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Jelinek J, Lee JT, Cesaroni M, Madzo J, Liang S, Lu Y, Issa JJ
Digital Restriction Enzyme Analysis of Methylation (DREAM)
Methods Mol Biol (2018) 1708:247-265.
Abstract
Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS) technology. The method is based on sequential cuts of genomic DNA with a pair of restriction enzymes (SmaI and XmaI) at CCCGGG target sites. Unmethylated sites are first digested with SmaI. This enzyme cuts the sites in the middle at CCC^GGG, leaving behind blunt ended fragments. CpG methylation completely blocks SmaI; therefore, only unmethylated sites are cleaved. The remaining methylated sites are digested with XmaI in the next step. This enzyme is not blocked by CpG methylation. It cuts the recognition site sideways at C^CCGGG forming 5'-CCGG overhangs. The sequential cuts thus create distinct methylation-specific signatures at the ends of restriction fragments: 5'-GGG for unmethylated CpG sites and 5'-CCGGG for methylated sites. The DNA fragments resulting from the digestions are ligated to NGS adapters. Sequencing libraries are prepared using hexanucleotide barcodes for sample identification. Individual libraries with distinct barcodes are pooled and sequenced using a paired ends protocol. The sequencing reads are aligned to the genome and mapped to unique CCCGGG target sites. Methylation at individual CpG sites is calculated as the ratio of sequencing reads with the methylated signature to the total number of reads mapping to the site. Sequencing of 25 million reads per sample typically yields 50,000 unique CpG sites covered with hundreds of reads enabling accurate determination of DNA methylation levels. DREAM does not require bisulfite conversion, has a very low background, and has high sensitivity to low levels of methylation. The method is simple, cost-effective, quantitative, highly reproducible, and can be applied to any species.
Note
Publication Date: 2018-01-01.
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Last updated on Tuesday, December 04, 2018