Ji Lexiang, Sasaki Takahiko, Sun Xiaoxiao, Ma Ping, Lewis Zachary A, Schmitz Robert J
Department of Genetics, University of Georgia, Athens, GA USA ; Institute of Bioinformatics, University of Georgia, Athens, GA USA.
Department of Microbiology, University of Georgia, Athens, GA USA.
Front Genet. 2014 Oct 21;5:341. doi: 10.3389/fgene.2014.00341. eCollection 2014.
The development of whole-genome bisulfite sequencing (WGBS) has resulted in a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced 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 reads from methylated DNA in 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. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.
全基因组亚硫酸氢盐测序（WGBS）的发展带来了许多关于DNA甲基化作用的令人兴奋的发现，从而产生了大量新的可测试假设。构建经亚硫酸氢钠转化和扩增的文库的方法最近已取得进展，以至于使用WGBS的实验的瓶颈已转移到数据分析和解释上。在此，我们提供了WGBS中甲基化DNA读数过度代表的经验证据。PCR循环数增加会加剧这种对甲基化DNA的富集，并且它受用于扩增测序文库的尿嘧啶不敏感DNA聚合酶类型的影响。未来在计算上校正这种富集偏差的努力对于提高确定单个胞嘧啶甲基化水平的准确性至关重要。对于试图准确量化可能因等位基因DNA甲基化状态而分离的群体中的DNA甲基化水平的研究而言，这尤其关键。
