Advanced Methylome Analysis after Bisulfite Deep Sequencing: An Example in Arabidopsis
Advanced Methylome Analysis after Bisulfite Deep Sequencing: An Example in Arabidopsis
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DOI：
10.1371/journal.pone.0041528
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发表时间：
2012-07-20
期刊：
影响因子：
3.7
通讯作者：
von Haeseler, Arndt
中科院分区：
文献类型：
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作者：
Dinh, Huy Q.;Dubin, Manu;von Haeseler, Arndt
亚硫酸氢盐转化后深度测序（BS-Seq）是以单碱基对分辨率生成胞嘧啶甲基化全基因组图谱的首选方法。将其应用于拟南芥花蕾组织的基因组DNA导致了第一个完整的甲基化组，确定该组织中的甲基化率为6.7%。应用所谓的3-字母基因组方法，将BS-Seq读数映射到计算机转换的参考基因组上。在这里，我们提出了BiSS（亚硫酸氢盐测序评分器），一种新的方法，应用史密斯-沃特曼比对映射亚硫酸氢盐转换的读数到参考基因组。此外，我们引入了一个全面的自适应误差估计，占测序错误，错误的亚硫酸氢盐转换，也错误映射读取。用BiSS对拟南芥甲基化组数据的重新分析将实质上更多的读段映射到基因组。因此，它确定了额外10%胞嘧啶的甲基化状态，并估计甲基化率为7.7%。我们通过对选定的基因组区域进行单独的传统亚硫酸氢盐测序来验证结果。除了预测每个胞嘧啶的甲基化状态，BiSS还提供了每个基因组位点甲基化程度的估计。因此，BiSS更广泛地探索BS-Seq数据，并为下游分析提供更多信息。
Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis.