Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR.
Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR.
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DOI：
10.12688/f1000research.13196.2
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Smyth GK
中科院分区：
文献类型：
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作者：
Chen Y;Pal B;Visvader JE;Smyth GK
胞嘧啶甲基化是一种重要的DNA表观遗传修饰。在脊椎动物中，甲基化发生在CpG位点，其是DNA序列中从5'到3'胞嘧啶紧接着鸟嘌呤的二核苷酸。当位于基因的启动子区域时，DNA甲基化通常与基因的转录沉默相关。DNA甲基化异常与癌症等多种疾病的发生有关。亚硫酸氢盐测序（BS-seq）是目前高分辨率DNA甲基化分析的“金标准”技术。简化代表性亚硫酸氢盐测序（RRBS）是BS-seq的有效形式，其靶向富含CpG的DNA区域以节省测序成本。一个典型的生物信息学目标是鉴定在实验条件之间差异甲基化（DM）的CpG。该工作流程表明，可以使用最初为RNA-seq数据开发的软件和方法进行RRBS数据的差异甲基化分析。通过向设计矩阵中添加额外的列以考虑每个CpG处的读段覆盖率，使RNA-seq管道适应甲基化，之后RRBS和RNA-seq管道几乎相同。这种方法在统计学上是自然的，并使分析人员能够使用丰富的分析工具，包括广义线性模型，基因集测试和途径分析。本文介绍了一个完整的开始完成的案例研究分析RRBS配置文件的不同细胞群从小鼠乳腺使用Bioconductor包edgeR。我们发现谱系定型细胞与祖细胞相比通常是高度甲基化的，这在所有常染色体上都是如此，但在性染色体上不是。我们证明了启动子区域的甲基化和基因表达之间的强负相关性，通过RNA-seq测量相同的细胞类型，表明甲基化是一种参与上皮线性定型的调控机制。
Cytosine methylation is an important DNA epigenetic modification. In vertebrates, methylation occurs at CpG sites, which are dinucleotides where a cytosine is immediately followed by a guanine in the DNA sequence from 5' to 3'. When located in the promoter region of a gene, DNA methylation is often associated with transcriptional silencing of the gene. Aberrant DNA methylation is associated with the development of various diseases such as cancer. Bisulfite sequencing (BS-seq) is the current "gold-standard" technology for high-resolution profiling of DNA methylation. Reduced representation bisulfite sequencing (RRBS) is an efficient form of BS-seq that targets CpG-rich DNA regions in order to save sequencing costs. A typical bioinformatics aim is to identify CpGs that are differentially methylated (DM) between experimental conditions. This workflow demonstrates that differential methylation analysis of RRBS data can be conducted using software and methodology originally developed for RNA-seq data. The RNA-seq pipeline is adapted to methylation by adding extra columns to the design matrix to account for read coverage at each CpG, after which the RRBS and RNA-seq pipelines are almost identical. This approach is statistically natural and gives analysts access to a rich collection of analysis tools including generalized linear models, gene set testing and pathway analysis. The article presents a complete start to finish case study analysis of RRBS profiles of different cell populations from the mouse mammary gland using the Bioconductor package edgeR. We show that lineage-committed cells are typically hyper-methylated compared to progenitor cells and this is true on all the autosomes but not the sex chromosomes. We demonstrate a strong negative correlation between methylation of promoter regions and gene expression as measured by RNA-seq for the same cell types, showing that methylation is a regulatory mechanism involved in epithelial linear commitment.