Chemical modifications of DNA have been recognized as key epigenetic mechanisms for maintenance of the cellular state and memory. Such DNA modifications include canonical 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Recent advances in detection and quantification of DNA modifications have enabled epigenetic variation to be connected to phenotypic consequences on an unprecedented scale. These methods may use chemical or enzymatic DNA treatment, may be targeted or non-targeted and may utilize array-based hybridization or sequencing. Key considerations in the choice of assay are cost, minimum sample input requirements, accuracy and throughput. This Review discusses the principles behind recently developed techniques, compares their respective strengths and limitations and provides general guidelines for selecting appropriate methods for specific experimental contexts.
At a glance
- In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases. PLoS Genet. 8, e1002750 (2012). et al.
- Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nature Genet. 45, 1198–1206 (2013). et al.
- Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009). et al.
- Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011). et al.
- Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013). et al.
- Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS Genet 7, e1002389 (2011). et al.
- Large conserved domains of low DNA methylation maintained by Dnmt3a. Nature Genet. 46, 17–23 (2014). et al.
- DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011). et al.
- Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134–1148 (2013). et al.
- A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006). et al.
- Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nature Rev. Genet. 13, 484–492 (2012).
- DNA methylation: roles in mammalian development. Nature Rev. Genet. 14, 204–220 (2013). &
- DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape. Nature Genet. 45, 836–841 (2013). et al.
- Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nature Protoc. 7, 2159–2170 (2012). et al.
- Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011). et al.
- Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013). et al.
- 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nature Neurosci. 14, 1607–1616 (2011). et al.
- A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119 (2013). et al.
- Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013). et al.
- Principles and challenges of genome-wide DNA methylation analysis. Nature Rev. Genet. 11, 191–203 (2010). ,
- 5-hydroxymethylcytosine: generation, fate, and genomic distribution. Curr. Opin. Cell Biol. 25, 289–296 (2013). &
- Mapping recently identified nucleotide variants in the genome and transcriptome. Nature Biotech. 30, 1107–1116 (2012). , &
- Analysing and interpreting DNA methylation data. Nature Rev. Genet. 13, 705–719 (2012).
- DNA methylome analysis using short bisulfite sequencing data. Nature Methods 9, 145–151 (2012). et al.
- Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, e65 (2001). , &
- Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J. Mol. Diagn. 8, 209–217 (2006). et al.
- Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res. 22, 467–477 (2012). et al.
- Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling. Genome Biol. 13, R92 (2012). et al.
- Double restriction-enzyme digestion improves the coverage and accuracy of genome-wide CpG methylation profiling by reduced representation bisulfite sequencing. BMC Genomics 14, 11 (2013). et al.
- Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Nucleic Acids Res. 41, e116 (2013). et al.
- Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 23, 2126–2135 (2013). et al.
- Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. Proc. Natl Acad. Sci. USA 109, 16196–16201 (2012). et al.
- Restriction landmark genome scanning. Methods Mol. Biol. 791, 101–112 (2011). et al.
- Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010). et al.
- Methylome analysis using MeDIP-seq with low DNA concentrations. Nature Protoc. 7, 617–636 (2012). et al.
- A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the infinium HumanMethylation450 BeadChip® for methylome profiling. PLoS ONE 7, e50233 (2012). et al.
- MBD-seq as a cost-effective approach for methylome-wide association studies: demonstration in 1500 case–control samples. Epigenomics 4, 605–621 (2012). et al.
- High resolution detection and analysis of CpG dinucleotides methylation using MBD-seq technology. PLoS ONE 6, e22226 (2011). et al.
- Whole-genome DNA methylation profiling using MethylCap-seq. Methods 52, 232–236 (2010). et al.
- AutoMeDIP-seq: a high-throughput, whole genome, DNA methylation assay. Methods 52, 223–231 (2010). &
- Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotech. 28, 1106–1114 (2010). et al.
- 5-hydroxymethylcytosine: a new kid on the epigenetic block? Mol. Syst. Biol. 7, 562 (2011). , &
- Application of microdroplet PCR for large-scale targeted bisulfite sequencing. Genome Res. 21, 1738–1745 (2011).
This paper demonstrates a fully automated method for quantification of DNA methylation on 2100 genes.
- High-throughput method for analyzing methylation of CpGs in targeted genomic regions. Proc. Natl Acad. Sci. USA 107, 12587–12592 (2010). et al.
- Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome Res. 20, 1279–1287 (2010). &
- Library-free methylation sequencing with bisulfite padlock probes. Nature Methods 9, 270–272 (2012).
This paper describes a method for high-throughput padlock probes that sequence methylated DNA.
- Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature Biotech. 27, 353–360 (2009). et al.
- Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing. Nucleic Acids Res. 39, e127 (2011). et al.
- High resolution profiling of human exon methylation by liquid hybridization capture-based bisulfite sequencing. BMC Genomics 12, 597 (2011). et al.
- In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes. Nucleic Acids Res. 41, e72 (2013). et al.
- Tet1 controls meiosis by regulating meiotic gene expression. Nature 492, 443–447 (2012). et al.
- High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome Res. 23, 616–627 (2013). et al.
- Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet. 9, e1003439 (2013). et al.
- DNA methylation analysis of germ cells by using bisulfite-based sequencing methods. Methods Mol. Biol. 825, 223–235 (2012). &
- Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Res. 22, 1139–1143 (2012).
