DREAM: A Simple Method for DNA Methylation Profiling by High-throughput Sequencing

Abstract

The digital restriction enzyme analysis of methylation (DREAM) is a simple method for DNA methylation analysis at tens of thousands of CpG sites across the genome. The method creates specific signatures at unmethylated and methylated CpG sites by sequential digests of genomic DNA with restriction endonucleases SmaI and XmaI, respectively. Both enzymes have the same CCCGGG recognition site; however, they differ in their sensitivity to CpG methylation and their cutting pattern. SmaI cuts only unmethylated sites leaving blunt 5'-GGG ends. XmaI cuts remaining methylated CC(me)CGG sites leaving 5'-CCGGG ends. Restriction fragments with distinct signatures at their ends are ligated to Illumina sequencing adaptors with sample-specific barcodes. High-throughput sequencing of pooled libraries follows. Sequencing reads are mapped to the restriction sites in the reference genome, and signatures corresponding to methylation status of individual DNA molecules are resolved. Methylation levels at target CpG sites are calculated as the proportion of sequencing reads with the methylated signature to the total number of reads mapping to the particular restriction site. Aligning the reads to the reference genome of any species is straightforward, since the method does not rely on bisulfite conversion of DNA. Sequencing of 25 million reads per human DNA library yields over 50,000 unique CpG sites with high coverage enabling accurate determination of DNA methylation levels. DREAM has a background less than 1 % making it suitable for accurate detection of low methylation levels. In summary, the method is simple, robust, highly reproducible, and cost-effective.

Age estimation in a long‐lived seabird (Ardenna tenuirostris) using DNA methylation‐based biomarkers

“…However, DNAm scores generated using this method are relative to the count of unmethylated EpiRADSeq reads only. This is avoided when using a dual methylation‐sensitive digest as in DREAM, as reads are generated for both methylated and unmethylated CpGs (Jelinek & Madzo, ). Reduced representation bisulphite sequencing can also be used to quantify CpG DNAm, but does require a higher quantity of initial genomic DNA (Meissner et al, ).…”

“…We examined DNAm at CpG sites throughout the genome using DREAM of 71 Short‐tailed shearwater whole blood DNA samples (Jelinek & Madzo, ). Briefly, genomic DNA (1 μg) extracted from shearwater blood FTA samples was sequentially cut with two enzymes that recognize the “CCCGGG” sequence motif in DNA (Figure ).…”

Mapping chromatin modifications at the single cell level

“…Classically, mapping DNA modifications relies on one of the following techniques: (1) chemical conversion of unmodified nucleobases to another nucleobase (Frommer et al, 1992); (2) recognition of the modified nucleobases by restriction enzymes (Jelinek and Madzo, 2016); or (3) recognition of the modified nucleobases by antibodies (Weber et al, 2005). The last two methods are less popular: the second is restricted to detecting modifications at the subset of DNA loci that contain the particular enzyme recognition sequence, and the third method is not ideal because existing antibodies do not discriminate well between DNA modifications, and it has low resolution (a few hundred base pairs, limited by fragment length).…”

TET1andTDGsuppress intestinal tumorigenesis by down-regulating the inflammatory and immune response pathways

“…The restriction fragments with distinct signatures, 5′-GGG at unmethylated sites or 5′-CCGGG at methylated sites, were analyzed by next generation sequencing. Sequencing reads were mapped to the restriction sites in the reference genome, and signatures corresponding to methylation status of individual DNA molecules were resolved (26). Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts.…”

“…Our goal was to identify the epigenomic changes associated with Tet1 and Tdg defects in the context of Apc Min/+ mutation. To this purpose, we conducted a genome-wide DNA methylation analysis by digital restriction enzyme analysis of methylation (DREAM) (26), which allows for identification of differentially methylated sites in CpG islands (CGI) and non-CpG islands (NCGI) at high resolution (it interrogates approximately 25K CpG sites in the murine genome). DREAM analysis was conducted on triplicate adenomas from mice within the four genotype groups, and on triplicate samples of colonic mucosa from C57BL6 mice.…”