Key Points
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Cytosine methylation has an important role in the regulation of mammalian gene expression. Additional forms of cytosine modification, including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are intermediates of 5mC that occur during DNA demethylation, and are hypothesized to have additional functional roles in cellular development.
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Recent efforts in method development for quantifying cytosine modifications and mapping their genomic location, preferentially at single-base resolution, have focused on improving accuracy, increasing throughput, lowering sample input and reducing costs.
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One major class of DNA methylation assays relies heavily on bisulphite treatment and sequencing approaches that provide single-base resolution. Whole-genome bisulphite sequencing (WGBS) and reduced representation bisulphite sequencing (RRBS) are widely used to generate genome-wide maps of DNA methylation. Application of low input methods such as tagmentation-based WGBS (T-WGBS) and post-bisulphite adaptor tagging (PBAT) allows for the detection of 5mC using input DNA from hundreds to thousands of cells.
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Various targeted methylation-sequencing approaches, such as liquid hybridization and parallel amplification, have been developed to characterize DNA methylation at selected regions. These approaches are more cost effective for analysing large numbers of samples than non-targeted methods.
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Improvements in several techniques, including restriction enzyme-based single-cell methylation assay (RSMA), limiting dilution bisulphite (pyro) sequencing and single-cell RRBS (scRRBS) and single-cell BS-seq (scBS-seq), have enabled 5mC detection in single cells.
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Array-based methods are widely used in many studies of large cohorts. A major improvement is the dramatic increase in features per array and, hence, genome coverage.
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The combination of chromatin immunoprecipitation (ChIP) assay and bisulphite sequencing (BS-seq) in sequential order allows for simultaneous detection of DNA methylation and other epigenetic marks. Profiling of fluorescence-labelled DNA fragments using nanofluidic devices has helped to correlate multiple chromatin marks with DNA methylation. In addition, the incidence of DNA methylation and nucleosome occupancy can be mapped with nucleosome occupancy and methylome sequencing (NOMe-seq).
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A major challenge in the quantification of 5mC oxidation derivatives is their rarity in mammalian genomes. The use of specific antibodies and chemical or enzymatic modifications has enabled the enrichment of 5mC oxidation derivatives and the determination of their relative abundance in the genome, albeit with limited resolution. Chemical modifications coupled with BS-seq can be used to identify 5mC oxidation derivatives at single-base resolution, as in the oxidative bisulphite sequencing (oxBS-seq) and Tet-assisted bisulphite sequencing (TAB-seq) approaches for 5hmC quantification, the 5fC chemical modification-assisted bisulphite sequencing (fCAB-seq) and redBS-seq approaches for 5fC quantification, and the chemical modification-assisted bisulphite sequencing (CAB-seq) approach for 5caC quantification.
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Third-generation DNA sequencing technologies, including single-molecule, real-time (SMRT) sequencing and nanopore sequencing, are very appealing for direct reading of 5mC and other DNA modifications on the same DNA molecule, with the potential advantages of speed, read length and the lack of chemical treatment. Nevertheless, the throughput and accuracy of these technologies are still not sufficient for routine use.
Abstract
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.
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References
Arand, J. et al. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases. PLoS Genet. 8, e1002750 (2012).
Hon, G. C. et al. Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nature Genet. 45, 1198–1206 (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).
Lister, R. et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).
Ziller, M. J. et al. Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS Genet 7, e1002389 (2011).
Jeong, M. et al. Large conserved domains of low DNA methylation maintained by Dnmt3a. Nature Genet. 46, 17–23 (2014).
Stadler, M. B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).
Xie, W. et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134–1148 (2013).
Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).
Jones, P. A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nature Rev. Genet. 13, 484–492 (2012).
Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nature Rev. Genet. 14, 204–220 (2013).
Xie, M. et al. DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape. Nature Genet. 45, 836–841 (2013).
Yu, M. et al. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nature Protoc. 7, 2159–2170 (2012).
Pastor, W. A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011).
Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013).
Szulwach, K. E. et al. 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nature Neurosci. 14, 1607–1616 (2011).
Iurlaro, M. et al. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119 (2013).
Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).
Laird, P. W., Principles and challenges of genome-wide DNA methylation analysis. Nature Rev. Genet. 11, 191–203 (2010).
Shen, L. & Zhang, Y. 5-hydroxymethylcytosine: generation, fate, and genomic distribution. Curr. Opin. Cell Biol. 25, 289–296 (2013).
Song, C.-X., Yi, C. & He, C. Mapping recently identified nucleotide variants in the genome and transcriptome. Nature Biotech. 30, 1107–1116 (2012).
Bock, C. Analysing and interpreting DNA methylation data. Nature Rev. Genet. 13, 705–719 (2012).
Krueger, F. et al. DNA methylome analysis using short bisulfite sequencing data. Nature Methods 9, 145–151 (2012).
Grunau, C., Clark, S. J. & Rosenthal, A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, e65 (2001).
Ogino, S. et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J. Mol. Diagn. 8, 209–217 (2006).
Nestor, C. E. et al. Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res. 22, 467–477 (2012).
Boyle, P. et al. Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling. Genome Biol. 13, R92 (2012).
Wang, J. 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).
Schillebeeckx, M. 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).
Guo, H. 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).
Ruiz, S. 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).
Okuizumi, H. et al. Restriction landmark genome scanning. Methods Mol. Biol. 791, 101–112 (2011).
Maunakea, A. K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).
Taiwo, O. et al. Methylome analysis using MeDIP-seq with low DNA concentrations. Nature Protoc. 7, 617–636 (2012).
Clark, C. 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).
Aberg, K. A. 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).
Lan, X. et al. High resolution detection and analysis of CpG dinucleotides methylation using MBD-seq technology. PLoS ONE 6, e22226 (2011).
Brinkman, A. B. et al. Whole-genome DNA methylation profiling using MethylCap-seq. Methods 52, 232–236 (2010).
Butcher, L. M. & Beck, S. AutoMeDIP-seq: a high-throughput, whole genome, DNA methylation assay. Methods 52, 223–231 (2010).
Bock, C. et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotech. 28, 1106–1114 (2010).
Matarese, F., Carrillo-de Santa Pau, E. & Stunnenberg, H. G. 5-hydroxymethylcytosine: a new kid on the epigenetic block? Mol. Syst. Biol. 7, 562 (2011).
Komori, H. K. et al. 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.
Nautiyal, S. et al. High-throughput method for analyzing methylation of CpGs in targeted genomic regions. Proc. Natl Acad. Sci. USA 107, 12587–12592 (2010).
Varley, K. E. & Mitra, R. D. Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome Res. 20, 1279–1287 (2010).
Diep, D. et al. 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.
Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature Biotech. 27, 353–360 (2009).
Lee, E.-J. et al. Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing. Nucleic Acids Res. 39, e127 (2011).
Wang, J. et al. High resolution profiling of human exon methylation by liquid hybridization capture-based bisulfite sequencing. BMC Genomics 12, 597 (2011).
Ivanov, M. et al. In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes. Nucleic Acids Res. 41, e72 (2013).
Yamaguchi, S. et al. Tet1 controls meiosis by regulating meiotic gene expression. Nature 492, 443–447 (2012).
Kobayashi, H. et al. High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome Res. 23, 616–627 (2013).
Shirane, K. 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).
Kobayashi, H. & Kono, T. DNA methylation analysis of germ cells by using bisulfite-based sequencing methods. Methods Mol. Biol. 825, 223–235 (2012).
Adey, A. & Shendure, J. 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.
Wang, Q. et al. Tagmentation-based whole-genome bisulfite sequencing. Nature Protoc. 8, 2022–2032 (2013).
Miura, F. et al. Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 40, e136 (2012).
Kantlehner, M. et al. A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res. 39, e44 (2011).
Lorthongpanich, C. 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.
El Hajj, N. et al. Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics 6, 1176–1188 (2011).
Guo, H. et al. The DNA methylation landscape of human early embryos. Nature 511, 606–610 (2014).
Smallwood, S.A. et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nature Methods 11, 817–820 (2014).
Irizarry, R. A. et al. Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18, 780–790 (2008).
Bibikova, M. et al. High density DNA methylation array with single CpG site resolution. Genomics 98, 288–295 (2011).
Yalcin, A. 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).
Gilson, E. & Horard, B. Comprehensive DNA methylation profiling of human repetitive DNA elements using an MeDIP-on-RepArray assay. Methods Mol. Biol. 859, 267–291 (2012).
Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126, 1189–1201 (2006).
Oliver, V. F. 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).
Dumenil, T. D. et al. Genome-wide DNA methylation analysis of formalin-fixed paraffin embedded colorectal cancer tissue. Genes Chromosomes Cancer 53, 537–548 (2014).
Brinkman, A. B. 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.
Statham, A. L. et al. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA. Genome Res. 22, 1120–1127 (2012).
Li, Y. & Tollefsbol, T. O. Combined chromatin immunoprecipitation and bisulfite methylation sequencing analysis. Methods Mol. Biol. 791, 239–251 (2011).
