Abbasova Leyla, Urbanaviciute Paulina, Hu Di, Ismail Joy N, Schilder Brian M, Nott Alexi, Skene Nathan G, Marzi Sarah J
UK Dementia Research Institute at Imperial College London, London, UK.
Department of Brain Sciences, Imperial College London, London, UK.
Nat Commun. 2025 Mar 27;16(1):2993. doi: 10.1038/s41467-025-58137-2.
DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Cleavage Under Targets & Tagmentation (CUT&Tag) is a rapidly expanding technique that enables the profiling of such interactions in situ at high sensitivity. However, thorough evaluation and benchmarking against established ChIP-seq datasets are lacking. Here, we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. We tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Overall, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments, providing a benchmarking framework for the experimental design and analysis of CUT&Tag studies.
传统上，DNA与蛋白质的相互作用是通过染色质免疫沉淀结合下一代测序（ChIP-seq）来进行分析的。靶向切割与标签化（CUT&Tag）是一种迅速发展的技术，能够在原位以高灵敏度分析此类相互作用。然而，目前缺乏针对已建立的ChIP-seq数据集进行的全面评估和基准测试。在此，我们针对K562细胞中来自ENCODE的已发表ChIP-seq图谱，对H3K27ac和H3K27me3的CUT&Tag技术进行了全面的基准测试。结合多个新的和已发表的CUT&Tag数据集，两种组蛋白修饰对已知ENCODE峰的平均召回率为54%。我们测试了峰值调用软件MACS2和SEACR，并确定了最佳的峰值调用参数。总体而言，CUT&Tag识别出的峰代表了最强的ENCODE峰，并且与ENCODE识别出的ChIP-seq峰显示出相同的功能和生物学富集。我们的工作流程系统地评估了方法调整的优点，为CUT&Tag研究提供了一个实验设计和分析的基准框架。
