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 CCmeCGG 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.
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References
Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13:484–492
Issa JP (2014) Aging and epigenetic drift: a vicious cycle. J Clin Invest 124:24–29
Maegawa S, Gough SM, Watanabe-Okochi N, Lu Y, Zhang N, Castoro RJ, Estecio MR, Jelinek J, Liang S, Kitamura T, Aplan PD, Issa JP (2014) Age-related epigenetic drift in the pathogenesis of MDS and AML. Genome Res 24:580–591
Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692
Mack SC, Witt H, Piro RM, Gu L, Zuyderduyn S, Stütz AM, Wang X, Gallo M, Garzia L, Zayne K, Zhang X, Ramaswamy V, Jäger N, Jones DT, Sill M, Pugh TJ, Ryzhova M, Wani KM, Shih DJ, Head R, Remke M, Bailey SD, Zichner T, Faria CC, Barszczyk M, Stark S, Seker-Cin H, Hutter S, Johann P, Bender S, Hovestadt V, Tzaridis T, Dubuc AM, Northcott PA, Peacock J, Bertrand KC, Agnihotri S, Cavalli FM, Clarke I, Nethery-Brokx K, Creasy CL, Verma SK, Koster J, Wu X, Yao Y, Milde T, Sin-Chan P, Zuccaro J, Lau L, Pereira S, Castelo-Branco P, Hirst M, Marra MA, Roberts SS, Fults D, Massimi L, Cho YJ, Van Meter T, Grajkowska W, Lach B, Kulozik AE, von Deimling A, Witt O, Scherer SW, Fan X, Muraszko KM, Kool M, Pomeroy SL, Gupta N, Phillips J, Huang A, Tabori U, Hawkins C, Malkin D, Kongkham PN, Weiss WA, Jabado N, Rutka JT, Bouffet E, Korbel JO, Lupien M, Aldape KD, Bader GD, Eils R, Lichter P, Dirks PB, Pfister SM, Korshunov A, Taylor MD (2014) Epigenomic alterations define lethal CIMP-positive ependymomas of infancy. Nature 506:445–450
Mayle A, Yang L, Rodriguez B, Zhou T, Chang E, Curry CV, Challen GA, Li W, Wheeler D, Rebel VI, Goodell MA (2015) Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation. Blood 125:629–638
Baylin SB, Jones PA (2011) A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer 11:726–734
Issa JP (2012) DNA methylation as a clinical marker in oncology. J Clin Oncol 30:2566–2568
Chung W, Bondaruk J, Jelinek J, Lotan Y, Liang S, Czerniak B, Issa J-PJ (2011) Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments. Cancer Epidemiol Biomarkers Prev 20:1483–1491
Foy JP, Pickering CR, Papadimitrakopoulou VA, Jelinek J, Lin SH, William WN, Frederick MJ, Wang J, Lang W, Feng L, Zhang L, Kim ES, Fan YH, Hong WK, El-Naggar AK, Lee JJ, Myers JN, Issa JP, Lippman SM, Mao L, Saintigny P (2015) New DNA methylation markers and global DNA hypomethylation are associated with oral cancer development. Cancer Prev Res (Phila) 8:1027–1035
Laird PW (2010) Principles and challenges of genomewide DNA methylation analysis. Nat Rev Genet 11:191–203
Plongthongkum N, Diep DH, Zhang K (2014) Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet 15:647–661
Jelinek J, Liang S, Lu Y, He R, Ramagli LS, Shpall EJ, Estecio MR, Issa JP (2012) Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique. Epigenetics 7:1368–1378
Risso D, Ngai J, Speed TP, Dudoit S (2014) The role of spike-in standards in the normalization of RNA-seq. In: Datta S, Nettleton D (eds) Statistical analysis of next generation sequencing data. Frontiers in Probability and the Statistical Sciences. Springer International Publishing, Switzerland, pp 169–190
Malouf GG, Taube JH, Lu Y, Roysarkar T, Panjarian S, Estecio MR, Jelinek J, Yamazaki J, Raynal NJ, Long H, Tahara T, Tinnirello A, Ramachandran P, Zhang XY, Liang S, Mani SA, Issa JP (2013) Architecture of epigenetic reprogramming following Twist1-mediated epithelial-mesenchymal transition. Genome Biol 14:R144
Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, Issa JP (2014) TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells. Nucleic Acids Res 42:6956–6971
Qin T, Si J, Raynal NJ, Wang X, Gharibyan V, Ahmed S, Hu X, Jin C, Lu Y, Shu J, Estecio MR, Jelinek J, Issa JP (2015) Epigenetic synergy between decitabine and platinum derivatives. Clin Epigenetics 7:97
Malouf GG, Tahara T, Paradis V, Fabre M, Guettier C, Yamazaki J, Long H, Lu Y, Raynal NJ, Jelinek J, Mouawad R, Khayat D, Brugières L, Raymond E, Issa JP (2015) Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features. Epigenetics 10:872–881
Yamazaki J, Jelinek J, Lu Y, Cesaroni M, Madzo J, Neumann F, He R, Taby R, Vasanthakumar A, Macrae T, Ostler KR, Kantarjian HM, Liang S, Estecio MR, Godley LA Issa JP (2015) TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor-binding sites in chronic myelomonocytic Leukemia. Cancer Res 75:2833–2843
Challen GA, Sun D, Jeong M, Luo M, Jelinek J, Berg JS, Bock C, Vasanthakumar A, Gu H, Xi Y, Liang S, Lu Y, Darlington GJ, Meissner A, Issa JP, Godley LA, Li W, Goodell MA (2012) Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 44:23–31
van Esterik JC, Vitins AP, Hodemaekers HM, Kamstra JH, Legler J, Pennings JL, Steegenga WT, Lute C, Jelinek J, Issa JP, Dollé ME, van der Ven LT (2014) Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A. Toxicol Lett 232:293–300
Acknowledgments
We thank Dr. Jean-Pierre Issa for the support and expert advice, Justin T. Lee and Bela Patel for the technical expertise in making of DREAM libraries, Dr. Yue-Sheng Li at Fox Chase Cancer Center Genomic Facility for the high-throughput sequencing, Dr. Matteo Cesaroni for the generous help with bioinformatics and data processing, and Dr. Amy B. Hart for the editorial help.
This work was supported by NIH grants R01-HD075203 from NICHD and P01-CA049639 from NCI.
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Jelinek, J., Madzo, J. (2016). DREAM: A Simple Method for DNA Methylation Profiling by High-throughput Sequencing. In: Li, S., Zhang, H. (eds) Chronic Myeloid Leukemia. Methods in Molecular Biology, vol 1465. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4011-0_10
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DOI: https://doi.org/10.1007/978-1-4939-4011-0_10
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