RNA 修饰对人类健康至关重要——修饰过多或过少都会导致从神经发育障碍到癌症等严重疾病。 RNA 修饰测序技术的进步开始揭示疾病中失调的多种 RNA 修饰酶的 RNA 靶点。然而，由这些酶安装的修饰核苷的新兴转录组范围图谱应被视为初稿。特别是，一系列技术制品会导致假阴性，即由于技术依赖且通常与序列上下文相关的“盲点”而被忽视的修改位点。在这篇综述中，我们讨论了基于测序的 RNA 修饰图谱中假阴性的潜在来源，提出了缓解策略，并提出了透明报告敏感性以在分析研究中检测修饰位点的指南。识别和避免假阴性的重要考虑因素包括评估和报告位置特异性测序深度、识别方案依赖性 RNA 捕获偏差以及应用假阴性和假阳性的对照。尽管有其局限性，新兴的 RNA 修饰图谱揭示了 RNA 功能各个方面的转录后控制令人兴奋且很大程度上未知的潜力。© 2024。Springer Nature Limited。

RNA modifications are essential for human health - too much or too little of them leads to serious illnesses ranging from neurodevelopmental disorders to cancer. Technical advances in RNA modification sequencing are beginning to uncover the RNA targets of diverse RNA-modifying enzymes that are dysregulated in disease. However, the emerging transcriptome-wide maps of modified nucleosides installed by these enzymes should be considered as first drafts. In particular, a range of technical artefacts lead to false negatives - modified sites that are overlooked owing to technique-dependent, and often sequence-context-specific, 'blind spots'. In this Review, we discuss potential sources of false negatives in sequencing-based RNA modification maps, propose mitigation strategies and suggest guidelines for transparent reporting of sensitivity to detect modified sites in profiling studies. Important considerations for recognition and avoidance of false negatives include assessment and reporting of position-specific sequencing depth, identification of protocol-dependent RNA capture biases and applying controls for false negatives as well as for false positives. Despite their limitations, emerging maps of RNA modifications reveal exciting and largely uncharted potential for post-transcriptional control of all aspects of RNA function.© 2024. Springer Nature Limited.