标题： 从技术条件的角度选择ChIP-seq归一化方法 显示英文标题

标题： Selecting ChIP-seq Normalization Methods from the Perspective of their Technical Conditions

摘要： 染色质免疫沉淀结合高通量测序（ChIP-seq）提供了关于感兴趣蛋白所占据的基因组位置以及实验状态下DNA占有率差异的见解。 鉴于ChIP-seq数据是通过实验收集的，确定状态间具有不同DNA占有率区域的重要步骤是在样本间的标准化。 虽然样本间标准化对于下游差异结合分析至关重要，但ChIP-seq中样本间标准化方法背后的技术条件尚未被检查。 我们确定了ChIP-seq样本间标准化方法背后的三个重要技术条件：平衡的差异DNA占有率、相等的总DNA占有率和跨状态相等的背景结合。 为了展示满足所选标准化方法的技术条件以用于下游差异结合分析，我们模拟了ChIP-seq读取计数数据，其中违反了不同的技术条件组合。 然后，我们使用实验数据外部验证我们的模拟结果。 基于我们的发现，我们建议研究人员利用他们对当前ChIP-seq实验的理解来指导他们选择样本间标准化方法。 或者，研究人员可以使用高置信度峰集，这是从使用不同的样本间标准化方法获得的不同结合峰集的交集。 在我们的两个实验分析中，每种标准化方法大约有一半的峰值被调用为差异结合。 高置信度峰对样本间标准化方法的选择不太敏感，并且当不确定满足哪些技术条件时，可能是识别实验状态下具有不同DNA占有率的基因组区域的一个更稳健的基础。 显示英文摘要

摘要： Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) provides insights into both the genomic location occupied by the protein of interest and the difference in DNA occupancy between experimental states. Given that ChIP-seq data is collected experimentally, an important step for determining regions with differential DNA occupancy between states is between-sample normalization. While between-sample normalization is crucial for downstream differential binding analysis, the technical conditions underlying between-sample normalization methods have yet to be examined for ChIP-seq. We identify three important technical conditions underlying ChIP-seq between-sample normalization methods: balanced differential DNA occupancy, equal total DNA occupancy, and equal background binding across states. To illustrate satisfying the selected normalization method's technical conditions for downstream differential binding analysis, we simulate ChIP-seq read count data where different combinations of the technical conditions are violated. We then externally verify our simulation results using experimental data. Based on our findings, we suggest that researchers use their understanding of the ChIP-seq experiment at hand to guide their choice of between-sample normalization method. Alternatively, researchers can use a high-confidence peakset, which is the intersection of the differentially bound peaksets obtained from using different between-sample normalization methods. In our two experimental analyses, roughly half of the called peaks were called as differentially bound for every normalization method. High-confidence peaks are less sensitive to choice of between-sample normalization method and could be a more robust basis for identifying genomic regions with differential DNA occupancy between experimental states when there is uncertainty about which technical conditions are satisfied.