What Is DNA Methylation?

A Quick Guide for DNA Methylation Profiling with NGS-based Methods

What is DNA Methylation – An Epigenetic Modification that Regulates Gene Expression

Did you know that the amount of cuddles a baby receives from a caregiver could affect the baby’s DNA methylation profile, and in turn, the developmental progress?1 In fact, DNA methylation affects various aspects of daily life that most people may not even think about, such as the ripening process of sweet orange fruits.2 So, what is DNA methylation?

DNA methylation is a chemical modification to DNA molecules. In most cases, DNA methylation refers to the covalent addition of a methyl group to the 5-carbon position of cytosine, forming 5-methylcytosine (5mC) (Figure 1, the two structures on the left). DNA methyltransferases perform this reaction to establish and maintain DNA methylation patterns across the genome. DNA methylation regulates gene expression without changing the sequences of the nucleotides. Hence, DNA methylation is an epigenetic mark, where “epi” implies that this feature is “on top of” genetics playing its roles without alterations in the DNA sequence.

Figure showing the chemical structures of cytosines and 5-methylcytosines
Figure 1. The chemical structures of cytosine, 5-methylcytosine, uracil and thymine. 5-methylcytosine is commonly referred to as DNA methylation.

DNA methylation is among the most extensively studied epigenetic marks.3 In eukaryotes, DNA methylation pervasively occurs at CpG dinucleotides. The patterns of DNA methylation at promoters, gene bodies and enhancers play vital roles in regulating gene expression.4-6

Commonly Recognized DNA Methylation Patterns

Some DNA methylation patterns have been widely studied and researchers have acquired a fair amount of knowledge on their regulation of gene expression. A few examples of such DNA methylation patterns are as follows.

  • DNA hypermethylation (substantially increased DNA methylation levels) in promoter regions is generally associated with gene suppression or silencing. A good example is that DNA hypermethylation serves to inactivate the X chromosome and silence repetitive DNA sites.4
  • The CpG sites in CpG islands (CGIs, regions with high frequency of GpG sites) associated with the promoters of housekeeping genes and developmental regulatory genes are usually resistant to DNA methylation. However, these promoter CGIs were found to be aberrantly hypermethylated in tumor suppressor genes and DNA mismatch repair genes, which in turn suppress the related genes’ activation and expression. Such aberrant DNA methylation pattern in promoter CGIs is perhaps the most widely studied epigenetic alteration in cancers.5, 6
  • CpGs in enhancers have been found to contain tissue- or cell type-specific DNA methylation patterns. Such differentially methylated regions (DMRs) may have contributed to the unique gene expression among different tissue and cell types.7

Given that precise gene regulation is important for many fundamental biological processes, DNA methylation is essential throughout organismal development and homeostasis. Therefore, elucidating DNA methylation patterns in samples from various species under diverse conditions enables further exploration of its roles in gene regulatory mechanisms. This will moreover enhance our understanding in how DNA methylation influences human diseases and aging, as well as the health of ecosystems. How then, do we profile DNA methylation?

How to Profile DNA Methylation

The gold-standard reaction for DNA methylation analysis is called bisulfite conversion. Bisulfite reagents convert unmethylated cytosine residues to uracils (Figure 1, the third structure) while leaving the methylated ones untouched. During PCR, DNA polymerase recognize uracils as thymines and the subsequent analysis takes “C” as methylated cytosines while “T” (Figure 1, the last structure) as unmethylated cytosines in the DNA sample. Many methods have been developed to investigate DNA methylation-based on bisulfite conversion. Since the demand of genome-wide DNA methylation profiling has increased, NGS-based methods have become much more popular.

Methods Based-on Next-Generation Sequencing (NGS). NGS-based methods are the most comprehensive and have emerged as the primary means of DNA methylation analysis recently. NGS-based methods produce results with high coverages across an entire genome and are compatible with any species. Bisulfite sequencing is thus gaining tremendous popularity among researchers for DNA methylation profiling, indicated by the increasing number of related publications over the years. DNA samples that are bisulfite converted must be constructed into libraries that contain adapters for sequencing on a compatible NGS instrument. The methylated cytosines are recognized as “C” in NGS readouts.

