Targeted DNA methylation analysis by next-generation sequencing.
Targeted DNA methylation analysis by next-generation sequencing.
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通过下一代测序进行靶向DNA甲基化分析。
DOI：
10.3791/52488
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发表时间：
2015-02-24
期刊：
影响因子：
--
通讯作者：
Freeman WM
中科院分区：
文献类型：
--
作者：
Masser DR;Stanford DR;Freeman WM
表观遗传过程在基因表达控制中的作用多年来已为人所知。胞嘧啶残基上的 DNA 甲基化对于表观遗传学研究特别重要，因为它已被证明是基因表达的持久且动态的调节因子。由于缺乏高通量、定量准确的方法，检查健康和疾病的表观遗传变化的努力受到阻碍。随着下一代测序 (NGS) 技术的出现和普及，除了现有的基因组和转录组方法之外，这些工具现在还应用于表观基因组学。对于胞嘧啶甲基化的表观遗传学研究，其中已识别出感兴趣的区域，例如特定基因启动子或 CpG 岛，并且需要以高定量精度检查大量样品，我们开发了一种称为亚硫酸氢盐扩增子测序 (BSAS) 的方法。该方法将亚硫酸氢盐转化与感兴趣区域的靶向扩增、转座体介导的文库构建和台式 NGS 相结合。 BSAS 提供了一种快速有效的方法，可一次分析多达 96 个样本中多达 10 kb 的目标区域，大多数具有基本分子生物学技能的研究小组都可以执行该方法。结果提供了具有碱基特异性的胞嘧啶甲基化的绝对定量。 BSAS 可应用于任何 DNA 来源的任何基因组区域。该方法可用于感兴趣目标区域的假设检验研究以及全基因组甲基化分析（例如全基因组亚硫酸氢盐测序、简化代表性亚硫酸氢盐测序和甲基化 DNA 免疫沉淀测序）中识别的区域的确认。
The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.
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影响因子：
14.9
作者：
WANG, RYH;GEHRKE, CW;EHRLICH, M
通讯作者：
EHRLICH, M
影响因子：
4.1
作者：
Mikeska, Thomas;Bock, Christoph;Waha, Andreas
通讯作者：
Waha, Andreas
影响因子：
15
作者：
HAYATSU, H;WATAYA, Y;KAI, K
通讯作者：
KAI, K
影响因子：
14.9
作者：
Ivanov M;Kals M;Kacevska M;Metspalu A;Ingelman-Sundberg M;Milani L
通讯作者：
Milani L
超低输入，基于标记的全基因组硫酸盐测序。
影响因子：
7
作者：
Adey A;Shendure J
通讯作者：
Shendure J