Development of a Droplet Digital (TM) PCR DNA methylation detection and quantification assay of prenatal tobacco exposure

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 (TM) PCR (ddPCR (TM)) 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. METHODS SUMMARY Differentially methylated gBlocks (TM) were designed and generated following Integrated DNA Technologies manufacturer guidelines. PCR products for next-generation sequencing (NGS) and Droplet Digital PCR (TM) (ddPCR) were generated with primer/probe sets spanning similar to 146 bps around the targeted sites. NGS amplicons were sent directly to University of Southern California's Molecular Core Facility for bisulfite sequencing, while analysis was performed using Bismark Bisulfite Mapper. ddPCR amplicons were analyzed using the Bio-Rad QX200 and AutoDG ddPCR system. [GRAPHICS] .