DNA甲基化是DNA被添加甲基()修飾影響基因功能或者表達尾菇。最常見的甲基化是胞嘧啶產(chǎn)生5-甲基胞嘧啶(5-methylcytosine, 5-mC)。有甲基化過程就有去甲基化過程，整個胞嘧啶甲基化循環(huán)如下圖所示枫虏。
在體細(xì)胞中 5-mC 幾乎只發(fā)生在 CpG 位點盾鳞，但是 CpG 島(CpG island)區(qū)域往往不是甲基化的，因為許多 CpG 島是靠近啟動子區(qū)域的诡挂，甲基化將導(dǎo)致基因無法表達碎浇。
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 技術(shù)利用亞硫酸氫鈉(Sodium Bisulfite)處理DNA導(dǎo)致未甲基化的C變成U并在后續(xù)PCR和測序過程成為T临谱，而甲基化的C不受影響。BS 處理 DNA 容易產(chǎn)生破壞作用奴璃，尤其是 CpG 島含有大量未甲基化的C悉默，因此在此區(qū)域容易覆蓋度低。建庫方法可以分為2種苟穆，1種是先進行 DNA 破碎與連接接頭抄课，然后亞硫酸鹽處理 C->T 轉(zhuǎn)化；另一種先進行 C->T 轉(zhuǎn)化然后再連接接頭擴展雳旅。后者對 DNA 投入要求更低跟磨。WGBS 技術(shù)無法區(qū)分 5-hmC 與 5-mC 。
下圖是 illumina 的建庫流程攒盈，由于只保留原始 BS 處理后鏈互補鏈作為測序模板抵拘，因此 read1 序列跟 BS 后鏈序列相同。
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 比對軟件是 Bismark沦童， Bismark 將參考基因組序列預(yù)先進行 C->T 和 G->A 2種轉(zhuǎn)換仑濒。比對時每一條 reads 同樣進行 C->T 和 G->A 2種轉(zhuǎn)換，這樣組合以后每條 reads 相當(dāng)于進行 4 種不同的比對偷遗，這些比對選出最佳比對墩瞳，就可以確定發(fā)生甲基化的鏈方向和可能甲基化位點。下圖所示氏豌。
[參考]
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.
