School of Medicine, Hunan Normal University, Tongzipo Road 371, Changsha, 410013, People's Republic of China.
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine One Baylor Plaza, Houston, TX, 77-30, USA.
BMC Bioinformatics. 2020 Jan 31;21(1):38. doi: 10.1186/s12859-019-3334-z.
Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS.
Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter.
Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.
全基因组亚硫酸氢盐测序（WGBS）也称为 BS-seq，已被广泛用于以单碱基分辨率测量全基因组的甲基化。该测定法的关键步骤之一是将未甲基化的胞嘧啶转化为胸腺嘧啶（BS 转化）。未甲基化胞嘧啶的不完全转化可能会引入假阳性甲基化调用。开发一种快速评估亚硫酸氢盐转化率（BCR）的方法既有利于 WGBS 的质量控制，也有利于数据分析。
在这里，我们提供了一种名为“BCREval”的计算方法，该方法使用端粒重复 DNA 作为天然内参来估计未转化率（UCR）。我们使用公共 WGBS 数据测试了该方法，发现它非常稳定，大多数 BS 转化测定法的效率都可以达到>99.5%。端粒处的非 CpG 二核苷酸甲基化符合二项式模型，可能是由随机过程引起的，可能性非常低（比率<0.4%）。BCREval 与广泛使用的 BCR 评估器 Bismark（Krueger 和 Andrews，Bioinformatics 27:1571-1572, 2011）的比较表明，我们的算法比后者快得多且效率更高。
我们的方法是一种简单但稳健的方法，可以对 WGBS 实验进行 QC 和推测 BCR，以确保其达到可接受的水平。它比当前的工具更快、更高效，并且可以轻松集成到现有的 WGBS 管道中。
