DNA甲基化是重要的表觀遺傳機制之一，DNA甲基化也涉及許多生物學過程，包括轉錄活性、基因組印記、發育和包括癌症在內的許多疾病。近幾年，隨著新一代測序技術的進步，為研究全基因組DNA甲基化提供了機會。另外，亞硫酸氫鹽測序為現在流行且強大的甲基化檢測方法，同時也是最多人研究的方法。全基因亞硫酸氫鹽測序和還原型亞硫酸氫鹽測序經常被用於生成DNA甲基化組，需要高效的工具來比對亞硫酸氫鹽測序數據。
現今雖然已經有許多亞硫酸氫鹽工具被研究出來，但由於缺少基準的評估，這讓使用者在判斷工具的準確度方面造成很多不便。因此本文將對現有的工具進行基準的評估，進而對預測DNA甲基化擬定出不同的策略，以及根據不同的情況選擇適合的工具操作。接下來將在此篇文章中分析EAGLE-­METH、BS-­Seeker2和Bismark這三種對齊工具，比較其評估甲基化程度的能力，並討論在不同物種的數據或是不同染色體片段中，對於分析甲基化水平的影響。
從實驗結果可以得知BS-­Seeker2和Bismark在甲基化水平的計算上準確度都非常高。但相較於BS­-Seeker2，在可視化甲基化分析方面Bismark有著更齊全的功能來可視化我們的結果。此外，我們認為EAGLE­-meth是能夠用來評估亞硫酸氫數據的比對工具。EAGLE-­meth已經可以預測出大部分CpG中發生甲基化的機率，這對DNA甲基化的研究提供了很大的幫助。

DNA methylation is one of the important epigenetic mechanisms and that are also involved in many biological processes, including transcriptional activity, genomic imprinting, development, and many diseases including cancer. In recent years, advances in next­generation sequencing technologies have provided opportunities to study genome­wide DNA methylation. In addition, bisulfite sequencing is now a popular and powerful method for methylation detection and is also the most studied method. Whole gene bisulfite sequencing and reduced representation bisulfite sequencing are often used to generate DNA methylation, and efficient tools are needed to compare bisulfite sequencing data.
Although many bisulfite tools have been developed, the lack of benchmarking makes it inconvenient for users to evaluate the accuracy of the tools. Therefore, in this article, we will benchmark the existing tools and develop different strategies for predicting DNA methylation and selecting the appropriate tool for each situation. In this paper, we will analyze three aligned tools, EAGLE-METH, BS­Seeker2, and Bismark, to compare their ability to evaluate the methylation level, and discuss the impact on the analysis of methylation level in different species of data or different chromosome fragments.
From the experimental results, we can see that both BS­-Seeker2 and Bismark are very accurate in the calculation of methylation levels. However, compared to BS­-Seeker2, Bismark has a more complete set of features to visualize our results in terms of visual methylation analysis. In addition, we consider EAGLE-­meth as a comparative tool to evaluate bisulfite data, and EAGLE­-meth has been able to predict the probability of methylation in most CpG, which is a great help for DNA methylation studies.

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