Cipriany, B. R. et al. Single molecule epigenetic analysis in a nanofluidic channel. Anal. Chem. 82, 2480–2487 (2010).
Cipriany, B. R. 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.
Murphy, P. J. et al. Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells. Proc. Natl Acad. Sci. USA 110, 7772–7777 (2013).
You, J. S. 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).
Kelly, T. K. 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.
Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).
Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).
Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).
Stroud, H. et al. 5-hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 12, R54 (2011).
Thomson, J. P. et al. Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res. 41, e206 (2013).
Inoue, A. et al. Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development. Cell Res. 21, 1670–1676 (2011).
Ito, S. 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).
Ko, M. et al. Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature 497, 122–126 (2013).
Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).
Szwagierczak, A. et al. Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181 (2010).
Song, C. X. et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nature Biotech. 29, 68–72 (2011).
Baskin, J. M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007).
Pastor, W. A. et al. The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine. Nature Protocols 7, 1909–1917 (2012).
Robertson, A. B. et al. Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nature Protocols 7, 340–350 (2012).
Michaeli, Y. et al. Optical detection of epigenetic marks: sensitive quantification and direct imaging of individual hydroxymethylcytosine bases. Chem. Commun. (Camb.) 49, 8599–8601 (2013).
Song, C.-X. 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.
Hu, J. et al. Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes. Chemistry 19, 5836–5840 (2013).
Kinney, S. M. et al. Tissue-specific distribution and dynamic changes of 5-Hydroxymethylcytosine in mammalian genomes. J. Biol. Chem. 286, 24685–24693 (2011).
Sun, Z. et al. High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep. 3, 567–576 (2013).
Shankaranarayanan, P. et al. Single-tube linear DNA amplification (LinDA) for robust ChIP–seq. Nature Methods 8, 565–567 (2011).
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. et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protoc. 8, 1841–1851 (2013).
Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).
Lu, X. et al. Chemical modification-assisted bisulfite sequencing (CAB-seq) for 5-Carboxylcytosine detection in DNA. J. Am. Chem. Soc. 135, 9315–9317 (2013).
Booth, M. J. 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).
Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).
Flusberg, B. A. 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.
Clark, T. et al. Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation. BMC Biol. 11, 4 (2013).
Song, C.-X. et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nature Methods 9, 75–77 (2012).
Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotech. 26, 1146–1153 (2008).
Purnell, R. & Schmidt, J. Measurements of DNA immobilized in the alpha-hemolysin nanopore. Methods Mol. Biol. 870, 39–53 (2012).
Stoddart, D. et al. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl Acad. Sci. USA 106, 7702–7707 (2009).
Butler, T. Z. et al. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl Acad. Sci. USA 105, 20647–20652 (2008).
Manrao, E. A. et al. Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS ONE 6, e25723 (2011).
Wanunu, M. et al. Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules. J. Am. Chem. Soc. 133, 486–492 (2011).
Laszlo, A. H. et al. Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl Acad. Sci. USA 110, 18904–18909 (2013).
Ross, M. G. et al. Characterizing and measuring bias in sequence data. Genome Biol. 14, R51 (2013).
Acknowledgements
The authors gratefully acknowledge members of the Zhang laboratory for their proofreading of the manuscript. We apologize to those authors whose works were not covered here due to space constraints. This work is funded by the US National Institutes of Health grants R01GM097253 and R01AG042187. D.H.D. is supported by a UCSD-CIRM pre-doctoral fellowship.
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Glossary
- Partially methylated domains
-
(PMDs). Large contiguous regions of the genome (mean length ~ 153 kb) that display an intermediate level of DNA methylation (average <70%).
- Ten-eleven translocation
-
(TET). DNA-binding enzymes that have been found to be methylcytosine dioxygenases in mammals and include TET1, TET2 and TET3. They were named for the genetic variant found in the TET1 gene sequence in acute myeloid and lymphocytic leukaemia.
- Short interspersed nuclear elements
-
(SINEs). A subtype of transposable elements reverse-transcribed from RNA molecules. They do not encode a functional reverse transcriptase protein and cover a substantial portion of primate genomes, including all Alu sequences.
- Long terminal repeats
-
(LTRs). Stretches of DNA sequences that are identical and repeat in hundreds to thousands of copies. LTRs were founds at the end of retrotransposons and act as insertion sites for viruses to insert their genetic material into the host genome.
- Type I errors
-
The errors that result when there are false positives or when falsely rejecting the null hypothesis.
- Type II errors
-
The errors that result when there are false negatives or incorrect failure to reject the null hypothesis
- Laser-capture microdissection (LCM)
-
A method for isolating specific cells or specific areas from cell, tissue or organism samples using laser cutting under microscopic visualization.
