCR imager for 3D light sheet scanning of positive partitions.

Figure 3

Ultrapartition characteristics. (A) Ultrapartitions settle in the bottom of the tube (shown in red brackets) and are optically clear for direct in-tube 3D imaging. (B) Slice of 3D light sheet scan of a sample with ∼500,000 counts of human RPP30 to showcase low DNA occupancy of the system.

Figure 4

6-Log dynamic range on the UltraPCR platform. Demonstration of a 6-log dynamic range in serial dilution experiments, measuring DNA-positive partitions vs target RPP30 input copies. For each dilution, four technical replicates are performed, with total error (%CV) represented in the error bars.

Figure 5

Orthogonal testing between UltraPCR and ddPCR. Scatterplots of RPP30 counts where the same serially diluted sets of samples were tested on (A) ddPCR with the Poisson 95% confidence interval (CI) as error bars as indicated by the manufacturer and (B) UltraPCR with positive partitions normalized to 50 μL. Note that “NTC” is defined as 0 expected RPP30 input from serial dilution but contains N1 input.

Figure 6

UltraPCR NIPT assay analytical study. (A–C) Boxplot of different chromosomal ratios. Chr21/18 and Chr21/13 are expected to increase with more %T21 spiked in, and Chr13/18 is expected to remain constant. (D) ROC curve using all spiked %T21 samples compared to 0% to determine the optimal threshold to separate control versus T21-spiked samples. (E) Accuracy plot vs cutoff where the blue line represents data using Chr13 as a reference chromosome and the orange line represents data using Chr18 as a reference chromosome. Dotted lines represent their respective optimal cutoff to maximize accuracy of the assay.