DNA methylation, one of the most studied epigenetic modifications, refers to the addition of a methyl group to the fifth carbon of cytosine (C) catalyzed by DNA methyltransferases (Dnmts), forming 5-methylcytosine (5mC). DNA methylation predominantly occurs in CpGs but is also found in non-CpG contexts.

1. Sequencing-Based
Approaches for Profiling
DNA Methylation

2. Introduction to DNA Methylation
DNA methylation, one of the most studied epigenetic modifications, refers to the
addition of a methyl group to the fifth carbon of cytosine (C) catalyzed by DNA
methyltransferases (Dnmts), forming 5-methylcytosine (5mC). DNA methylation
predominantly occurs in CpGs but is also found in non-CpG contexts.

3. Introduction to DNA Methylation
Transcriptional repression
Transposon inactivation
X chromosome
inactivation
Embryonic development
Genomic imprinting
Alteration of chromatin
structure

4. Next-generation sequencing (NGS) has been widely used for genome-wide DNA
methylation analysis:
Next-Generation Sequencing

5. Next-Generation Sequencing
MeDIP-Seq
RRBS
WGBS

6. Next-Generation Sequencing
Method
s
Strength Weakness Resolution Quantitativ
e Nature
Cost
WGBS Evaluate methylation state of
almost every CpG sites
-High cost
-Substantial DNA degradation after
bisulfite treatment
-Cannot discriminate between 5mC and
5hmC
Single
base
Digital High
RRBS -High CGI coverage
-High sensitivity
-Cost-effective comparing to
WGBS
May exhibit a poor coverage at
intergenic and distal regulatory elements
-Substantial DNA degradation after
bisulfite treatment
-Limited to regions in proximity to
enzymes’ recognition sites
-Cannot discriminate between 5mC and
5hmC
Single
base
Digital Moderat
e
MeDIP-
seq
-Cost-effective
-No mutation introduced
-Specific to 5mC/5hmC
depending on the antibody
specificity
-More sensitive in regions with
-Biased toward hypermethylated regions
-Cannot identify individual 5mC sites
-Inability to predict absolute methylation
level
~100 bp Abundance Moderat
e
Table. A comparison among WGBS, RRBS, and MeDIP-
seq.

7. Emerging 3rd generation sequencing technologies, including PacBio SMRT and
Oxford nanopore sequencing, have been recently applied in epigenetics research.
Advantages:
Third-Generation Sequencing
Minimal chemical
modification
Eliminate steps
for DNA
amplification
Reduced input Generate
longer reads
Detect different
types of
epigenetic
modifications

8. • PacBio SMRT allows the direct
detection of base modifications by
monitoring the activity of DNA
polymerase during the
incorporation of different
fluorescently labeled nucleotides
into complementary DNA strands.
• The detection of diverse base
modifications involves the
measurement of the kinetics
variation in the time between base
incorporations.
Third-Generation Sequencing

9. • Single-stranded DNA is pulled by a
phage DNA polymerase through a
bacterial pore in single-nucleotide
steps, and the ion current through the
pore is recorded.
• C can be distinguished from 5mC and
5hmC based on differences in the
current traces.
Third-Generation Sequencing

10. Disadvantages:
Third-Generation Sequencing
Higher error rate Higher costLower throughput

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