A text read: Comparison of DNA methylation effects and various high throughput assay

DNA methylation related definitions

DNA methylation has always been one of the apparent focus on the field of genetics. Of DNA methylation (Methylation) refers to the DNA methyltransferase (DNA methyltransferase, abbreviation of DNMT) action, the genomic DNA sequence of dinucleotide CpG islands 5 'end of the cytosines 5' methylcytosine (5 'methylcytosine, abbreviated 5mC). Such a modified DNA sequence of the gene does not change the way, but it can inhibit the expression of certain genes. In mammals, methylation of the genomic DNA can be divided into two types: methylated maintain (maintenance DNA methylation) and de novo methylation (de Novo by methylation) .

 

Maintaining methylation refers to a role in the methyl transferase enzyme, the DNA of the semi-conservative replication process will be modified by methylation process in the corresponding position of the daughter strand. Novo methylation refers to a role in the methyltransferase, the original double-stranded DNA is not methylated, the methylation procedure, then to maintain the stability of the DNA methylation status is maintained methylase. For both methyl mechanism, there are two types of the corresponding methylase: methyl transferases maintain and re-methyltransferase. In mammals, the more common DNA methyltransferase (of DNMT) mainly: DNMTl, DNMT3a, DNMT3b. Away methylation can induce gene expression, and reactivation.

 

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Cytosine methylation and demethylation domain and machine activity (Wu et al. 2014, Cell )

 

The role of DNA methylation

Biological function of DNA methylation can be divided into three categories:

1) genomic sequence C -> T mutation, such as the p53 tumor suppressor gene mutations, many patients with solid tumors, and many of the mutant gene is induced after deamination C CpG methylation -> T mutation;

2）影响基因组错配的修复，研究表明DNA错配修复系统(mismatchrepairsystem, MMR)与DNA甲基化有关；

3）基因沉默，即影响基因的表达。

 

在真核生物中，大约60%~90%的CpG 二核苷酸中的胞嘧啶都呈现出了甲基化状态(Ehrlich et al., 1982)。DNA甲基化在维持正常细胞的功能、雌性个体X染色体失活、寄生DNA序列的抑制、基因组结构稳定、遗传印记、胚胎发育、及肿瘤和疾病的发生、发展紧密相关，具有至关重要的作用。已有的研究表明胚胎的正常发育非常依赖于基因组DNA的适当甲基化，任何一种甲基转移酶的缺失，都可导致小鼠胚胎发育的中止而死亡。此外，各种肿瘤中都普遍存在DNA甲基化状态的异常改变，且异常的DNA甲基化状态是肿瘤的重要特征之一。小鼠的体外实验和体内实验都已表明，全基因组水平的去甲基化可能导致整个基因组的不稳定，从而增加肿瘤的发生几率。而且，抑癌基因启动子区域CpG 岛的高甲基化，是诸多癌症发生早期的重要事件之一。因此，探索肿瘤中DNA 的去甲基化的机制对于了解肿瘤的发生、发展至关重要。

DNA甲基化与癌症的关系（Robertson KD, Nat Rev Genet. 2005）

 

DNA甲基化的检测

至今，DNA甲基化检测技术已经有了突飞猛进的变化，若按照“样品前处理”的方式，则可分为：

1）酶切法(Enzyme digestion)；

2）亲和富集法(Affinity enrichment)；

3）亚硫酸盐处理(Sodium bisulphite)；

三种类型方法的综合比较（Barros-Silva et al. Genes, 2018）

 

而按照“分析步骤”则又可细分为：

1）位点特异性分析(Locus-specific analysis)；

2）凝胶分析(Gel-based analysis)；

3）全基因组高覆盖率的芯片分析(Array—based analysis)；

4）基于新一代测序技术的甲基化分析(NGS-based analysis)。

 

具体如下图所示：

主要的DNA甲基化研究方法（Peter W. Laird, Nat Rev Genet. 2010）。缩写与全称为：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 arraysfor 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 representationbisulphite sequencing; –seq, followed by sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.

