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首页> 美国卫生研究院文献>Genome Research >Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization
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Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization

机译：定量ChIP-seq数据：一种加标方法为样品间标准化提供内部参考

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摘要

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This “spike” chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes.
机译：染色质免疫沉淀后再进行深度测序（ChIP-seq）实验被广泛用于确定整个基因组内任何目标蛋白质的占据位点，包括例如转录因子，RNA聚合酶或具有或没有各种修饰的组蛋白。除了可以确定一种细胞类型内和一种条件下的占用位点外，该方法原则上还可以建立和比较各种细胞类型，组织和条件下的占用图。但是，此类比较需要对样本进行归一化。当因子占有率在一个位点的子集处变化时，包括分位数归一化步骤在内的广泛使用的归一化方法会很好地执行，但是可能会错过全基因组范围内位点占有率的均匀增加或减少。我们描述了一个峰值调整程序（SAP），与在分析阶段介入的常用归一化方法不同，该步骤需要在免疫沉淀之前进行实验。将来自单批外源基因组染色质的恒定，少量添加到实验染色质中。然后，这种“尖峰”染色质可以作为内部对照，可以将实验信号调节到该内部对照。我们表明，该方法提高了重复之间的相似性，并揭示了生物学差异，包括全局和很大程度上统一的变化。

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