Table 1 Protocol comparison
From: “Same difference”: comprehensive evaluation of four DNA methylation measurement platforms
DNA requirement | ERRBS | SSMethylSeq | CpGiant | TruSeqEpic | WGBS-PBAT | ||
|---|---|---|---|---|---|---|---|
75 ng > 40 kb | 1 µg | 3 µg | 0.25 µg | 1 µg | 0.5 µg | 100 ng > 40 kb | |
DNA processing | MspI digestion to completion followed by fractionation of 84–334 bp | Sonication to 150–200 bp | Sonication to 180–220 bp | Sonication to 180–220 bp | Single stranded and fragmented during bisulfite conversion | ||
Enrichment method | Size fractionation of 84–334 bp sizes | Hybridization to RNA capture probes | Hybridization to oligo probes containing fully, partially and unmethylated cytosines from both strands | Hybridization to oligo probes of stranded design | None | ||
Bisulfite conversion step | Post-adapter ligation | Post-hybridization capture | Pre-hybridization capture | Post-hybridization capture | Pre-adapter tagging | ||
Zymo Research Bisulfite Conversion kit | EZ DNA Methylation (50 °C, 55 cycles) | EZ DNA Methylation-Gold (64 °C, 2.5 h) | EZ-Methylation Lightning (54 °C, 1 h) | EZ-Methylation Lightning (54 °C, 2 h) | EZ DNA Methylation-Gold | ||
Total PCR amplification cycles | 18; Post enrichment and bisulfite conversion | 14; Post enrichment and bisulfite conversion (8 for amplification and 6 for Indexing) | 29; 13 post-bisulfite conversion and 16 post enrichment | 27; 11 post-bisulfite conversion and 16 post enrichment | 11; Post enrichment and bisulfite conversion | 10; Post-bisulfite conversion | |
DNA Polymerase (uracil tolerant) | FastStart Taq (Roche) | Taq2000 (Agilent Technologies) | HiFi HotSart Uracil + (Kapa Biosystems) | HiFi HotSart Uracil + (Kapa Biosystems) | FailSafe Enzyme (Epicentre) | ||
Predicted number of targeted CpG sites | 6.6 M | 3.7 M | 5.6 M | 3.3 M | 56 M | ||
Relative price (library preparation + PE100 sequencing @300 M reads) | 13.5% | 15.5% | 15.5% | 3.3% (@75 M reads) | 100% | ||