This study shows that whole-genome bisulphite sequencing can be performed on ~1–10 ng of genomic DNA.
- Tagmentation-based whole-genome bisulfite sequencing. Nature Protoc. 8, 2022–2032 (2013). et al.
- Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 40, e136 (2012). et al.
- A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res. 39, e44 (2011). et al.
- Single-cell DNA-methylation analysis reveals epigenetic chimerism in preimplantation embryos. Science 341, 1110–1112 (2013).
The first paper to use single-cell DNA methylation analysis to address an important biological problem.
- Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics 6, 1176–1188 (2011). et al.
- The DNA methylation landscape of human early embryos. Nature 511, 606–610 (2014). et al.
- Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nature Methods 11, 817–820 (2014). et al.
- Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18, 780–790 (2008). et al.
- High density DNA methylation array with single CpG site resolution. Genomics 98, 288–295 (2011). et al.
- MeDIP coupled with a promoter tiling array as a platform to investigate global DNA methylation patterns in AML cells. Leukemia Res. 37, 102–111 (2013). et al.
- Comprehensive DNA methylation profiling of human repetitive DNA elements using an MeDIP-on-RepArray assay. Methods Mol. Biol. 859, 267–291 (2012). &
- Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126, 1189–1201 (2006). et al.
- A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples. Epigenetics Chromatin 6, 1–11 (2013). et al.
- Genome-wide DNA methylation analysis of formalin-fixed paraffin embedded colorectal cancer tissue. Genes Chromosomes Cancer 53, 537–548 (2014). et al.
- Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 22, 1128–1138 (2012).
Together with reference 69, these papers describe DNA precipitation followed by bisulphite treatment to create a map of DNA methylation patterns associated with chromatin modifications.
- Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA. Genome Res. 22, 1120–1127 (2012). et al.
- Combined chromatin immunoprecipitation and bisulfite methylation sequencing analysis. Methods Mol. Biol. 791, 239–251 (2011). &
- Single molecule epigenetic analysis in a nanofluidic channel. Anal. Chem. 82, 2480–2487 (2010). et al.
- Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel. Proc. Natl Acad. Sci. USA 109, 8477–8482 (2012).
This study discusses a nanofluidic device for sorting single methylated DNA molecules.
- Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells. Proc. Natl Acad. Sci. USA 110, 7772–7777 (2013). et al.
- OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes. Proc. Natl Acad. Sci. USA 108, 14497–14502 (2011). et al.
- Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res. 22, 2497–2506 (2012).
This study describes NOMe-seq, a method using M. Cvi PI treatment and bisulphite conversion to produce a genome-wide base resolution map of nucleosome positioning and DNA methylation on the same DNA molecules.
- Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011). et al.
- Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011). et al.
- TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011). et al.
- 5-hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 12, R54 (2011). et al.
- Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res. 41, e206 (2013). et al.
- Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development. Cell Res. 21, 1670–1676 (2011). et al.
- Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010). et al.
- Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature 497, 122–126 (2013). et al.
- The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010). et al.
- Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181 (2010). et al.
- Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nature Biotech. 29, 68–72 (2011). et al.
- Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007). et al.
- The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine. Nature Protocols 7, 1909–1917 (2012). et al.
- Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nature Protocols 7, 340–350 (2012). et al.
- Optical detection of epigenetic marks: sensitive quantification and direct imaging of individual hydroxymethylcytosine bases. Chem. Commun. (Camb.) 49, 8599–8601 (2013). et al.
- Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691 (2013).
This study describes the first genome-wide base resolution map of 5fC in mESCs.
- Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes. Chemistry 19, 5836–5840 (2013). et al.
- Tissue-specific distribution and dynamic changes of 5-Hydroxymethylcytosine in mammalian genomes. J. Biol. Chem. 286, 24685–24693 (2011). et al.
- High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep. 3, 567–576 (2013). et al.
- Single-tube linear DNA amplification (LinDA) for robust ChIP–seq. Nature Methods 8, 565–567 (2011). et al.
- Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012). et al.
- Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protoc. 8, 1841–1851 (2013). et al.
- Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012). et al.
- Chemical modification-assisted bisulfite sequencing (CAB-seq) for 5-Carboxylcytosine detection in DNA. J. Am. Chem. Soc. 135, 9315–9317 (2013). et al.
- Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chem. 6, 435–440 (2014).
Together with reference 98 this paper describes a method for producing a whole-genome base resolution map of 5hmC (in mice and humans).
- Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009). et al.
- Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature Methods 7, 461–465 (2010).
Together with reference 96 this paper describes a method for producing a whole-genome base resolution map of 5hmC.
- Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation. BMC Biol. 11, 4 (2013). et al.
- Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nature Methods 9, 75–77 (2012). et al.
- The potential and challenges of nanopore sequencing. Nature Biotech. 26, 1146–1153 (2008). et al.
- Measurements of DNA immobilized in the alpha-hemolysin nanopore. Methods Mol. Biol. 870, 39–53 (2012). &
- Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl Acad. Sci. USA 106, 7702–7707 (2009). et al.
- Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl Acad. Sci. USA 105, 20647–20652 (2008). et al.
- Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS ONE 6, e25723 (2011). et al.
- Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules. J. Am. Chem. Soc. 133, 486–492 (2011). et al.
- Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl Acad. Sci. USA 110, 18904–18909 (2013). et al.
- Characterizing and measuring bias in sequence data. Genome Biol. 14, R51 (2013). et al.