Methylated DNA is over-represented in whole-genome bisulfite sequencing data. Arabidopsis thaliana

The development of whole-genome bisulfite sequencing (WGBS) has led to a number of exciting discoveries about how genomes utilize DNA methylation and has led to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently excelled to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of methylated DNA from WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. Overall design: MethylC-Seq of Arabidopsis thaliana

全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGBS）的发展，推动了诸多关于基因组如何利用DNA甲基化的突破性发现，同时催生了大量可验证的全新科学假说。近年来，构建亚硫酸氢盐转化扩增文库的实验方法已日趋成熟，使得WGBS实验的瓶颈已从实验环节转向数据分析与结果解读。本研究提供了WGBS中甲基化DNA过度富集的实证依据：该甲基化DNA富集效应会因PCR循环数增加而加剧，同时受用于扩增测序文库的尿嘧啶不敏感DNA聚合酶（uracil-insensitive DNA polymerase）类型的影响。若要精准测定单个胞嘧啶的甲基化水平，未来对该富集偏差开展计算校正的相关工作将至关重要。整体实验设计：拟南芥（Arabidopsis thaliana）的MethylC-Seq测序。