Abstract
Ribosome profiling, or Ribo-seq, provides precise information about the position of actively translating ribosomes. It can be used to identify open reading frames (ORFs) that are translated in a given sample. The RiboTaper pipeline, and the ORFquant R package, leverages the periodic distribution of such ribosomes along the ORF to perform a statistically robust test for translation which is insensitive to aperiodic noise and provides a statistically robust measure of translation. In addition to accounting for complex loci with overlapping ORFs, ORFquant is also able to use Ribo-seq as a tool for distinguishing actively translated transcripts from non-translated ones, within a given gene locus.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Calviello L, Mukherjee N, Wyler E, Zauber H, Hirsekorn A, Selbach M, Landthaler M, Obermayer B, Ohler U (2016) Detecting actively translated open reading frames in ribosome profiling data. Nat Methods 13(2):165–170. https://doi.org/10.1038/nmeth.3688
Bazzini AA, Johnstone TG, Christiano R, Mackowiak SD, Obermayer B, Fleming ES, Vejnar CE, Lee MT, Rajewsky N, Walther TC, Giraldez AJ (2014) Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J 33(9):981–993. https://doi.org/10.1002/embj.201488411
Gonzalez-Porta M, Frankish A, Rung J, Harrow J, Brazma A (2013) Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol 14(7):R70. https://doi.org/10.1186/gb-2013-14-7-r70
Calviello L, Hirsekorn A, Ohler U (2019) Quantification of translation uncovers the functions of the alternative transcriptome. bioRxiv:608794. https://doi.org/10.1101/608794
Calviello L, Sydow D, Harnett D, Ohler U (2019) Ribo-seQC: comprehensive analysis of cytoplasmic and organellar ribosome profiling data. bioRxiv:601468. https://doi.org/10.1101/601468
Ahmed N, Sormanni P, Ciryam P, Vendruscolo M, Dobson CM, O'Brien EP (2019) Identifying A- and P-site locations on ribosome-protected mRNA fragments using integer programming. Sci Rep 9(1):6256. https://doi.org/10.1038/s41598-019-42348-x
Weinberg DE, Shah P, Eichhorn SW, Hussmann JA, Plotkin JB, Bartel DP (2016) Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation. Cell Rep 14(7):1787–1799. https://doi.org/10.1016/j.celrep.2016.01.043
Fang H, Huang YF, Radhakrishnan A, Siepel A, Lyon GJ, Schatz MC (2018) Scikit-ribo enables accurate estimation and robust modeling of translation dynamics at codon resolution. Cell Syst 6(2):180–191. e184. https://doi.org/10.1016/j.cels.2017.12.007
Acknowledgments
This work supported by Deutsche Forschungsgemeinschaft (grant TR175) and the Federal Ministry for Education and Research (BMBF, grant de.NBI/RBC). The authors acknowledge helpful feedback and support from Antje Hirsekorn and Dilmurat Yusuf.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Harnett, D., Meerdink, E., Calviello, L., Sydow, D., Ohler, U. (2021). Genome-Wide Analysis of Actively Translated Open Reading Frames Using RiboTaper/ORFquant. In: Labunskyy, V.M. (eds) Ribosome Profiling. Methods in Molecular Biology, vol 2252. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1150-0_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1150-0_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1149-4
Online ISBN: 978-1-0716-1150-0
eBook Packages: Springer Protocols