Fig. 4

cirDNA Methylation Detection Methods. Methods for the detection of cirDNA methylation can be divided into two groups: (1) Methods based on bisulfite conversion. These techniques are presented as the gold standard for DNA methylation studies. Bisulfite conversion typically causes significant deterioration of DNA, resulting in the loss of some essential information, especially when the input cirDNA is low [183]. Bisulfite conversion methods include: (a) Whole-Genome Bisulfite Sequencing (WGBS), initially established to map the human DNA methylomes [223]. WGBS is currently the most comprehensive DNA methylation profiling technology [224] with the ability to identify cytosines’ methylation state, including regions with low CpG density as well as non-CpG sites (CpA, CpT, and CpC). It is an expensive technique, especially when producing high-depth data, which requires only 30 ng of human input DNA. Other approaches, such as single-cell bisulfite sequencing (scBS-Seq) [225] and single-cell whole-genome bisulfite sequencing (scWGBS), have been established to address the rising demand for low-input DNA analysis [226]. (b) Post-bisulfite adaptor tagging (PBAT) is a very effective method for creating libraries for WGBS with low input DNA. PBAT adds adapters to bisulfite-converted genomic DNA to bypass the DNA degradation inherent in standard WGBS protocols, triggered by bisulfite. PBAT is a PCR-free approach that adds adaptors after bisulfite treatment to avoid bisulfite-induced DNA library degradation, thus it can create a library starting from 125 pg of DNA [227, 228]. (c) Reduced-Representation Bisulfite Sequencing (RRBS) was employed to make DNA methylome analysis cost-effective by combining MspI digestion and bisulfite conversion for the study of regions enriched in CpGs, which requires extremely low input DNA (10–300 ng) [229, 230]. (d) Methylated CpG Tandems Amplification and Sequencing (MCTA-Seq) is a highly sensitive method for the identification of hypermethylated CpG islands [231] that uses a primer harboring a semi-random sequence, a special molecular identifier (UMI) sequence, and an anchor sequence, to amplify the DNA transformed with bisulfite at the 3'-end.. This highly sensitive technique will work with as little as 7.5 pg of genomic DNA, which is equal to 2.5 copies of the haploid genome [232]. (e) Bisulfite padlock probes (BSPP), where bisulfite-converted DNA is isolated using specific probes which contain two short capture sequences connected via a standard linker sequence [233, 234]. This method can be performed on low DNA input as low as 10–15 ng [235]. A unique feature of BSPP is its ability to be incorporated into a capture reaction across hundreds of samples resulting in more than 97% specificity. (f) Methylation array: Illumina’s MethylationEPIC ‘850 K’ Bead Chip is an excellent tool for quantitative methylation analysis at a single CpG site level. It enables the interrogation of over 850,000 methylation sites, selected by experts [236]. One drawback of array-based approaches, however, is the low genome-wide coverage of entire methylation regions, leading to the lack of some genome methylation contents [237]. (g) Methylation-specific PCR (MSP) where two different methylation-specific primer sets against target DNA are used to amplify methylated DNA converted with bisulfite and untreated DNA. In an unmethylated state, the unmethylated primer is unique to bisulfite converted DNA [238]. Several quantitative MSP (qMSP) methods have been established using real-time PCR [239,240,241]. For methylation detection, the methylation-sensitive high-resolution melting analysis (MS-HRM) technique has also been created [242]. However, our group has found that optimizing this method can be laborious and, in some cases, may have unreliable. (2) Methods based on non-bisulfite conversion. In order to overcome the limitations of bisulfite conversion, several non-bisulfite conversion techniques have been developed. The restriction enzyme-based method is a conventional approach for methylation analysis, which commonly uses two types of methylation restriction enzymes (MREs): methylation-sensitive enzymes, which cleave only unmethylated DNA; and methylation-insensitive enzymes, which cleave DNA regardless of the methylation status at the recognition sites [172]. Non-bisulfite conversion methods are categorized as (2.1) Affinity enrichment-based methods, where anti-methylcytosine antibodies or methyl-CpG binding proteins are used to pull down the methylated genomic fragments, while stringent washing eliminates unmethylated fractions. These enrichment-based approaches have not only demonstrated comparable sensitivity to and marginally greater precision than WGBS methods but have also demonstrated additional benefits [243]. They include: (a) Nanopore sequencing technology makes significant advances in the detection of DNA methylation with an accuracy of 92–98%. It works based on immersing a high-resistance film with protein nanopores in an aqueous ion solution[244,245,246]. (b) Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) was initially established as a method for the immunoprecipitation of methylated DNA [203]. Due to inadequate methylation enrichment, a minimum input of 50 ng DNA is recommended [247] and in practice, the DNA amount in plasma limits the application of this method. Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-Seq) has been established to employ MeDIP-Seq for low-input cirDNA [67]. (c) Methyl-CpG Binding Domain Protein Capture Sequencing (MBD-Seq). The methyl-CpG binding domain in methyl-CpG binding proteins may be used instead of immunoprecipitation to pulldown DNA methylated regions using magnetic beads [204]. MBD-based enrichment has been shown to outperform MeDIP in regions with a high CpG density and to classify the largest proportion of CGIs [248]. (d) Methylated CpG Island Recovery Assay (MIRA-Seq) that works based on the MBD2b/MDB3L1 complex high affinity for double-stranded methylated DNA, enabling the isolation of methylated DNA without the use of bisulfite conversion or antibodies. MIRA can detect methylated CpG nucleotides with low methylation density and can be used in combination with microarrays or NGS [249]. (2.2) 5-Hydroxymethylation profiling methods. Emerging data suggest that 5hmC not only serves as a generally stable epigenetic marker [250], but also interacts with tumorigenesis and tumor development [251]. They include: (a) 5hmC-Seal (aka hMe-Seal) where azide-modified glucose is produced by β-glucosyltransferase and then biotinylated through click chemistry. Since 5hmC-Seal can work with ultra-low amounts of input DNA (about 5 ng), this technology is very useful for liquid biopsy [252, 253]. (b) hmC-CATCH is a bisulfite-free method for genome-wide identification of 5hmC requiring only nanoscale input genomic DNA samples [254]. This approach is based on the 5-formylcytosine (5fC) blocking method, 5hmC to 5fC selective oxidation, newly created 5fC chemical labeling, and C to T transformation during PCR amplification [254]. (c) Hydroxymethylated DNA Immunoprecipitation Sequencing (hMeDIP-Seq) method is an updated version of MeDIP that enables the unique enrichment of DNA fragments harboring 5hmC [255]. hMeDIP requires immunoprecipitation using anti-5hmC antibodies accompanied by downstream approaches including NGS, microarray, or PCR. (d) Oxidative Bisulfite Conversion. Cytosines in 5fC and 5-carboxylcytosine (5caC) are not preserved after deamination by sodium bisulfite; this led to the development of oxidative bisulfite sequencing (OxBS-Seq) [256] and TET-assisted bisulfite sequencing (TAB-Seq) [257]