Development of a Droplet Digital™ PCR DNA Methylation Detection and Quantification Assay of Prenatal Tobacco Exposure

Abstract

DNA methylation is a labile modification associated with gene expression control and environmental adaptations. High throughput, scalable and quantitative assessments of specific DNA methylation modifications in complex genomic regions for use in large population studies are needed. The performance of Droplet Digital™ PCR (ddPCR™) was investigated for DNA methylation detection against next-generation bisulfite sequencing (NGS) to demonstrate the ability of ddPCR to detect and validate DNA methylation levels and complex patterns among neighboring CpGs in regions associated with prenatal tobacco exposure. While both techniques are reproducible, ddPCR demonstrates a unique advantage for high-throughput DNA methylation analysis in large-scale population studies and provides the specificity to accurately measure DNA methylation of target CpGs in complex regions.

A validated restriction enzyme ddPCR cg05575921 (AHRR) assay to accurately assess smoking exposure

“…In this study we report a technically accurate restriction enzyme digestion ddPCR assay, which appears to have similar performance to that of previously reported ddPCR assays (3,4) without the need for the chemically harsh bisul te conversion process (6), which can be an issue particularly when working with samples containing limited amounts of input DNA. Bravo-Gutierrez and colleagues have also reported a HpaII restrction enzyme digest method, but using qPCR rather than ddPCR as the detection platform.…”

“…It has been previously demonstrated that a bisul te converted DNA ddPCR assay can accurately determine the CpG methylation status of cg05575921 within AHRR (3,4,13). Furthermore, strong concordance between BIS ddPCR and Illumina Epic array measures of cg05575921 have been reported (4,13).…”

“…A second ddPCR assay, based on the bisul te (BIS) ddPCR assay published by Arroyo et al (3), was used for comparison with the RE ddPCR approach. The primers and probes were as previously published (3) with the exception of the forward primer which was redesigned in Primer3Plus (9) to achieve a smaller ampli cation product of 126bp. Neither primers nor probes contained polymorphic bases with global population minor allele frequencies greater than 0.1%.…”

“…Numerous epigenome-wide association studies have reported robust and reproducible DNA methylation changes correlated with smoking exposure, with the strongest association consistently being cg05575921, within the gene body of AHRR (1,2). Sensitive PCR methods have subsequently been developed to assess the methylation status of cg05575921 (3)(4)(5). These techniques typically utilise bisul te conversion, which is a harsh chemical process resulting in signi cant DNA degradation and can be associated with issues regarding conversion e ciency if not carefully conducted (6).…”

A validated restriction enzyme ddPCR cg05575921 (AHRR) assay to accurately assess smoking exposure.

“…Numerous epigenome-wide association studies have reported robust and reproducible DNA methylation changes correlated with smoking exposure, with the strongest association consistently being cg05575921, within the gene body of AHRR (1,2). Sensitive PCR methods have subsequently been developed to assess the methylation status of cg05575921 (3)(4)(5). These techniques typically utilise bisul te conversion, which is a harsh chemical process resulting in signi cant DNA degradation and can be associated with issues regarding conversion e ciency if not carefully conducted (6).…”