Table 2.
Comparison of methods for DNA methylation analysis.
| Method | Availability as a Kit | Coverage | Sensitivity | Specificity | Amount of Starting Material | § Price | References |
|---|---|---|---|---|---|---|---|
| Methods for Whole Genome Profiling | Whole Genome Assessment without Identification of Differentially-Methylated Regions | ||||||
| Mass spectrometry (LC-MS/MS) | No | Whole genome assessment | *** detect 5% difference in methylation between samples | *** | 100 ng–1 μg | $80/sample (Millis Scientific) | [18,19,20,21] |
| LUMA (Luminometric-Based Assay) | No | Whole genome assessment | ** | ** | 250–500 ng | [34,38] | |
| LINE-1 + pyrosequencing | No | 17% of the whole genome | *** | ** | 50 ng | [39] | |
| HPLC-UV | No | Whole genome assessment | * | ** | 3–10 μg | [17] | |
| Methods for Identification of Differentially-Methylated Regions | |||||||
| # Enrichment of 5metC regions by pulldown with MBD protein (needs to be followed by NGS or microarray) | MethylCap kit (Diagenode) | ~80% of all 5–5 mCpG | *** | *** | 200 ng–1.2 μg | $550/48 rxns | [3,40] |
| MethylMiner Methylated DNA Enrichment Kit (Thermo Fisher Scientific) | * | * | 5 ng–1 μg | $485/25 rxns | |||
| # Enrichment for 5 mC regions by MBD2b/MBD3L1 proteins pulldown (MIRA-based assay) (needs to be followed by NGS or microarray) | MethylCollector Ultra (Active Motif) | No info | ** | ** | 1 ng–1 μg | $495/10 rxns | [40,41] |
| # Enrichment for 5 mC regions with antibodies (MeDIP) (needs to be followed by NGS) | MeDIP (Active Motif) | No info | No info | No info | 100 ng–1 μg | $395/10 rxns | [42,43] |
| MagMeDIP (Diagenode) | No info | No info | No info | 1.2 μg | $595/48 rxns; $325/10 rxns | [44] | |
| # Enrichment for CpG rich regions with RNA baits (needs to be followed by NGS) | SureSelect Human Methyl-Seq (Agilent) | 3.7 × 106 CpGs | *** | *** | 3 μg | ~$320 (baits); +$70 (library prep) | [5] |
| # Enrichment for CpG rich regions by hybridisation with bait oligonucleotides (needs to be followed by NGS) | SeqCap Epi CpGiant Enrichment Kit (Roche) | 5.5 × 106 CpGs | *** | *** | 1 μg | ~$450; + bisulfite conversion kit | [45,46] |
| HumanMethylation450 BeadChip array | Illumina | 482.000 CpG sites (99% of known genes) | *** | *** | 0.5–1 μg | ~$9000/24 samples (2 chips) | [47] |
| Sequencing of methylation-enriched fraction of the genome (after MeDIP or MIRA) | Illumina | varies, depending on the sample | varies, as number of reads correlates with the amount of DNA | Depends on the enrichment method | > 50 ng | $360/sample | |
| Whole genome bisulfite sequencing (WGBS) | Illumina | 100% | *** | 50–100 ng | $6000/sample | [48,49] | |
| Reduced representation bisulfite sequencing (RRBS) | Illumina | ~60% of promoters, 85% of CGI (~1.5 × 106 CpGs) | varies, as sequencing depth correlates with the amount of DNA (20X coverage is recommended) | *** | 1 μg | $2700–5000/sample (sequencing and library prep) | [50,51] |
| Methyl-MiniSeq (improved RRBS) | Zymoresearch | >85% coverage of all CpG islands and >80% of all gene promoters (~3 × 106 CpGs) | varies, as sequencing depth correlates with the amount of DNA (20X coverage is recommended) | 100 ng–5 μg | $2800 (all steps and bioinformatics included) | [44,52] | |
| Methyl-sensitive cut counting (MSCC) (needs to be followed by NGS) | No | 1% of all CpG | depends on the sequencing coverage | *** | 1–5 μg | [53] | |
| Improved MSCC (needs to be followed by NGS) | No | 30% of CpGs (~58% of CpG-rich regions) | *** | 1–5 μg | [54,55] | ||
| Low Throughput Methods | |||||||
| PCR-based (digestion followed by PCR) | OneStep qMethyl Kit (Zymoresearch) | Gene-specific 22 samples/ 96-well plate | *** | *** | ≥20 ng | [56] | |
| EpiTect II DNA methylation enzyme Kit (Qiagen) | 1–4 μg | [57] | |||||
# Enriched fractions are normally used for NGS. § All prices are approximate and have been obtained during 2015. *** Defines the best sensitivity or specificity (*** > ** > *).