A robust internal control for high-precision DNA methylation analyses by droplet digital PCR.

Clinical Epigenetics
Heidi D PharoGuro E Lind

Abstract

Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated CDO1, SEPT9, and VIM. A 4Plex panel consisting of EPHA3, KBTBD4, PLEKHF1, and SYT10 was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was deve...Continue Reading

References

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Citations

Sep 8, 2019·Clinical Epigenetics·Francesco NataleTiziana Angrisano
Apr 17, 2020·Journal of Clinical Microbiology·April AralarStephanie I Fraley
Jun 26, 2020·BMC Biology·Yang HanWolfgang Wagner
Aug 27, 2021·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Hege Marie VedeldGuro E Lind

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Methods Mentioned

BETA
PCR
biopsy
PCRs
chromosomal aberrations
chromosomal aberration

Software Mentioned

4Plex
QuantaSoft
PoDCall
MethyLight
R
LESS
R package “ mclust

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