RT Journal Article SR Electronic T1 A spectral analysis approach to detect actively translated open reading frames in high-resolution ribosome profiling data JF bioRxiv FD Cold Spring Harbor Laboratory SP 031625 DO 10.1101/031625 A1 Lorenzo Calviello A1 Neelanjan Mukherjee A1 Emanuel Wyler A1 Henrik Zauber A1 Antje Hirsekorn A1 Matthias Selbach A1 Markus Landthaler A1 Benedikt Obermayer A1 Uwe Ohler YR 2015 UL http://biorxiv.org/content/early/2015/11/13/031625.abstract AB RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions by means of the characteristic three-nucleotide periodicity of Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of periodic Ribo-seq reads via spectral analysis methods.We applied RiboTaper on newly generated, deep Ribo-seq data in HEK293 cells, to derive an extensive map of translation that covers Open Reading Frame (ORF) annotations for more than 11,000 protein-coding genes. We also find distinct ribosomal signatures for several hundred detected upstream ORFs and ORFs in annotated non-coding genes (ncORFs). Mass spectrometry data confirms that RiboTaper achieves excellent coverage of the cellular proteome and validates dozens of novel peptide products. Collectively, RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/) is a powerful method for comprehensive de novo identification of actively used ORFs in the human genome.