Table 3.
Summarised methods for the detection of DNA methylation in liquid biopsy.
| Method name | Class* | Sub-class | Bisulfite-based | Description | Citation |
|---|---|---|---|---|---|
| Whole Genome Bisulfite Sequencing (WGBS) | GW | High-throughput sequencing | Y | Various approaches for the tagging and sequencing of bisulfite converted DNA. Adapter tagging can be done before or after conversion. Different approaches may introduce biases. | (Lister et al., 2009) |
| Bisulfite Sanger Sequencing (Bis-Seq) | TGT | Bisulfite conversion specific amplification | Y | Bisulfite-treated genomic DNA subjected to amplification with conversion-specific PCR primers. Primers are unbiased and contain no CpGs | (Frommer et al., 1992) |
| Nested PCR | TGT | Bisulfite conversion specific amplification | Y | Bisulfite-treated genomic DNA subjected to amplification with conversion-specific PCR primers, followed by secondary PCR with primers targets within the first PCR fragment to enhance specificity or sensitivity. | (Herman et al., 1996) |
| Methylation-specific PCR | TGT | Bisulfite conversion specific amplification | Y | Bisulfite-treated DNA subjected to amplification. PCR primers intentionally biased by including multiple CpGs in binding sites. | (Herman et al., 1996) |
| MethylLight | TGT | Fluorescence probe PCR | Y | Conversion or Methylation specific PCR with the addition of a TaqMan probe. Methylation specificity obtained by including CpGs in the primers, probe or both. | (Eads et al., 2000) |
| Quantitative Allele-specific Real-time Target and Signal amplification (QuARTS) | TGT | Fluorescence probe PCR | Y | Bisulfite-treated genomic DNA subjected to PCR with probes targeting alternate methylation states. Probe fluorescence activated by an additional oligo binding immediately upstream. | (Zou et al., 2012) |
| HeavyMethyl | TGT | PCR with blocker | Y | Competitive inhibition of PCR using primers combined with a blocker oligo that target alternate methylation states. | (Cottrell et al., 2004) |
| Cold-PCR | TGT | Preferential denaturation temperature PCR | N | The first few cycles are conventional PCR. Subsequent cycles use a lower denaturation temperature to enrich for DNA molecules that contain mismatches, which occur if there are mutant DNA sequences in the sample. | (Milbury et al., 2011; Castellanos-Rizaldos et al., 2014) |
| Ice-COLD-PCR | TGT | Preferential denaturation temperature PCR | N | The same as cold-PCR but with the addition of a further blocker oligonucleotide to inhibit amplification of unwanted targets. | (Milbury et al., 2011; Mauger et al., 2018) |
| High-resolution melt (HRM) curve analysis | TGT | Melt-curve analysis | Y | Following traditional PCR with an intercalating dye (e.g. SYBR green), the PCR product is gradually warmed until the DNA strands denature (melt) apart. DNA melting is detectable by shifts in the level of fluorescent signal over time. | (Wittwer, 2003; Wojdacz and Dobrovic, 2007) |
| Bis-seq (pyrosequencing) | TGT | Bisulfite conversion specific amplification | Y | “Sequencing by synthesis” method. Sequence readout is obtained by detecting pyrophosphate released during base incorporation during synthesis of the complementary DNA to the target fragment. | (Ronaghi, 1998; Fraga and Esteller, 2002) |
| EpiTYPER | TGT | Mass-spectrometry | Y | Utilizes mass spectrometry to accurately measure the methylation of PCR-derived amplicons. DNA is converted and amplified by PCR. Incorporation of C/G or T/A bases (methylated or unmethylated) during amplification leads to measurable shifts in molecular weight. | (Ehrich et al., 2005) |
| Reduced representation bisulfite sequencing (RRBS) | RGW | Enzymatic digest | Y | Genomic DNA digested with methylation-insensitive restriction enzyme (with CpG in the recognition site), followed by size selection prior to bisulfite conversion. | (Meissner et al., 2005) |
| Combined Bisulfite Restriction Analysis (COBRA) | RGW | Enzymatic digest | Y | Bisulfite-treated genomic DNA is subjected to methylation-insensitive restriction enzyme digest targeting the unconverted amplicon (i.e. originally methylated). Ratio of digested fragments to total fragments correlates with methylation level. | (Xiong and Laird, 1997) |
| Digital Restriction Enzyme Analysis of Methylation (DREAM) | RGW | Enzymatic digest | N | Methylation specific restriction enzyme (MRSE) variant. DNA digested with two enzymes, one methylation sensitive and one not. Methylation readout based on ratio of cutting. | (Jelinek and Madzo, 2016) |
| Methylation-sensitive restriction enzyme (MSRE) + qPCR | TGT | Enzymatic digest | N | Unconverted DNA is digested with MRSE. Quantitative PCR is used to establish the efficiency of digestion, which indicates the level of methylation at the target site. | (Hashimoto et al., 2007) |
| Helper-dependent chain reaction (HDCR) | TGT | Enzymatic digest | N | Genomic DNA digested with methylation dependent restriction enzyme such as GlaI. Gene-specific sequence fragments are tagged with “helper” oligos, while “driver” oligos maintains preferential amplification of tagged fragments. | (Rand et al., 2013) |
| End-specific PCR (ES-PCR) | TGT | Enzymatic digest | N | MRSE variant for detecting unmethylated sequences. DNA is digested with a methylation sensitive enzyme, then specialized oligos are used to add priming sites to the target sequence. Highly useful method for targeting repetitive sequences that are difficult to assay by other methods. | (Rand and Molloy, 2010) |
| MeDIP | RGW | Affinity capture | N | DNA capture using antibody specific to methylated cytosine. Captured DNA suitable of PCR, array and sequencing based methods. | (Weber et al., 2005) |
| Various methyl-CpG binding domain (MDB)-based assays | RGW | Affinity capture | N | DNA capture using methylated DNA binding protein (MBD2). Use of salts during elution from MBD can facilitate fractionation on methylation level. Captured DNA suitable of PCR, array and sequencing based methods. | (De Meyer et al., 2013; Aberg et al., 2015) |
| SuBLiME | RGW | Affinity capture | Y | Biotinylated bases are incorporated into DNA fragments using a PCR-like approach following bisulfite conversion. Biotinylated fragments are captured to enrich for methylated targets. Can be performed in a targeted or genome-wide method. | (Ross et al., 2013) |
| Bisulfite Specific Padlock Probes (BSPP) | RGW | Molecular inversion probes | Y | BSPP utilizes bisulfite converted DNA and specialized DNA probes. Probes bind two sites in target sequences to form circular DNA structures that can be amplified and sequenced. | (Diep et al., 2012) |
| Infinium HumanMethylation | RGW | Infinium assay | Y | Bisulfite treated DNA is hybridized to the BeadArray chip, detection using single-base extension and fluorescence ratio between converted and unconverted probes. | (Bibikova et al., 2011) |
*GW, genome-wide; RGW, representative genome-wide; TGT, targeted.