Specialized RNA-seq methods are required to identify the 5′ ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the ‘cap analysis of gene expression’ (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

需要专门的 RNA-seq 方法来识别转录物的 5' 末端，这对于基因调控的研究至关重要，但这些方法尚未得到系统的基准测试。我们直接比较了六种这样的方法，包括五种方法在单个人类细胞 RNA 样本上的性能，以及一种新的尖峰 RNA 测定法，有助于规避注释和 RNA 处理中的不确定性带来的挑战。我们发现“基因表达的上限分析”（CAGE）方法对 mRNA 表现最好，并且其大多数未注释的峰得到了其他基因组方法的证据的支持。我们将 CAGE 应用于八个与大脑相关的样本，并确定了样本特异性转录起始位点 (TSS) 的使用，以及胎儿和成人大脑之间 TSS 使用的转录组范围内的变化。