title: 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.
authors: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Rm 339 F, PAHB, Philadelphia, PA, 19140, USA. jjelinek@temple.edu.
authors: Jaroslav
authors: J
authors: Jelinek
authors: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, 19140, USA.
authors: Jozef
authors: J
authors: Madzo
mesh_terms: D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D015183:Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: CpG sites; DNA methylation; DREAM; High-throughput sequencing; Restriction endonuclease; SmaI; XmaI