The Significance of DNA Methylation and Sequencing Methods Comparison

 

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The Significance of DNA Methylation and Sequencing Methods Comparison

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What is DNA methylation?

DNA methylation has always been one of the research focuses in the field of epigenetics. DNA methylation refers to the conversion of the 5′-terminal cytosine of the CpG island’s dinucleotide to the 5′-methylcytosine under the action of DNA methyltransferase (DNMT). This DNA modification method does not change the sequence of genes, but it can inhibit the expression of certain genes. In mammals, genomic DNA methylation can be divided into two types: maintenance DNA methylation and de novo methylation. Maintaining DNA methylation refers to the process of methylation modification at the corresponding position of the daughter strand during the semi-retained replication of DNA under the action of methyltransferase. De novo methylation refers to the process of methylation on the double strand of DNA that has not been methylated under the action of methyltransferase, and then the methylation enzyme maintains the stable DNA methylation state. For these two methylation mechanisms, there are two corresponding types of methylases: maintenance methyltransferase and remethylase. In mammals, the more common DNA methyltransferases (DNMT) are: DNMT1, DNMT3a, DNMT3b. And demethylation can induce gene reactivation and expression.

What is the role of DNA methylation?

The biological functions of DNA methylation can be divided into three categories:

1) Genomic sequence C-->T mutations, such as the mutation of the tumor suppressor gene p53 in many patients with solid tumors, and many mutations of this gene are C-->T mutations caused by deamination after CpG methylation;

2) Affect the repair of genomic mismatches. Studies have shown that DNA mismatch repair system (MMR) is related to DNA methylation;

3) Gene silencing, which affects gene expression.

In eukaryotes, approximately 60% to 90% of the cytosines in CpG dinucleotides are shown to be methylated (Ehrlich et al., 1982). DNA methylation is closely related to the maintenance of normal cell function, female individual X chromosome inactivation, suppression of parasitic DNA sequences, stable genome structure, genetic imprinting, embryonic development, and the occurrence and development of tumors and diseases. Existing studies have shown that the normal development of an embryo is very dependent on the proper methylation of genomic DNA. The loss of any methyltransferase can lead to the termination of mouse embryonic development and death. In addition, abnormal changes in DNA methylation status are common in various tumors, and abnormal DNA methylation status is one of the important features of tumors. Both in vitro and in vivo experiments in mice have shown that demethylation at the whole genome level may lead to instability of the entire genome, thereby increasing the incidence of tumors. Moreover, hypermethylation of CpG in the promoter region of tumor suppressor genes is one of the important events in the early stage of many cancers. Therefore, exploring the mechanism of DNA demethylation in tumors is essential for understanding tumor occurrence and development.

What are the methods of detection of DNA methylation?

So far, DNA methylation detection technology has undergone rapid changes. According to the "sample pretreatment" method, it can be divided into:

1) Enzyme digestion;

2) Affinity enrichment;

3) Sodium bisulphite;

According to the "analysis steps", it can be subdivided into:

1) Locus-specific analysis;

2) Gel-based analysis;

3) Array-based analysis;

4) NGS-based analysis.

The main DNA methylation research methods (Peter W. Laird, Nat Rev Genet. 2010) include: AIMS, amplification of inter-methylated sites; BC–seq, bisulphite conversion followed by capture and sequencing; BiMP, bisulphitemethylation profiling; BS, bisulphite sequencing; BSPP, bisulphite padlock probes; CHARM, comprehensive high-throughput arrays for relative methylation; COBRA, combined bisulphite restriction analysis; DMH, differential methylation hybridization; HELP, HpaIItiny fragment enrichment by ligation-mediated PCR; MCA, methylated CpG island amplification; MCAM, MCA with microarrayhybridization; MeDIP, mDIP and mCIP, methylated DNA immunoprecipitation; MIRA , methylated CpG island recovery assay; MMASS, microarray-based methylation assessment of single samples; MS-AP-PCR, methylation-sensitive arbitrarily primed PCR; MSCC, methylation-sensitive cut counting; MSP, methylation-specific PCR; MS-SNuPE, methylation-sensitive single nucleotideprimer extension; NGS, next-generation sequencing; RLGS, restriction landmark genome scanning; RRBS, reduced representationbisulp hite sequencing; –seq, followed by sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.

With the development of sequencing technology, the price of sequencing is getting lower and lower, and the throughput is getting higher and higher, and sequencing can achieve single-base resolution, sequencing-based methylation detection technology has gradually become mainstream.

Development history of DNA methylation detection technology based on second-generation sequencing (Barros-Silva et al. Genes, 2018) include: BS-Seq: bisulfite sequencing; MeDIP-Seq: methylated DNA immunoprecipitation sequencing; RRBS-Seq: reduced representation bisulfite sequencing; WGBS: whole genome bisulfite sequencing; MethylCap-Seq: methylation capture sequencing; MBD-Seq: methyl- CpG binding domain sequencing; oxBS.Seq: oxidative bisulfite sequencing; TAB-Seq: TET-associated bisulfite sequencing; BSAS: bisulfiteamplicon sequencing.

What are the commonly used methylation detection methods?

1) Methylated DNA Binding Domain Sequencing (MBD-Seq or MBDCap-seq);

It is a DNA methylation detection method that uses a combination of methylated DNA enrichment and high-throughput sequencing technology. Mainly through the specific binding of methylated DNA protein MBD2b to enrich hypermethylated DNA fragments, and then combine high-throughput sequencing technology to sequence the enriched DNA fragments, so that methylation can be detected throughout the genome

2) Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq)

It uses methylated DNA immunoprecipitation technology. First, the 5'-methylcytosine antibody was used to specifically enrich the methylated DNA fragments on the genome, and then the CpG-intensive hypermethylated regions were studied at the genome-wide level by high-throughput sequencing. This technique can find highly methylated regions in the genome, such as CpG islands, but cannot perform single-base analysis.

3) TET-assisted bisulfite sequencing (TAB-seq)

The principle is to use glucose imine and 5'hydroxymethylcytosine (5'hmC) to protect it from the oxidation of TET protein. 5'methylcytosine and unmethylated cytosine are deaminated to uracil, so that 5'hydroxymethylcytosine (5'hmC) can be identified from the single base level.

4) Reduced Representation Bisulfite Sequencing (RRBS)

RRBS digests the genomic DNA sequence with restriction enzymes, then enriches the promoter and CpG island regions, and then performs Bisulfite transformation, so that a single base containing the most CpG sites can be obtained with a smaller amount of data Horizontal methylation profile. In general, it is an accurate and cost-effective DNA methylation detection method, suitable for large-scale clinical sample research.

5) Whole-genome bisulfite sequencing (WGBS)

The principle is to use Bisulfite to process the DNA sequence, first convert the unmethylated C base in the genome to U(T), so as to distinguish it from the base C with methylation modification, and then perform PCR amplification, combined with high-throughput sequencing technology, is suitable for mapping DNA methylation maps with single-base resolution throughout the genome.

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