Vargas-Landin Dulce B, Pflüger Jahnvi, Lister Ryan
Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia.
Harry Perkins Institute of Medical Research, Nedlands, WA, Australia.
Methods Mol Biol. 2018;1767:291-298. doi: 10.1007/978-1-4939-7774-1_16.
Whole genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high quality libraries amplified with fewer PCR cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer cleanup steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with few PCR cycles to reach the yield needed for sequencing.
全基因组亚硫酸氢盐测序（WGBS）能够在单碱基对分辨率下检测DNA甲基化。用亚硫酸氢钠处理DNA可区分甲基化和未甲基化的胞嘧啶，但由于脱硫过程导致的DNA损伤，该技术的效能可能会受到DNA输入量和DNA片段长度的限制。在此，我们描述了一种WGBS文库制备方案，该方案可最大限度地减少DNA的损失和损伤，以较少的PCR循环数扩增生成高质量文库，从而从较少量的输入材料中获得较少PCR重复的数据。简而言之，基因组DNA被剪切、末端修复、3'端腺苷酸化，并在其间进行较少的纯化步骤连接接头，最大限度地减少DNA损失。然后将连接接头的DNA用亚硫酸氢钠处理，并用较少的PCR循环进行扩增，以达到测序所需的产量。
