Table 2.
Methods for Methylation Measurement.
| Methods | Advantages | Disadvantages | |
|---|---|---|---|
| Candidate Gene Approach | |||
| Sanger Sequencing | Chain-termination sequencing using capillary gel electrophoresis | High quality | High cost; small scale sequencing |
| Pyrosequencing | Sequencing by synthesis method that detects how many complementary nucleotides have been incorporated into a strand | High quality; low cost; faster than Sanger Sequencing | Small scale sequencing |
| Methylation-Specific PCR (e.g., MethyLight) | Determines DNAm status of target loci with specific primers and probes | ||
| Mass Spectrometry (e.g., EpiTyper) | After bisulfite conversion, uses base-specific cleavage, and mass spectrometry to quantify® DNA fragments | Reliable; cost-effective | |
| Whole Genome Approach | |||
| Enzyme Digestion (e.g., Met-seq1; MSCC2) | Uses restriction endonucleases, to detect and cleave specific DNA sequences | Low cost | Limited to sequences recognized by selected enzymes |
| Affinity-Based Antibody Enrichment (e.g., Me-DIP-seq3; MBD-seq4) | Anti-5methylcytosine antibodies are used to isolate methylated DNA followed by microarray | Low cost | Low resolution |
| Microarray (e.g., Illumina 850k EPIC BeadChip Array) | Quantification of all known genes, promoter sites, and enhancer regions of bisulfite converted DNA | Single CpG site resolution with small samples | High cost; limited to known CpG sites and subject to probe-type biases |
| Whole Genome Bisulfite Sequencing (WGBS) | Uses bisulfite converted DNA for DNAm analysis across the genome | High-quality interrogation w/o bias; DNAm status of single CpG sites | Higher cost |
| Reduced Representation Bisulfite Sequencing (RRBS) | Uses restriction enzymes and bisulfite conversion for DNAm analysis of the subset of the genome | High-quality interrogation w/o bias; DNAm status of single CpG sites | Lower cost; focuses on smaller regions instead of precise CpG sites |
| Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) | Uses restriction enzymes and bisulfite conversion for DNAm analysis of biologically relevant loci | High-quality interrogation w/o bias; DNAm status of single CpG sites | Lower cost; focuses on biologically relevant loci |
1
Methyl-Seq = methylation sequencing;
2
MSCC = Methylation Sensitive Cut Counting;.
3
Me-DIP-seq = Methylated DNA Immunoprecipitation;
4
MBD-seq = methylated CpG-binding.