虽然我们现在完全没有甲基化的数据，但还是可以了解一下。
什么是DNA甲基化，与组蛋白修饰有什么联系？
DNA Methylation and Its Basic Function
表观的定义就是DNA序列不变、表型变了。最重要的就是DNA的修饰（甲基化）和组蛋白的修饰（chip-seq里面的那几种）
为什么说甲基化比SNP在早筛方面要靠谱得多？
DNA甲基化测序的基本原理？Bisulfite Treatment，会特异的改变原来的甲基化和非甲基化的序列，无甲基化的C全部变为U。
Pan-Cancer Landscape of Aberrant DNA Methylation across Human Tumors
New England Biolabs® Launches NEBNext® Enzymatic Methyl-seq (EM-seq™) for Bisulfite-Free Methylation Analysis
This new enzyme-based system allows scientists to accurately and sensitively assess DNA methylation without bisulfite treatment, for epigenetics research.
亚硫酸氢盐很快将成为过去时
实战
基准医疗 - 官网 （了解产业界如何利用甲基化来做早筛）
Google - DNA methylation cancer screen review - 看看文献
A DNA Methylation-Based Test for Breast Cancer Detection in Circulating Cell-Free DNA- 选其中一篇，看懂研究方案
Epigenetics in cancer - 里程碑综述
问题：
1. DNA甲基化是什么时候发现的？看看wiki，DNA甲基化通常会改变DNA活性，更具体的说，当一个基因的promoter区被甲基化了，那么该基因的转录就会被抑制。4种碱基种仅有两种可以被甲基化：cytosine and adenine（C和A），不是完全的CG和AT。我们一般重点关注C的甲基化，哺乳动物中，甲基化主要集中在CpG dinucleotides上。75% of CpG dinucleotides are methylated in somatic cells，这说明体细胞中的大部分基因表达都被抑制了。甲基化的保持和denovo改变主要由DNMT，DNA甲基化转移酶来完成。
2. 汤富酬在做胚胎发育的甲基化，他有哪些成果？主要做早期胚胎发育、卵细胞，单细胞转录组和DNA甲基化。
3. DNA甲基化与癌症有什么关系？gene promoter CpG islands acquire abnormal hypermethylation，Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms. Typically, there is hypermethylation of tumor suppressor genes and hypomethylation of oncogenes. Generally, in progression to cancer, hundreds of genes are silenced or activated. Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation. DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes. Silencing of DNA repair genes through methylation of CpG islands in their promoters appears to be especially important in progression to cancer。说人话就是，癌症的核心点就是基因突变的积累，导致癌细胞的出现，癌细胞有一系列特性，生长不受控制、metastasis等。目前发现癌症发生时，DNA的甲基化水平也有巨大的变化。突变与DNA甲基化的共同作用导致大量正常基因激活或失活，从而激发细胞癌化。
A DNA Methylation-Based Test for Breast Cancer Detection in Circulating Cell-Free DNA
这篇文章的研究思路是什么？其实也就是DNA甲基化的癌症早筛研究思路。
癌症biomarker的寻找，这里不看DNA突变了，只看DNA甲基化。首先需要找一些靠谱的基因，然后检查它们promoter区域的甲基化程度，设定阈值，利用ROC和cox回归等方法来评估这些biomarker的早筛和预后分层的利用潜力。
一些细节的问题：
1. 为什么用cfDNA？优势和短板分别是什么？
2. DNA甲基化早筛的优劣？传统的mammography，乳房x射线诊断会导致很多假阳性，有些subtype无法检测。
3. 癌症研究三部曲：early detection, patients’ stratification and treatment。致死率的两大原因：disease recurrence and/or metastasis development。
4. 此文是如何阐述DNA甲基化与癌症的关系的？aberrant DNA methylation is considered a cancer-associated event。aberrant promoter methylation of several tumor suppressor genes was found in BrC precursor lesions, indicating that DNA methylation is an early event in breast carcinogenesis。DNA methylation has been proposed as a valuable cancer detection and prognosis biomarker owing to its link with tissue-specific gene silencing。Tumor-specific DNA methylation may also be detected in circulating cell-free DNA (ccfDNA) from liquid biopsies。翻译一下：就是发现DNA甲基化的异常在cancer中是普遍存在的（虽然机理仍然非常不明确），而且DNA甲基化是具有组织特异性的，可以很好的解释cancer中组织特异性的基因沉默。最后，cfDNA里检测到的甲基化异常也能反映癌症的发生。
5. 什么是cfDNA？有何特性，如何分析cfDNA来的数据？（可以看下文中参考文献）
缩写：
- Normal breast tissue (NBr)
- Breast cancer (BrC)
- Asymptomatic controls (AC)
Two independent cohorts of BrC patients were included in this study.
结果：
3.1. Clinical and Pathological Data of Tissue Cohort
summary一下临床的数据，检查里面的差异，做显著性分析。
两个cohorts之间的age没有显著差异，但是BrC和control之间差异显著。
3.2. Assessment of BrC and NBr Tissue Samples Methylation Levels
通过cohort #1来检查甲基化水平的差异。
3.3. Association between Promoter Methylation Levels, Molecular Subtypes and Standard Clinicopathological Parameters in Cohort #1
把Brc分成subtype来看甲基化水平的差异。
3.4. Survival Analysis
通过cohort #1来做生存分析
3.5. Biomarker Detection Performance in ccfDNA Liquid Biopsies (Cohort #2)
检查cohort #1中的biomarker在cfDNA中的表现。
有许多细节需要搞清楚：
1. 统计显著性的问题
Non-parametric tests were performed to determine statistical significance in all the comparisons made. Specifically, Kruskall-Wallis test was used for comparisons between three or more groups, whereas Mann-Whitney U test was used for comparisons between two groups.
2. 相关性分析
Spearman nonparametric correlation test was used to assess the association of methylation levels and age.
3. 生存分析
Disease-specific survival curves and disease-free survival curves (Kaplan–Meier with log rank test) were computed for standard clinicopathological variables and for categorized methylation status. A Cox-regression model comprising all significant variables (multivariable model) was computed to assess the relative contribution of each variable to the follow-up status.
4. age的confounder
Due to age’s difference between patients and controls, a multivariable model was constructed using logistic regression with the most informative genes and age.
5. multivariable analysis
6. panel
7. tissue
- primary BrC tissues
- circulating cell-free DNA (ccfDNA)
8. Molecular Subtypes
9. others
- disease-free survival
- disease-specific survival
- plasma or serum samples
Epigenetics in cancer - 里程碑综述
Epigenetic features of a normal cell
DNA hypomethylation in tumors
Inactivation of tumor suppressor genes
Histone modifications of cancer cells
Epigenetic factors and miRNA
Epigenetics in cancer management
Epigenetic therapy of cancer
说起cfDNA和癌症早筛就不得不看卢煜明
Yuk Ming Dennis Lo
利用EBV的DNA来筛查NPC鼻咽癌
这是个会议摘要，文章2017年发表过，这次增加了片段长度和表观方面的，增加PPV。
什么是PPV？
参考：