#表观全局#Epigenomic Assays：表观遗传检测方法荟萃（Cell SnapShot）中英互译V1.2
原创
2017-02-12
Huan
生物信息
生物信息
BioinforCN 生物信息 是研究生命或医学的“生产力”。 生物信息 是分析高通量生命数据的“指路灯”。 生物信息 是开发生物工程设备的“CPU”。 Epigenomic Assays 表观遗传检测方法荟萃 Canada’s Michael Smith Genome Sciences Centre Massively parallel sequencing technologies provided the foundation from which the field of epigenomics has been built. 大规模并行DNA测序技术（高通量测序）提供了研究“表观遗传学”方法论，并在为该领域的发展建立坚实的基础。 This SnapShot depicts key sequencing-based methods used in the analysis of epigenomes, including 本PDF文档描述的是以高通量测序为理论基础的表观遗传学关键实验技术的总结。 (1) bisulfite sequencing, 分析DNA甲基化的“重硫酸盐测序技术” (2) chromatin immunoprecipiation sequencing (ChIP-seq), 分析蛋白质与DNA结合的“染色质免疫共沉淀测序技术” (3) determination of open chromatin, and 分析染色体高级结构的“染色质开放状态分析技术” (4) 3D chromatin capture. 分析细胞核高级结构的“染色质三维结构捕获分析” Bisulfite Sequencing Genome-wide 5-methyl-cytosine (5mC) is measured by enrichment-based methods and direct sequencing of bisulfi te-converted DNA. 基因组尺度的重亚硫酸盐测序分析“5位甲基化的胞嘧啶” 是通过富集DNA方法和直接测序“重亚硫酸盐还原为受甲基化保护的DNA”。 Enrichment methods provide qualitative measures of 5mC (Down et al., 2008), are capable of distinguishing oxidative derivatives (e.g., hmC), and can be combined with methyl-restriction-based assays to improve detection of unmethylated cytosines (Maunakea et al., 2010). 富集的方法提供了定性检测甲基化胞嘧啶的措施，能够区分氧化还原的衍生品，可以结合甲基化被保护未发生反应来提高检测到未甲基化的胞核嘧啶的精度。 Enrichment-based methods require 50–100 million sequence reads per sample. 富集方法的最低要求：每个样本需要50 - 100万高通量Read（读取）的覆盖。 Direct sequencing of bisulfi te-treated DNA provides quantitative measurements of 5mC genome wide but is unable to distinguish oxidized derivatives. 直接测序可以定量检测测定重硫酸盐处理后的DNA，提供了定量测量5位甲基化胞嘧啶基因组的分布，但无法区分氧化还原的衍生产物。 Two main strategies for library construction have emerged for bisulfite sequencing. 对应重硫酸盐处理的DNA高通量测序的 文库构建有两个重要的策略 In the first method (shown), a genomic library with adapters added is subjected to bisulfite treatment (Lister et al., 2009). 第一个策略如图所示：文库的接头序列对于重硫酸盐的反应必须是惰性的，结合到样品DNA片段上接头才能准确反应样本的编号。 In the second (not shown), genomic DNA is first bisulfi te-converted and then subjected to library construction (Miura et al., 2012). 第二个策略：基因组DNA的文库构建的处理必须在重硫酸盐处理之后。 For conversion methods 1 billion 100nt sequence reads are generated for each sample and library construction methods introduce distinct library specific biases in genome coverage. 这样的方法进行测序：对于每个样本如果达到10亿条每条100Nt长度的测序Read，那就是文库构建方法过程中产生了“基因组特异性序列偏好误差”。 ChIP-Seq Genomic locations of modified histones are detected genome wide by ChIP-seq (Barski et al., 2007). 基因组尺度ChIP-Seq定位组蛋白修饰情况。 Typically, 25–50 million immunoprecipitated fragments are sequenced for a histone mark. 一般来说，2千5百万到5千万的免疫沉淀反应获取的DNA片段作为 一个组蛋白标记标记情况的测序集。 The two main strategies that have emerged to release nucleosomes from chromatin associated with 100–300 bp genomic fragments are shown. 图中表示了两个主要策略分析：松散的核小体结合的100 - 300bp长度的基因片段。 In addition, strategies that utilize transposon-based fragmentation of chromatin have recently become available (Schmidl et al., 2015). 此外：通过利用转座子碎片的分析染色质情况的方法也得到了公认。 Mapping Open Chromatin
Genomic positions of open chromatin regions are measured by directly sequencing genomic DNA fragments that are released from chromatin either by transposon insertion
(Buenrostro et al., 2013) or enzymatic digestion (Boyle et al., 2008; Crawford et al., 2006). 基因组尺度的开放染色质位置作图，是通过直接测序DNA片段测量基因组的染色质松弛区域是否转座子插入或核酸酶消化的情况。 A variant of this protocol (not shown) chemically cross-links chromatin and uses sonication to release non-cross-linked regions that are subsequently enriched by phenol-chloroform extraction (Boyle et al., 2008). 有几种方法但未统一，主要是通过化学交叉连接染色质和使用声波打断序列释放未交联区域。 Sequencing requirements depend on experimental parameters and resolution requirements and range from 10s to 100 s millions of fragments per sample. 高通测序需求取决于实验参数和精度的需要，范围从每样例1千万到1亿个Read不等。 Chromosome Conformation Capture Chromosome conformation capture (3C) technologies provide the location of DNA fragments that interact together on the basis of their proximity in three-dimensional (3D) space (Bonev and Cavalli, 2016). 染色体高级结构捕获技术提供交互的DNA片段的位置的基础上他们的距离在三维空间架构。 To measure chromatin interactions genome-wide a single experiment typically requires 500 million sequence reads. 全基因组尺度范围内测量染色质DNA片段间互作的高通量测序需要 单个样品5亿的Read。 原文链接为“技术方法总结”PDF
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