WGBS 全基因组甲基化测序

DNA甲基化是DNA被添加甲基()修饰影响基因功能或者表达。最常见的甲基化是胞嘧啶产生5-甲基胞嘧啶(5-methylcytosine, 5-mC)。有甲基化过程就有去甲基化过程，整个胞嘧啶甲基化循环如下图所示。

C甲基化循环

在体细胞中 5-mC 几乎只发生在 CpG 位点，但是 CpG 岛(CpG island)区域往往不是甲基化的，因为许多 CpG 岛是靠近启动子区域的，甲基化将导致基因无法表达。

The CpG dinucleotides are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG is shorthand for 5'—C—phosphate—G—3' , that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA.

CpG islands are usually defined as regions with 1) a length greater than 200bp, 2) a G+C content greater than 50%, 3) a ratio of observed to expected CpG greater than 0.6, although other definitions are sometimes used.

WGBS 技术利用亚硫酸氢钠(Sodium Bisulfite)处理DNA导致未甲基化的C变成U并在后续PCR和测序过程成为T，而甲基化的C不受影响。BS 处理 DNA 容易产生破坏作用，尤其是 CpG 岛含有大量未甲基化的C，因此在此区域容易覆盖度低。建库方法可以分为2种，1种是先进行 DNA 破碎与连接接头，然后亚硫酸盐处理 C->T 转化；另一种先进行 C->T 转化然后再连接接头扩展。后者对 DNA 投入要求更低。WGBS 技术无法区分 5-hmC 与 5-mC 。

2种建库策略

下图是 illumina 的建库流程，由于只保留原始 BS 处理后链互补链作为测序模板，因此 read1 序列跟 BS 后链序列相同。

Illumina建库流程

In this procedure, bisulfite-treated single-stranded DNA is randomprimed using a polymerase able to read uracil nucleotides, to synthesize DNA containing a specific sequence tag. The 3′ ends of the newly synthesized DNA strands are then selectively tagged with a second specific sequence, resulting in di-tagged DNA molecules with known sequence tags at their 5′ and 3′ ends (Figure 1). These tags are then used to add Illumina P7 and P5 adapters by PCR at the 5′ and 3′ ends, respectively, of the original DNA strand.

常用的 WGBS 比对软件是 Bismark， Bismark 将参考基因组序列预先进行 C->T 和 G->A 2种转换。比对时每一条 reads 同样进行 C->T 和 G->A 2种转换，这样组合以后每条 reads 相当于进行 4 种不同的比对，这些比对选出最佳比对，就可以确定发生甲基化的链方向和可能甲基化位点。下图所示。

Bismark比对

[参考]
DNA Methylation | What is Epigenetics?
DNA methylation - Wikipedia
How does bisulfite sequencing (WGBS/RRBS) work?
Grehl, Claudius, et al. "How to design a whole-genome bisulfite sequencing experiment." Epigenomes 2.4 (2018): 21.
Krueger, Felix, and Simon R. Andrews. "Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications." bioinformatics 27.11 (2011): 1571-1572.