TABLE 2.
Overview of methods for DNA methylation analysis (at the time of this original writing)
| Methylation-dependent treatment method | Detection method | Type of analysis | Comments | Cost and time | Protocols |
|---|---|---|---|---|---|
| Bisulfite conversiona | Sanger sequencing of clones | Locus specific | Single-base resolution | Inexpensive, time consuming and laborious | Protocol: DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA Methylation Detection [Lizardi et al. 2017a] |
| Sanger sequencing of PCR products | Locus specific | Semiquantitative evaluation of average level of DNA methylation; not very sensitive | Inexpensive, quick | ||
| Methylation-specific PCR | Locus specific | Semiquantitative | Inexpensive, quick | Protocol: Methylation-Specific Polymerase Chain Reaction (PCR) for Gene-Specific DNA Methylation Detection [Lizardi et al. 2017c] | |
| Hairpin-bisulfite sequencing | Locus specific | Determination of methylation on both strands | Inexpensive, quick | ||
| Methylation microarray | Multiple loci | Medium-to-high multiplexing, single-base resolution, potential hybridization bias | Expensive; analysis is time consuming | ||
| Direct pyrosequencing | Multiple loci | Medium multiplexing, high-throughput single-base resolution | Expensive, quick | ||
| Next-generation sequencing after targeted capture | Multiple loci | Expensive; analysis is time consuming | |||
| Next-generation sequencing of reduced redundancy | Genome-wide | Genome-wide coverage, single-base resolution at high number of locations | Expensive, analysis is time consuming | Protocol: Illumina Sequencing of Bisulfite-Converted DNA Libraries [Lizardi et al. 2017b] | |
| Next-generation sequencing of whole genome | Genome-wide | Whole genome sequencing, single-nucleotide resolution, expensive | Protocol: Illumina Sequencing of Bisulfite-Converted DNA Libraries [Lizardi et al. 2017b] | ||
| Mass spectrometry | Multiple loci | High multiplexing | Inexpensive, quick | ||
| Affinity enrichmentb | Quantitative PCR | Locus specific | Inexpensive, quick | Protocol: Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis [Lizardi et al. 2017d] | |
| Microarray | Genome-wide | Expensive; analysis is time consuming | |||
| Next-generation sequencing | Genome-wide | Expensive; analysis is time consuming | |||
| Methylation-sensitive enzyme digestionc | Gel-based methods | Genome-wide | Low resolution | Inexpensive, quick | |
| Microarray | Multiple loci or whole genome | Subject to hybridization artifact | Expensive; analysis is time consuming | ||
| Next-generation sequencing | Genome-wide | Expensive; analysis is time consuming | Protocol: High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation [Lizardi et al. 2017e] |
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aCompatible with ssDNA, accurate and reproducible, provides single-base resolution, could result in significant degradation of sample.
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bCompatible with ssDNA (MeC IP) or dsDNA (MBD), requires a large amount of DNA, relatively low resolution, cannot distinguish 5mC and 5hmC.
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cRequires dsDNA of high quality, prone to false positives caused by incomplete digestion.
