【作者】 吴金雨；

【导师】 史庆华；

【作者基本信息】 中国科学技术大学 ， 遗传学， 2014， 博士

【摘要】 DNA甲基化作为一种重要的表观遗传学修饰,在x染色体失活、胚胎发育、细胞分化、疾病发生和癌症形成等方面发挥着重要的作用。新一代高通量测序技术的兴起使得DNA甲基化研究在深度和广度上得到了很大的拓展,涌现出一批基于高通量测序的全基因组甲基化分析方法,例如全基因组重亚硫酸盐测序(BS-seq)、甲基化DNA免疫共沉淀测序(MeDIP-seq)以及简化的重亚硫酸盐测序技术(RRBS)。这些新技术在推动甲基化研究的同时,也产生了大量甲基化测序数据。如何从这些海量的数据中快速挖掘出有效的信息对于研究者们提出了巨大的挑战。本研究主要从甲基化测序数据模拟、差异甲基化区域(DMR)分析、甲基化分析整合平台的开发以及甲基化可视化软件这几个相互辅助的方面开发基于高通量测序的甲基化分析软件,为DNA甲基化研究者提供工具上的支撑。首先,我们开发了甲基化测序序列模拟工具BSSim。该模拟软件适用于各大主流测序平台的测序格式,可观测多种错误因素和不同层次对甲基化数据的影响,同时能为后续的分析选择合适参数并减小错误因素提供帮助。BSSim的开发可为我们评估随后开发软件的性能提供了基础。其次,我们开发了以滑窗方式,在基因组水平的单碱基精度甲基化数据上扫描查找DMR (differentially methylated regions)的软件swDMR。swDMR整合多种常用的统计学方法,适用于配对样本或多个样本的重亚硫酸盐处理的DNA甲基化测序数据的DMR查找、注释和可视化。通过swDMR,我们可以更快捷的发现潜在的参与表观调控的功能区域。随后,我们基于RRBS测序数据开发了整合的甲基化分析平台RRBS-Analyser。RRBS-Analyser可以对原始的测序数据进行质量评估,产生基本的统计信息,把质量控制之后产生的短序列比对到参考基因组上,确定并注释甲基化胞嘧啶的位点,并能对多个样品进行差异甲基化区间的检测及注释。此外,RRBS-Analyser还支持除人以外的其它多个物种的基因组DNA甲基化分析。最后,我们开发了全基因组甲基化可视化软件iMethy。iMethy拥有数据比对、甲基化位点寻找、甲基化可视化等多种功能,具有使用便捷、功能强大、界面友好,可以有效地发现隐藏在信息内部的特征和规律。iMethy适用于多种操作系统,适用性强。iMethy可作为研究人员进行甲基化数据分析的一个强有力的可视化工具。更多还原

【Abstract】 DNA methylation, as an important epigenetic modification, plays an crucial role in X-chromosome inactivation, embryonic development, cell differentiation, disease and cancer formation and other diseases. With the advent of high-throughput sequencing, researches on DNA methylation were facilitated both in the depth and scope, and a large number of genome-wide methylation analysis methods appeared, such as whole-genome bisulfite sequencing (BS-seq), methylated DNA immunoprecipitation sequencing (MeDIP-seq) and reduced representation bisulphite sequencing (RRBS). These new technologies did prompt the methylation studies, but also generated massive methylation sequencing data. Therefore, a huge challenge has emerged for researchers:how to quickly dig out the useful information from these massive data. With the hope of providing facilitations for researchers on DNA methylation, we developed the following analysis tools based on high-throughput sequencing, including simulation of methylation sequencing data, differentially methylated region (DMR) analysis, integrated platform for methylation analysis and visualization software.Firstly, we developed BSSim, a simulation software applied to generate short reads with sequencing format of main sequencing platforms. BSSim can evaluate the effects of multilevel factors on the sequencing data, also act as a reference for selecting the appropriate parameters in subsequent analysis and reduce the effect of error factors on the sequencing data.Secondly, we developed the swDMR, which is based on sliding window, to scan DMR at single-base resolution on the genome-wide scale. swDMR integrates a variety of commonly used statistical methods for detection, annotation and visualization of DMR from either paired samples or multiple samples treated by bisulfite. By using swDMR, users can identify potential functional regions involved in epigenetic regulation.Thirdly, we developed RRBS-Analyser:an integrated analysis platform which can comprehensively analyze RRBS sequencing data in genome-wide. It can perform quality assessment for the original sequencing data, produce basic statistical information, align the clean reads after quality control to the reference genome, identify and annotate sites of methylated cytosine, and identity and annotate DMR in multiple samples. Additionally, RRBS-Analyser supports genomic DNA methylation analysis of other species.Finally, we developed iMethy, a genome-wide methylation visualization software. It is characterized by the following functionalities:alignment, methylation identification and methylation visualization. It is powerful, userfriendly and can be used to effectively discover hidden features and patterns. Furthermore, iMethy is applicable to multiple operating systems, almost importantly, it has a fast rate in visualization refreshing and provide a powerful visualization tool for researchers on methylation analysis.更多还原