Thus far, researchers have implemented a myriad of bisulfite sequencing methods to uncover DNA methylation information for diverse biological questions. The most extensively used NGS methods include reduced representation bisulfite sequencing (RRBS), whole-genome bisulfite sequencing (WGBS), and targeted bisulfite sequencing. These methods share common steps in the procedure of library preparation such as bisulfite conversion, adapter ligation and PCR amplification, but differ slightly at the beginning of the workflows preparing the input DNA materials.

Reduced Representation Bisulfite Sequencing (RRBS)
  • RRBS uses restriction enzyme digestion to enrich for CpG-rich regions across the genome from the input DNA before proceeding to the common steps in bisulfite sequencing library preparation.
  • Features of RRBS.
    • RRBS reduces the number of required sequencing reads to achieve similar data quality compared to sequencing the entire bisulfite converted genome as in WGBS, and reveals DNA methylation information on most important regulatory regions such as promoters, CpG islands, and in gene bodies. With approximately 10-20% of the sequencing reads that are normally required by WGBS, RRBS can cover ≥70% of promoters, CpG islands, gene bodies, and around 35% of enhancers.
    • RRBS is more cost-effective compared to WGBS in sequencing and is ideal for scientists to screen genome-wide DNA methylation information in large-scale studies.
  • Many variations of RRBS have emerged over the years and an example of the basic RRBS workflow in Figure 2 covers the main steps to prepare RRBS libraries from input genomic DNA: MspI digestion, adapter ligation, bisulfite conversion, and PCR amplification with indexed primers. This specific kit has a simple protocol and is compatible with as low as 10 ng of genomic DNA, making RRBS more accessible to all users.
Figure showing the workflow of Zymo-Seq RRBS Library Kit
Figure 2. The workflow of Zymo-Seq RRBS Library Kit for RRBS as one of the NGS-based methods for DNA methylation profiling.
Whole-Genome Bisulfite Sequencing (WGBS)
  • Multiple workflows are available for WGBS. Though details can be different from each other, the workflows usually consist of optional fragmentation of input genomic DNA, bisulfite treatment of DNA, adapter ligation, and PCR amplification with indexed primers.
  • Features of WGBS.
    • WGBS detects the methylation status of all the cytosines in the input sample, thus it presents scientists a comprehensive profile of DNA methylation levels across the entire genome. Scientists are more likely to obtain de novo discovery of new DNA methylation patterns using WGBS data.
    • Depending on the size of the sample genome, WGBS can require several hundreds of millions of sequencing reads to obtain significant depth for accurate methylation calling. Therefore, the sequencing cost can be exceptionally high for WGBS.
  • A general WGBS workflow is shown in Figure 3. This kit is particularly outstanding in its compatibility with small amount of gDNA input as low as 10 pg.
Figure showing the workflow of Pico Mthyl-Seq Library Prep Kit for WGBS
Figure 3. The workflow of Pico Methyl-Seq Library Prep Kit for WGBS as one of the NGS-based methods for DNA methylation profiling.
Targeted Bisulfite Sequencing
  • Besides the two previously mentioned methods for genome-wide DNA methylation profiling, targeted bisulfite sequencing enables site-specific DNA methylation detection in desired loci.
  • Features of targeted bisulfite sequencing.
    • The most direct way is to design NGS-compatible PCR primers for specific gene regions of interest to generate libraries. This allows the investigation of DNA methylation levels in only these selected genomic regions, therefore, reducing per sample sequencing costs as compared to genome-wide based strategies.
    • Targeted bisulfite sequencing is a great option for validation of data generated by other genome-wide methods as well as screening on multi-targeted gene regions in large sample cohorts. Scientists can examine from 1 to 192 regions and beyond in the same sample using this method.
  • A general workflow of the targeted bisulfite sequencing available from Zymo Research is shown in Figure 4.
Figure showing the workflow of Zymo’s MethylCheck platform for targeted bisulfite sequencing
Figure 4. The workflow of Zymo’s MethylCheck platform for targeted bisulfite sequencing as one of the NGS-based methods for DNA methylation profiling.
NGS-Based DNA Methylation Study is Powerful

NGS-Based DNA Methylation Study Has Been Fruitful. Scientists have continually improved the wet lab techniques as well as related bioinformatic packages of all the bisulfite sequencing methods to expand their applications.

