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Methods Mol Biol. 2018;1768:173-190. doi: 10.1007/978-1-4939-7778-9_11.

Detection and Quantification of Mosaic Genomic DNA Variation in Primary Somatic Tissues Using ddPCR: Analysis of Mosaic Transposable-Element Insertions, Copy-Number Variants, and Single-Nucleotide Variants.

Author information

1
Department of Psychiatry and Behavioral Sciences, Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine, Palo Alto, CA, USA.
2
Program on Genetics of Brain Function, Department of Genetics, Stanford Center for Genomics and Personalized Medicine, Palo Alto, CA, USA.
3
Department of Health Sciences Research, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA.
4
Department of Psychiatry and Behavioral Sciences, Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine, Palo Alto, CA, USA. aeurban@stanford.edu.
5
Program on Genetics of Brain Function, Department of Genetics, Stanford Center for Genomics and Personalized Medicine, Palo Alto, CA, USA. aeurban@stanford.edu.

Abstract

Here, we describe approaches using droplet digital polymerase chain reaction (ddPCR) to validate and quantify somatic mosaic events contributed by transposable-element insertions, copy-number variants, and single-nucleotide variants. In the ddPCR assay, sample or template DNA is partitioned into tens of thousands of individual droplets such that when DNA input is low, the vast majority of droplets contains no more than one copy of template DNA. PCR takes place in each individual droplet and produces a fluorescent readout to indicate the presence or absence of the target of interest allowing for the accurate "counting" of the number of copies present in the sample. The number of partitions is large enough to assay somatic mosaic events with frequencies down to less than 1%.

KEYWORDS:

Copy number variations (CNVs); Droplet digital PCR (ddPCR); Mobile elements; Single nucleotide variations (SNVs); Somatic mosaicism

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