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DNA Methylation Methods & Selection Guide

DNA methylation is a crucial epigenetic regulator of gene expression and genomic organization in nearly all organisms, leading to the emergence of numerous methods for mapping 5-mC and 5-hmC. The number of options for analysis can make it difficult to know where to start. However, the best tactic can be determined based on the goals and resources for a particular experiment, with each method having advantages and drawbacks.

The following outline presents a comparison of methods.

Resource Overview to Common DNA Methylation Methods

Method Name Method Overview Input Resolution of Methylation Coverage Advantages Disadvantages Cost Per Sample Best Application
Global Quantification of DNA Methylation
Global 5mC/5hmC ELISA Isolated sample DNA is bound to wells of ELISA plate. A capture antibody for 5mC/5hmC binds to methylated DNA. Secondary antibody and developer is used for colorimetric detection. >10‑100 ng Genome Global quantitation of overall all methylation in DNA. Easy and inexpensive for many samples. Does not tell you which regions of the genome have methylation changes. $ Useful for screening/monitoring samples for changes in DNA methylation related to disease, aging, or environmental factors like smoking or obesity.
DNA Methylation Enrichment
MeDIP-Seq or hMeDIP-Seq Uses a methylation (5mC/5hmC) specific antibody to perform immunoprecipitation on fragmented DNA. >100 ng 100-500 bp ~88% CpG sites Covers both CpG and non CpG sites. MeDIP requires ssDNA. Higher background than enzymatic based methods. $$ Cost effective method for determining differently methylated regions of the genome related to disease or environmental exposure.
MBD-Seq A tagged methyl-CpG binding domain (MBD) protein is used to capture methylated CpG from fragmented DNA. >5‑10 ng 100-500 bp Up to 27 million CpGs Requires less sample than MeDIP. More sensitive and specific to CpG than MeDIP. Misses non-CpG methylation. $$ Cost effective method for determining differently methylated regions of the genome related to disease or environmental exposure.
5hmC-Seal Uses beta-Glucosyltransferase to selectively transfer modified glucose from UDP-Azide-Glucose donor to 5hmC on fragmented DNA. A chemical labeling reaction then attaches a biotin conjugate to the modified glucosyl-5-hmC so that it can be captured by streptavidin beads. >10 ng 100-500 bp N/A Requires less sample than hMeDIP. More sensitive and specific than antibody based detection of 5hmC. Can be difficult to consistently manufacture UDP-Azide-Glucose. $$ Commonly used to detect changes in hydroxymethylation in cfDNA samples that could be used for biomarkers for disease.
Single-Nucleotide Resolution Analysis of DNA Methylation
WGBS - Whole-Genome Bisulfite Sequencing DNA is bisulfite treated and PCR amplified to reveal the methylation pattern of the DNA. The DNA is then used to prepare a sequencing library. >500 ng Single base 28 million CpGs Comprehensive coverage of methylated and unmethylated regions. Expensive & time consuming. Does not distinguish 5mC from 5hmC. $$$$ Ideal when interested in low-CpG-density regions, intergenic “gene deserts”, or partially methylated domains and distal regulatory elements, that would be missed by RRBS.
RRBS - Reduced Representation Bisulfite Sequencing A restriction enzyme, typically MspI, is first used to cut DNA fragments in regions of high CG, which are common in CpG islands and promotors. DNA is then size selected for 40-220 bp fragments, adapter ligated, and bisulfite converted. >200 ng Single base 3-5 million CpGs
80-85% CpG Islands
50-60% promoters
density >= 3 CpG/100 bp
Can sequence regions dense in CpG methylation genome wide at single-base resolution for lower cost than WGBS. Biased towards regions enriched in CCGG. Genes which have no or sparse CCGG motif not covered. Does not distinguish 5mC from 5hmC. $$$ Profiling CpG islands and promoters.
Targeted Bisulfite Sequencing DNA is bisulfite treated and PCR amplified to reveal the methylation pattern of the DNA. PCR primers are then used to amplify targeted loci. These amplicons then undergo library construction and are sequenced. >1‑50 ng Single base Regions of selected amplicons; typically <8. Once primers are designed, high sequencing depth at low cost for regions of interest. Primers must be designed for every region. Does not distinguish 5mC from 5hmC. $$ Great when interested in methylation in a small number (<8) of regions of interest, especially if not in CpG islands.
Bead Array DNA is bisulfite treated and PCR amplified to reveal the methylation pattern of the DNA. It is then combined with DNA probes which target specific methylation sites. 250 ng Single base 850,000 CpGs
>96% CpG islands
Easy, reproducible, works with FFPE samples For human and mouse only. Misses 95% of CpG sites. Does not distinguish 5mC from 5hmC. $$$ Screening of clinical samples for aberrant methylation in CpG islands.
EM-seq TET2 enzyme is used to selectively oxidize 5mC/5hmC in adapter ligated DNA so that they are protected from APOBEC deamination in the next step, therefore revealing the methylation pattern of the DNA. Libraries are amplified and sequenced. >10 ng Single base 28 million CpGs Avoids the degradation of bisulfite conversion. Requires less material than WGBS. Does not distinguish 5mC from 5hmC. $$$$ Ideal when interested in low-CpG-density regions, intergenic “gene deserts”, or partially methylated domains and distal regulatory elements, that would be missed by RRBS. Better for cfDNA or FFPE DNA than WGBS.
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