| DNA methylation |
| PCR-based bisulfite sequencing |
The candidate gene and/or the targeted region are known. |
Cost-effective Allow detection of all CpG sites from the PCR products Single-base resolution |
Inconsistent results due to degradation of DNA template after bisulfite treatment Require multiple replications (>10) to confirm the results |
34 |
| MSP |
The candidate gene and/or the targeted CpGs are known. |
Cost-effective Single-base resolution |
Only few CpG sites can be detected through MSP. Low throughput |
35, 37
|
| Pyrosequencing |
The candidate gene and/or the targeted region are known. |
High quantitative and accurate resolution of DNA sequencing results through bioluminometric detection during nucleotide incorporation Cost-effective Single-base resolution |
Degradation of DNA template due to bisulfite treatment Validated primers are essential for the success of pyrosequencing |
36 |
| WGBS |
De novo DNA methylation exploration |
The most comprehensive method to evaluate global DNA methylation state of almost every CpG site Single-base resolution |
Expensive Require sophisticated bioinformatics analysis Require large amounts of input DNA due to degradation of DNA template after harsh bisulfite treatment |
38, 42
|
| HumanMet hylation450 |
De novo DNA methylation (exploration |
Cover most of CpG islands in human epigenome Relatively cost-effective Single-base resolution |
Available only in human samples Coverage is highly dependent on predesigned array |
38, 40, 41
|
| RRBS |
De novo DNA methylation exploration |
Cover the most representative CpG islands in the gene regulatory regions Detect DNA methylation in different species. Single-base resolution Relatively cost-effective |
Only detect 1–3% of genome May lose coverage at intergenic and distal regulatory regions |
43, 44
|
| MRE-Seq |
De novo DNA methylation exploration |
Cost-effective No harsh chemical treatment on DNA, thus avoid complicated experimental consequences |
Coverage relies on restriction enzymatic activities and recognition sites Only detect enrichment abundance Can not identify individual CpG site Can not detect absolute methylation level |
46 |
| MeDIP |
De novo DNA methylation exploration |
Cost-effective No harsh chemical treatment on DNA, thus avoid complicated experimental consequences Specific antibody against 5mC leads to sensitivity in regions with low CpG density |
Abundance resolution in ~100bp Can not identify individual CpG site Can not detect absolute methylation level The quality of the results relies on the quality of the antibody |
47 |
| ELISA-based assay |
Broad prediction of global DNA methylation changes |
Cost-effective Commercially-available kit targeting 5mC |
Only for rough estimation of global DNA methylation change High variability and unreliable results |
32, 48
|
| Single-cell bisulfite sequencing |
De novo DNA methylation exploration in single-cell level |
Provide methylation information into individual cells Particularly useful for specific cell types such as germ cells, embryonic stem cells. Have different sequencing options such as scWGBS and scRRBS |
Same weaknesses in WGBS or RRBS assays |
39, 49, 50
|
| SMRT sequencing |
De novo DNA methylation exploration |
Ability to sequence native DNA through single molecule long-read sequencing No harsh bisulfite treatment Detect both nucleotide sequence and major types of DNA methylation patterns such as 5mC, 5hmC, 6mA and 4mC simultaneously Particularly recommend to detect bacterial genomes |
Because DNA cannot be amplified, large input DNA is required. Mostly applied in bacterial genome |
38, 39, 54
|
| Nanopore sequencing |
De novo DNA methylation exploration |
Ability to sequence native DNA through single molecule long-read sequencing No harsh bisulfite treatment Applied in all species |
Because DNA cannot be amplified, large input DNA is required. Lack of generalized algorithms makes it hard to understand stability of performance across species and sequencing batches |
38, 39, 55
|
| OxBS-seq |
De novo DNA methylation exploration for 5hmC |
The most commonly used method to evaluate global 5hmC status
|
Multiple bisulfite treatments requires high amount of input DNA and high sequencing depths for confident determination of scarcely abundant modifications
|
65 |
| Histone modifications |
| ChIP-PCR |
Confirmatory studies |
Standard methods for detection of specific enrichment of histone modification patterns or binding ability of regulatory factors to the chromatin regions. Cost-effective |
Only detect enrichment abundance Can not identify enrichment peak via single nucleotide resolution |
14 |
| ChIP-chip |
De novo exploratory studies |
Microarray or hybridization techniques-based global histone modification detection Custom designed coverage specifically targeting certain genomic regions Relatively cost-effective |
Subject to microarray artifacts such as probe performance and hybridization efficiency The quality of the results relies on the quality of the antibody |
68 |
| ChIP-seq |
De novo exploratory studies |
The most comprehensive method to evaluate global histone modification state Enable single-base resolution |
High cost Require high quality antibody |
69 |
| ELISA-based assay |
Broad prediction of histone modification changes |
Cost-effective Commercially-available kits targeting different histone modification patterns |
Only for rough estimation of total enrichment changes of specific histone modification High variability and unreliable results |
48 |
| ncRNAs |
| qRT-PCR |
Confirmatory studies or small-scale experiments |
Standard or modified methods for detection of most types of ncRNAs Commercially-available customized kits for detecting different sizes of ncRNAs High sensitivity and specificity Cost-effective |
Validated primers are essential Require quality annotation |
18, 19, 84
|
| RNA-Seq |
De novo exploratory studies |
Whole genome analysis Single-base resolution |
Less sensitive than qPCR Require most input materials Require sophisticated bioinformatics analysis |
84, 88
|
| HITS-CLIP |
Genome-wide
functional
analysis |
Whole genome analysis that combines IP and RNA-seq Functional analysis for interaction of ncRNAs with specific proteins Single-base resolution |
Less sensitive than qPCR Require high quality antibody Require sophisticated bioinformatics analysis |
22 |
| Integrative analysis |
| ChIP-BMS |
Confirmatory studies |
Combine ChIP and bisulfite techniques to detect methylation status of specific chromatin regions Single-base resolution |
Validated primers are essential Degradation of ChIP DNA template after bisulfite treatment requires high amounts of input DNA Cloning techniques are normally required to obtain high quality PCR sequencing results |
71 |
| BisChIP-seq |
Confirmatory studies and de novo studies |
Combine ChIP and bisulfite techniques to detect whole-genome methylation status on allele-specific histone modification patterns Single-base resolution |
High cost Complicated procedures require both ChIP and bisulfite treatment Degradation of ChIP DNA template after bisulfite treatment requires high amounts of input DNA Require sophisticated bioinformatics analysis |
72 |
| Methyl-HiC |
Confirmatory studies and de novo studies |
Combining in situ Hi-C and WGBS to simultaneously capture chromosome conformation and DNA methylome in a single assay |
Expensive Complicated procedures require both in situ Hi-C and bisulfite treatment Require sophisticated bioinformatics analysis |
79 |