  • Researchers have applied WGBS to identify the mouse liver methylation heterogeneity among single cells.8 Averagely, 21.6 million CpG sites per bulk sample and 2.2 million CpG sites per single cell sample were covered.
  • Scientists have used RRBS to map the DNA methylation in samples from chicken, opossum, and platypus.9 Their data were the first to demonstrate the participation of DNA methylation in the X chromosome inactivation in marsupial mammals.
  • RRBS was also used to discover DNA methylation biomarkers for Barrett’s esophagus. This syndrome is an important risk factor for the development of esophageal cancer.10 Once they identified a CpG patch as the biomarker from the RRBS data of 46 biopsy samples, the researchers relied on targeted bisulfite sequencing for validation and expansion of the enrolled CpG sites in fresh and archived clinical samples.

Methods using Microarrays. Before the rapid rise of NGS-based methods, the conventional profiling method for DNA methylation has been microarray-based methods. Many datasets have been generated from these microarray platforms. Briefly, bisulfite converted DNA are denatured to hybridize with the CpG-specific probes immobilized on the array. The tagged dideoxynucleotides are incorporated during single-base extension to provide unmethylated and methylated signals that are used to call methylation levels on each CpG.

  • Limitations of array-based methods. While robust to use and easy to compare data from different datasets, array-based methods have several limitations. The top limitations are:
    • The coverage of CpG sites in the genome of interest is largely limited. The MethylationEpic array covers only ~3% of the total CpG sites in the human genome.
    • Little flexibility exists in terms of sample species - as one array is only compatible with one specific species.

As a result, NGS-based DNA methylation methods have surpassed the microarray-based methods and are gaining an increasing amount of attention and impact. NGS-based methods for measuring DNA methylation have expanded our knowledge of the multifaceted roles of DNA methylation. As the methodologies keep improving, the power of NGS-based DNA methylation analysis will undoubtedly be further harnessed to generate more informative pieces to help assemble the puzzles in epigenetics and beyond.

See below for a selection of Zymo Research products and services for DNA methylation study. We have a comprehensive solution for DNA methylation profiling, for every application and workflow.


  1. Moore, S. R.; McEwen, L. M.; Quirt, J.; Morin, A.; Mah, S. M.; Barr, R. G.; Boyce, W. T.; Kobor, M. S., Epigenetic correlates of neonatal contact in humans. Dev Psychopathol 2017, 29 (5), 1517-1538.
  2. Huang, H.; Liu, R.; Niu, Q.; Tang, K.; Zhang, B.; Zhang, H.; Chen, K.; Zhu, J. K.; Lang, Z., Global increase in DNA methylation during orange fruit development and ripening. Proc Natl Acad Sci U S A 2019, 116 (4), 1430-1436.
  3. Smith, Z. D.; Meissner, A., DNA methylation: roles in mammalian development. Nat Rev Genet 2013, 14 (3), 204-20.
  4. Greenberg, M. V. C.; Bourc'his, D., The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 2019, 20 (10), 590-607.
  5. Flavahan, W. A.; Gaskell, E.; Bernstein, B. E., Epigenetic plasticity and the hallmarks of cancer. Science 2017, 357 (6348).
  6. Ehrlich, M., DNA hypermethylation in disease: mechanisms and clinical relevance. Epigenetics 2019, 14 (12), 1141-1163.
  7. Song, Y.; van den Berg, P. R.; Markoulaki, S.; Soldner, F.; Dall'Agnese, A.; Henninger, J. E.; Drotar, J.; Rosenau, N.; Cohen, M. A.; Young, R. A.; Semrau, S.; Stelzer, Y.; Jaenisch, R., Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs. Mol Cell 2019, 75 (5), 905-920.e6.
  8. Gravina, S.; Dong, X.; Yu, B.; Vijg, J., Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome. Genome Biol 2016, 17 (1), 150.
  9. Waters, S. A.; Livernois, A. M.; Patel, H.; O'Meally, D.; Craig, J. M.; Marshall Graves, J. A.; Suter, C. M.; Waters, P. D., Landscape of DNA Methylation on the Marsupial X. Mol Biol Evol 2018, 35 (2), 431-439.
  10. Moinova, H. R.; LaFramboise, T.; Lutterbaugh, J. D.; Chandar, A. K.; Dumot, J.; Faulx, A.; Brock, W.; De la Cruz Cabrera, O.; Guda, K.; Barnholtz-Sloan, J. S.; Iyer, P. G.; Canto, M. I.; Wang, J. S.; Shaheen, N. J.; Thota, P. N.; Willis, J. E.; Chak, A.; Markowitz, S. D., Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett's esophagus. Sci Transl Med 2018, 10 (424).