Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein–DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Becker, C. et al. Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480, 245–249 (2011).
Calarco, J.P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).
Schmitz, R.J. et al. Transgenerational epigenetic instability is a source of novel methylation variants. Science 334, 369–373 (2011).
Law, J.A. & Jacobsen, S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).
Ziller, M.J. et al. Charting a dynamic DNA methylation landscape of the human genome. Nature 500, 477–481 (2013).
Lister, R. et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).
Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).
Lister, R. et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011).
Ramsahoye, B.H. et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl. Acad. Sci. USA 97, 5237–5242 (2000).
Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126, 1189–1201 (2006).
Zilberman, D., Gehring, M., Tran, R.K., Ballinger, T. & Henikoff, S. Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat. Genet. 39, 61–69 (2006).
Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).
Clark, S.J., Harrison, J., Paul, C.L. & Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997 (1994).
Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).
Hayatsu, H. Bisulfite modification of nucleic acids and their constituents. Prog. Nucleic Acid Res. Mol. Biol. 16, 75–124 (1976).
Hayatsu, H., Wataya, Y., Kai, K. & Iida, S. Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9, 2858–2865 (1970).
Shapiro, R., Braverman, B., Louis, J.B. & Servis, R.E. Nucleic acid reactivity and conformation. II. Reaction of cytosine and uracil with sodium bisulfite. J. Biol. Chem. 248, 4060–4064 (1973).
Shapiro, R., DiFate, V. & Welcher, M. Deamination of cytosine derivatives by bisulfite. Mechanism of the reaction. J. Am. Chem. Soc. 96, 906–912 (1974).
Dowen, R.H. et al. Widespread dynamic DNA methylation in response to biotic stress. Proc. Natl. Acad. Sci. USA 109, E2183–E2191 (2012).
Gu, H. et al. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat. Protoc. 6, 468–481 (2011).
Bibikova, M. et al. High density DNA methylation array with single CpG site resolution. Genomics 98, 288–295 (2011).
Schmitz, R.J. et al. Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population. Genome Res. 23, 1663–1674 (2013).
Schmitz, R.J. et al. Patterns of population epigenomic diversity. Nature 495, 193–198 (2013).
Xie, W. et al. Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome. Cell 148, 816–831 (2012).
Ibarra, C.A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).
Zhong, S. et al. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat. Biotechnol. 31, 154–159 (2013).
Potok, M.E., Nix, D.A., Parnell, T.J. & Cairns, B.R. Reprogramming the maternal zebrafish genome after fertilization to match the paternal methylation pattern. Cell 153, 759–772 (2013).
Jiang, L. et al. Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos. Cell 153, 773–784 (2013).
Stroud, H. et al. Plants regenerated from tissue culture contain stable epigenome changes in rice. eLife 2, e00354 (2013).
Takuno, S. & Gaut, B.S. Gene body methylation is conserved between plant orthologs and is of evolutionary consequence. Proc. Natl. Acad. Sci. USA 110, 1797–1802 (2013).
Gent, J.I. et al. CHH islands: de novo DNA methylation in near-gene chromatin regulation in maize. Genome Res. 23, 628–637 (2013).
Regulski, M. et al. The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA. Genome Res. 23, 1651–1662 (2013).
Zemach, A. et al. Local DNA hypomethylation activates genes in rice endosperm. Proc. Natl. Acad. Sci. USA 107, 18729–18734 (2010).
Vollmers, C. et al. Circadian oscillations of protein-coding and regulatory RNAs in a highly dynamic mammalian liver epigenome. Cell Metab. 16, 833–845 (2012).
Eichten, S.R. et al. Epigenetic and genetic influences on DNA methylation variation in maize populations. Plant Cell 25, 2783–2797 (2013).
Greaves, I.K. et al. Trans chromosomal methylation in Arabidopsis hybrids. Proc. Natl. Acad. Sci. USA 109, 3570–3575 (2012).
Groszmann, M. et al. Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor. Proc. Natl. Acad. Sci. USA 108, 2617–2622 (2011).
Barber, W.T. et al. Repeat associated small RNAs vary among parents and following hybridization in maize. Proc. Natl. Acad. Sci. USA 109, 10444–10449 (2012).
Li, X. et al. High-resolution mapping of epigenetic modifications of the rice genome uncovers interplay between DNA methylation, histone methylation, and gene expression. Plant Cell 20, 259–276 (2008).
Wang, X. et al. Genome-wide and organ-specific landscapes of epigenetic modifications and their relationships to mRNA and small RNA transcriptomes in maize. Plant Cell 21, 1053–1069 (2009).
Stroud, H., Greenberg, M.V., Feng, S., Bernatavichute, Y.V. & Jacobsen, S.E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).
Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).
Kurihara, Y. et al. Surveillance of 3′ noncoding transcripts requires FIERY1 and XRN3 in Arabidopsis. G3 (Bethesda) 2, 487–498 (2012).
Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).
Booth, M.J. et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012).
Booth, M.J., Marsico, G., Bachman, M. & Beraldi, D. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat. Chem. 6, 435–440 (2014).
Cokus, S.J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).
Miura, F., Enomoto, Y., Dairiki, R. & Ito, T. Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 40, e136 (2012).
This work was supported by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (GMBF3034) and grants from the National Science Foundation (MCB1344299 and MCB122246) to J.R.E. R.L. was supported with an Australian Research Council Future Fellowship (FT120100862). R.J.S. was supported by the US National Institutes of Health (R00GM100000) and the US National Science Foundation (IOS-1339194).
The authors declare no competing financial interests.
About this article
Cite this article
Urich, M., Nery, J., Lister, R. et al. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10, 475–483 (2015). https://doi.org/10.1038/nprot.2014.114
Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome
BMC Genomics (2021)
Genome Biology (2021)
Biotechnology for Biofuels (2021)
BMC Genomics (2021)
Prenatal testosterone triggers long-term behavioral changes in male zebra finches: unravelling the neurogenomic mechanisms
BMC Genomics (2021)