BACKGROUND Chromatin dysregulation is associated with developmental disorders and cancer. Numerous methods for measuring genome-wide chromatin accessibility have been developed in the genomic era to interrogate the function of chromatin regulators. A recent technique which has gained widespread use due to speed and low input requirements with native chromatin is the Assay for Transposase-Accessible Chromatin, or ATAC-seq. Biologists have since used this method to compare chromatin accessibility between two cellular conditions. However, approaches for calculating differential accessibility can yield conflicting results, and little emphasis is placed on choice of normalization method during differential ATAC-seq analysis, especially when global chromatin alterations might be expected. RESULTS Using an in vivo ATAC-seq data set generated in our recent report, we observed differences in chromatin accessibility patterns depending on the data normalization method used to calculate differential accessibility. This observation was further verified on published ATAC-seq data from yeast. We propose a generalized workflow for differential accessibility analysis using ATAC-seq data. We further show this workflow identifies sites of differential chromatin accessibility that correlate with gene expression and is sensitive to differential analysis using negative controls. CONCLUSIONS We argue that researchers should systematically compare multiple normalization methods before continuing with differential accessibility analysis. ATAC-seq users should be aware of the interpretations of potential bias within experimental data and the assumptions of the normalization method implemented.

中文翻译：

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ATAC-seq归一化方法会严重影响差异可访问性分析和解释。

背景技术染色质失调与发育障碍和癌症有关。在基因组时代，已经开发了许多用于测量全基因组染色质可及性的方法，以检验染色质调节剂的功能。由于速度快和对天然染色质的低输入要求而获得广泛应用的最新技术是转座酶可及染色质测定法（ATAC-seq）。此后，生物学家使用这种方法比较了两种细胞条件之间的染色质可及性。但是，用于计算差异可访问性的方法可能会产生矛盾的结果，并且在差异ATAC-seq分析期间，特别是在可能期望整体染色质改变的情况下，很少强调标准化方法的选择。结果使用我们最近的报告中生成的体内ATAC-seq数据集，我们观察到了染色质可及性模式的差异，这取决于用于计算差异可及性的数据标准化方法。这项观察结果已在酵母中发表的ATAC-seq数据上得到进一步证实。我们为使用ATAC-seq数据的差异可访问性分析提出了一种通用的工作流。我们进一步显示，此工作流程可识别与基因表达相关且对使用阴性对照进行差异分析敏感的差异性染色质可及性的位点。结论我们认为研究人员应在继续进行差异可及性分析之前，系统地比较多种归一化方法。