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Virtual partition digital PCR for high precision chromosomal counting applications

Jacky, Lucien and Yurk, Dominic and Alvarado, John and Leatham, Bryan and Schwartz, Jerrod and MacDonald, Chris and Rajagopal, Aditya (2021) Virtual partition digital PCR for high precision chromosomal counting applications. . (Unpublished)

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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 fluorescent 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 non-invasive 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 variance of chromosome 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 ultra-high-precision quantitation.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper ItemData ItemData ItemData
Jacky, Lucien0000-0001-5570-7925
Yurk, Dominic0000-0002-2276-4189
Rajagopal, Aditya0000-0002-7768-2463
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted April 29, 2021. The authors would like to thank Jeff Gole and Mimi Wang of ChromaCode, Inc. for making software available to select conserved genomic regions for primer design, Sheila Rosenburg of ChromaCode, Inc for critique and lively statistical discussions and Molly Smith of ChromaCode, Inc for procuring the sheared DNA from UCSD. The following cell lines/DNA samples were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research: NA12878DNA, NANA15453DNA and NA04965DNA. Covaris DNA Shearing was conducted at the IGM Genomics Center, University of California, San Diego, La Jolla, CA. Competing Interest Statement: The authors of this manuscript have the following competing interests: L.J., D.Y., J.A., B.L., J.S., C.M. and A.R. are employed by ChromaCode Inc., a company which commercializes molecular diagnostic assays. In addition, D.Y., C.M. and A.R. are inventors on patents that include claims related to the digital PCR multiplexing methods described in this manuscript.
Record Number:CaltechAUTHORS:20210430-132803852
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Official Citation:Virtual partition digital PCR for high precision chromosomal counting applications. Lucien Jacky, Dominic Yurk, John Alvarado, Bryan Leatham, Jerrod Schwartz, Chris MacDonald, Aditya Rajagopal. bioRxiv 2021.04.29.441975; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108907
Deposited By: Tony Diaz
Deposited On:30 Apr 2021 20:46
Last Modified:30 Apr 2021 20:46

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