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Ribosome Profiling

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Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing

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Abstract

Ribosome profiling (also called Ribo-Seq) is a relatively new method for studying translational regulation of biological processes. Based on the assumption that ribosomes bound to mRNA molecules are all actively decoding the nucleotide sequence to produce polypeptides, ribosome profiling uses next-generation sequencing of mRNA fragments protected by the translating ribosome to determine translational efficiency. It has been, among others, used to study miRNA regulation mechanisms, open reading frames and differential translation of specific genes. Although the method is already 6 years old, it is still in the development phase, and although a detailed protocol has been published, individual wet lab and computational analysis steps are still being optimized. In the following pages, we present different applications of ribosome profiling and the current state-of-the-art experiment guidelines, with special attention put to alternative protocols and open questions.

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Acknowledgements

The authors would like to thank Dr. Julie Aspden for critically reviewing the manuscripts and suggesting several improvements.

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Authors and Affiliations

  1. Department of Environmental Toxicology, Eawag – Swiss Federal Institute for Aquatic Research and Technology, Überlandstrasse 133, Dübendorf, 8600, Switzerland

    Anze Zupanic Ph.D.

  2. School of Biological and Biomedical Sciences, Durham University, Mountjoy Science Site, Durham, DH1 3LE, UK

    Sushma Nagaraja Grellscheid Ph.D.

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  1. Anze Zupanic Ph.D.
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Correspondence to Anze Zupanic Ph.D. .

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Editors and Affiliations

  1. Genome Analysis Platform, CIC bioGUNE, Derio, Spain

    Ana M. Aransay

  2. Genome Analysis Platform, CIC bioGUNE, Derio, Spain

    José Luis Lavín Trueba

Annex: Quick Reference Guide

Annex: Quick Reference Guide

Fig. QG8.1
figure a

Representation of the wet lab procedure workflow

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Fig. QG8.2
figure b

Main steps of the computational analysis pipeline

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Table QG8.1 Experimental design considerations
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Table QG8.2 Available software recommendations
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Zupanic, A., Grellscheid, S.N. (2016). Ribosome Profiling. In: Aransay, A., Lavín Trueba, J. (eds) Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing. Springer, Cham. https://doi.org/10.1007/978-3-319-31350-4_8

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