Comprehensive comparative analysis of 5′-end RNA-sequencing methods

Xian Adiconis, Adam L. Haber, Sean K. Simmons, Ami Levy Moonshine, Zhe Ji, Michele A. Busby, Xi Shi, Justin Jacques, Madeline A. Lancaster, Jen Q. Pan, Aviv Regev, Joshua Z. Levin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

51 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)505-511
Number of pages7
JournalNature Methods
Issue number7
StatePublished - Jul 1 2018

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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