High-throughput methods such as RNA-seq, ChIP-seq, and ATAC-seq have well-established guidelines, commercial kits, and analysis pipelines that enable consistency and wider adoption for understanding genome function and regulation. STARR-seq, a popular assay for directly quantifying the activities of thousands of enhancer sequences simultaneously, has seen limited standardization across studies. The assay is long, with more than 250 steps, and frequent customization of the protocol and variations in bioinformatics methods raise concerns for reproducibility of STARR-seq studies. Here, we assess each step of the protocol and analysis pipelines from published sources and in-house assays, and identify critical steps and quality control (QC) checkpoints necessary for reproducibility of the assay. We also provide guidelines for experimental design, protocol scaling, customization, and analysis pipelines for better adoption of the assay. These resources will allow better optimization of STARR-seq for specific research needs, enable comparisons and integration across studies, and improve the reproducibility of results.

中文翻译：

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STARR-seq 检测重现性的挑战和注意事项

RNA-seq、ChIP-seq 和 ATAC-seq 等高通量方法拥有完善的指南、商业试剂盒和分析流程，可实现一致性和更广泛的采用，以了解基因组功能和调控。STARR-seq 是一种同时直接量化数千个增强子序列活性的流行检测方法，但各项研究的标准化程度有限。该检测过程很长，有超过 250 个步骤，并且频繁定制实验方案和生物信息学方法的变化引起了人们对 STARR-seq 研究的可重复性的担忧。在这里，我们从已发表的来源和内部检测中评估方案和分析流程的每个步骤，并确定检测重现性所需的关键步骤和质量控制 (QC) 检查点。我们还提供实验设计、方案扩展、定制和分析流程的指南，以更好地采用该检测。这些资源将有助于针对特定研究需求更好地优化 STARR-seq，实现研究之间的比较和整合，并提高结果的可重复性。