Abstract
Many genomic segments vary in copy number among individuals of the same species, or between cancer and normal cells within the same person. Correctly measuring this copy number variation is critical for studying its genetic properties, its distribution in populations and its relationship to phenotypes. Droplet digital PCR (ddPCR) enables accurate measurement of copy number by partitioning a PCR reaction into thousands of nanoliter-scale droplets, so that a genomic sequence of interest—whose presence or absence in a droplet is determined by end-point fluorescence—can be digitally counted. Here, we describe how we analyze copy number variants using ddPCR and review the design of effective assays, the performance of ddPCR with those assays, the optimization of reactions, and the interpretation of data.
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Acknowledgment
Our understanding of CNVs and assays has benefited greatly from interactions with our colleagues Robert Handsaker, Aswin Sekar, and Linda Boettger. We also thank Katherine Tooley for helpful discussions of this protocol. This work was supported by a grant from the National Human Genome Research Institute (R01 HG006855, to S.A.M.).
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Bell, A.D., Usher, C.L., McCarroll, S.A. (2018). Analyzing Copy Number Variation with Droplet Digital PCR. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_9
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_9
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