 

随着测序技术的发展，测序价格越来越低，而通量越来越高，且测序可实现单碱基的分辨率，基于测序的甲基化检测技术已逐渐成为主流。下图展示了基于测序的DNA甲基化解析技术的发展流程：

基于二代测序的DNA甲基化检测技术发展历程（Barros-Silva et al. Genes, 2018）。缩写与全称：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.

 

常用的甲基化检测方法具体如下图所示：

常用的甲基化检测方法示意图（Yong et al. Epigenetics & Chromatin, 2016）

 

1）蛋白质富集全基因组甲基化测序（Methylated DNA Binding Domain Sequencing, MBD-Seq或MBDCap-seq）

为一种采用甲基化DNA富集与高通量测序技术相结合的DNA甲基化检测方法。主要通过特异性结合甲基化DNA的蛋白MBD2b去富集高甲基化的DNA片段，然后结合高通量测序技术，对富集到的DNA片段进行测序，从而可在全基因组范围内检测甲基化位点。

2）甲基化DNA免疫共沉淀测序（Methylated DNA Immunoprecipitation Sequencing, MeDIP-Seq）

其采用的甲基化DNA免疫共沉淀技术。首先通过5'-甲基胞嘧啶抗体去特异性富集基因组上发生甲基化的DNA片段，接着通过高通量测序在全基因组水平上对CpG密集的高甲基化区域进行研究。这种技术可以发现基因组中高度甲基化的区域，如CpG岛，但不能进行单个碱基水平的分析。

3）TET辅助的重亚硫酸盐测序(TAB-seq) 

原理是采用葡萄糖亚胺与5’羟甲基胞嘧啶(5’hmC)作用使其免受TET蛋白的氧化。而5’甲基胞嘧啶和未甲基化的胞嘧啶被脱氨基成尿嘧啶，从而可从单碱基水平鉴定5’羟甲基胞嘧啶(5’hmC)。

4）限制性内切酶-重亚硫酸盐靶向测序（Reduced Representation Bisulfite Sequencing，RRBS）

RRBS通过限制性内切酶对基因组DNA序列进行酶切，然后富集启动子及CpG岛区域，接着再进行Bisulfite转化，从而用较小的数据量就可得到包含最多CpG位点的单碱基水平的甲基化图谱。总的来说，是一种准确且高性价比的DNA甲基化检测方法，适用于大规模临床样本的研究。

5）全基因组DNA甲基化测序（Whole-genome bisulfite sequencing，WGBS）

其原理是用 Bisulfite 处理DNA序列，首先将基因组中未发生甲基化的 C 碱基转换成 U（T），从而与原本具有甲基化修饰的  碱基C区分开来，然后进行PCR扩增，结合高通量测序技术，适用于全基因组 范围内绘制单碱基分辨率的DNA 甲基化图谱。

 

下表列出了不同甲基化检测技术的特点，及可能存在的bias：

不同甲基化检测技术的特点及可能的bias（Peter W. Laird, Nat Rev Genet. 2010）。标注含义：‘•’ indicates that the method has this feature or potentially has this bias; ‘(•)’ indicates that the method has this feature to a limited extent or in some circumstances.BC–seq, bisulphite conversion followed by capture and sequencing; BSPP, bisulphite padlock probes; –chip, followed by microarray; MeDIP, methylated DNAimmunoprecipitation; RRBS, reduced representation bisulphite sequencing; –seq, followed by sequencing; WGSBS, whole-genome shotgun bisulphite sequencing.

 

此外，不同甲基化检测技术，拥有不同通量和基因组甲基化检测覆盖度，具体如下图所示：

甲基化检测技术的样本通量及基因组覆盖度比较（Peter W. Laird, Nat Rev Genet. 2010）

 

所以，各种甲基化检测技术都有自己的优点和缺点，其中全基因组DNA甲基化测序（WGBS）可获得全基因组范围内单碱基分辨率的所有DNA甲基化情况，信息最为丰富和全面。

 

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