DNA methylation occurs when DNA methyltransferase transfers a methyl group from S-adenosyl-methionine to cytosine in CpG dinucleotides. The methylation of 5’ methyl cytosine (5mC) nucleotides is an important epigenetic change that regulates gene activity and impacts several cellular processes including differentiation, transcriptional control and chromatin remodeling.

Genome wide analysis of 5mC, histone modifications and DNA accessibility are possible with next generation sequencing approaches and provide unique insight to complex phenotypes where primary genomic sequence is not sufficient.

Methods for methyl DNA sequencing can be broken down into three global approaches:

1) bisulfite sequencing

2) restriction enzyme based sequencing

3) targeted enrichment of methyl sites

We’ve outlined several library preparation techniques under each category.

1) Bisulfite Sequencing

Bisulfite-seq (1, 2) is a well-established protocol that provides single base resolution of methylated cytosine in the genome. Genomic DNA is bisulfite treated, deaminating un-methylated cytosines to uracils, which are later converted to thymidines. Methylated cytosines are protected from deamination, allowing researchers to identify methylation sites by comparing the sequence of bisulfite and non-bisulfite treated samples.

1-Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning

2-Highly integrated single-base resolution maps of the epigenome in Arabidopsis

2) Post Bisulfite Adapter Tagging (PBAT)

To avoid loss of template during bisulfite treatment, with PBAT (3) bisulfite treatment follows adapter ligation (tagging) and two rounds of random primer extension.

3- Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging.

3) Reduced Representation Bisulfite Sequencing (RRBS)

RRBS (4) is a method aimed at targeting sequencing coverage toward CpG islands or regions of the genome with dense CpG methylation. Sample is digested with one more restriction enzymes and then is treated with bisulfite prior to sequencing. This method offers single nucleotide methylation

4- Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

4) Oxidative Bisulfite Sequencing (oxBS-Seq)

5-hydroxymethylcytosine (5’hmC), an intermediate of the demethylation of 5-methylcytosine (5’mC) to cytosine cannot be distinguished using the bisulfite-seq approach. With oxBS-Seq (5), 5’hmC is oxidized, causing a deamination to uracil, while leaving 5’mC. Sequencing of both treated and untreated samples allows for single base resolution of 5’hmC and 5’mC modifications.

5-Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution

5) TET-Assisted Bisulfite Sequencing (TAB-Seq)

TAB-Seq (6) utilizes glucose moieties to interact with 5’hmC protecting it from TET protein oxidation. 5’mC and non-methylated cytosines are deaminated to uracil and sequenced as thymidines, allowing for the specific identification of 5’hmC.

6- Base-resolution analysis of 5-hydroxymethylcytosine in the Mammalian genome

6) Methylation Sensitive Restriction Enzyme Sequencing (MRE-Seq)

MRE-Seq (7) utilizes a combination of methyl sensitive and insensitive restriction enzymes to identify regions of CpG methylation status.

7- Genome-scale DNA methylation analysis

7) HpaII tiny fragment-Enrichment by Ligation-mediated PCR (HELP-Seq)

HELP-Seq (8) allows for intragenomic profiling and intergenomic comparisons of cytosine methylation by using HpaII and its methylation insensitive isoschizomer MSPI.

8- Comparative isoschizomer profiling of cytosine methylation: the HELP assay

8) Methylated DNA Immunoprecipitation Sequencing (MeDIP)

MeDIP (9) is a technique based on affinity enrichment of methylated DNA using either antibodies or other protein capable of binding methylated DNA. This technique pulls down heavily methylated regions of the genome, such as CpG islands. It does not offer single nucleotide resolution.

9- Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells

9) Methyl Binding Domain Capture (MBD-CAP)

MBD-CAP (10) uses methyl DNA binding proteins MeCP2, MBD1-2 and MBD3LI to immunoprecipitate methylated DNA. Similar to MeDIP, this approach pulls down regions that are heavily methylated and does not offer single nucleotide methylation resolution.

10- High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer

10) Probe Based Targeted Enrichment

Methyl-Seq Targeted enrichment – involves the use of synthetic, biotinylated oligonucleotides designed to CpG islands, shores, gene promoters and differentially methylated regions (DMRs). Kits are commercially available through Agilent and Roche Nimblegen.  

Finally, it’s worth mentioning Single Molecule Real Time (SMRT) DNA Sequencing. SMRT sequencing by Pacific Biosciences uses the kinetics of base incorporation to allow for direct detection of methylated cytosines. Unlike any of the protocols mentioned, this does not require the use of restriction enzymes or bisulfite reagent.

Several service providers on Genohub offer targeted bisulfite-seq, reduced representation bisulfite-seq (RRBS), methylated DNA immunoprecipitation seq (MeDIP) and whole genome bisulfite-seq (WGBS) library preparation and sequencing services. Simply click on one of these application types to get started.