- Ligation capture
-
A method for capturing restriction enzyme-digested DNA molecules via the annealing of an oligonucleotide containing complementary sequences to adaptor oligonucleotides to the DNA molecules and to the adaptor oligonucleotides. The adaptors and DNA molecules are then ligated together, allowing for PCR amplification of only the ligated products.
- Bisulphite padlock probe (BSPP) capture
-
A method for capturing the target CpG sites of bisulphite treated genomic DNA with bisulphite padlock probes (BSPP). The two capturing arms of the BSPPs are designed to flank the region of interest. After annealing padlock probes to target regions, polymerization is preformed to fill the gap and two ends of the padlock probe are joined together to form circularized DNA. The captured regions are amplified with barcoded adaptor primers and sequenced.
- Liquid hybridization
-
A method for capturing fragmented DNA molecules via the annealing of biotinylated oligonucleotides to the DNA molecules. The binding of biotin to streptavidin beads allows for washing and removal of uncaptured DNA molecules, and subsequent elution of the captured DNA molecules.
- Microdroplet PCR
-
Massively parallel PCR amplification of target sequences in microdroplets. The process involves the preparation of template and PCR mix in picoliter volume and primer droplets, combination of individual template and primer pair droplet, pooling the fused droplets for thermal cycling, and releasing of PCR amplicons for purification and sequencing.
- Barcoded primers
-
Unique DNA sequences that are incorporated into adaptor sequences for tagging of different samples before sample pooling and shotgun sequencing.
- Pyrosequencing
-
A sequencing-by-synthesis method based on the detection of phyrophosphate released upon nucleotide incorporation.
- Shotgun library construction
-
The generation of a sequencing library involving random fragmentation of DNA and addition of adaptor sequences to both ends of DNA fragments before sequencing.
- Transposase-based library construction
-
A procedure to generate a sequencing library using the transposase Tn5 to insert common transposon sequences to DNA. DNA segments are then amplified by annealing of primers to the transposon sequences.
- Tn5 transposase
-
A member of the RNase superfamily of proteins that harbours retroviral integrases to catalyse random insertion of transposon DNA into target DNA.
- Binning
-
A computational technique frequently used to reduce noise by grouping sequencing reads mapped to contiguous genomic segments.
- Nucleosome
-
A basic unit of DNA packaging in eukaryotes that consists of section of DNA (~ 166 bp) wrapping around a histone core. Nucleosome structure helps to condense DNA into smaller volume. Nucleosomes are subunits of chromatin.
- GpC methyltransferase (M.CviPI)
-
An enzyme from Chlorella virus that methylates all cytosines within the double-stranded dinucleotide recognition sequence 5′... GC...3′.
- CCCTC-binding factor
-
(CTCF). A chromatin binding factor with highly conserved zinc finger domains that control binding to consensus DNA target sequences. CTCF regulates transcription by binding to chromatin insulators and preventing interaction between the promoter and enhancers or silencers.
- Third-generation sequencing
-
A new progression of sequencing technology that aims to improve throughput and reduce sequencing cost and time. The main goals of third-generation sequencing are to eliminate the DNA amplification step before sequencing and to enable real-time signal monitoring.
- β-glucosyltransferase
-
(βGT). An enzyme that transfers the glucose residue of uridine diphosphosphate glucose (UDP-Glc) specifically to the hydroxyl group of 5-hydroxymethylcytosine to generate β-glucosyl-5hmC (5gmC).
- Click chemistry
-
A nonspecific chemical reaction that combines small modular units and is used to generate or label a substance. Azide alkyne Huisgen cycloaddition, in which an azide and alkyne interact to form a 1,2,3-triazole (with 5-membered ring) is the most popular click chemistry reaction. Click chemistry has been used for selectively labelling biomolecules.
- Glucosylation
-
The process of transferring a glucose residue from a nucleotide sugar derivative, such as from uridine diphosphate glucose (UDP-Glc) to a target molecule.
- J-binding proteins
-
Proteins that specifically bind to the base J (β-D-glucopyranosyloxymethyluracil), a modified form of uracil found in the DNA of a number of organisms, such as human pathogen Trypanosoma and the kinetoplastids. Base J is formed by hydroxylation of thymidine and subsequent glycosylation by glycosyltransferase enzyme.
- Isoschizomer
-
Restriction enzymes that have the same recognition sequences and cleave at the same positions.
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Plongthongkum, N., Diep, D. & Zhang, K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet 15, 647–661 (2014). https://doi.org/10.1038/nrg3772
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DOI: https://doi.org/10.1038/nrg3772
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