title: Dual randomization of oligonucleotides to reduce the bias in ribosome-profiling libraries.
abstract: Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Muenster Graduate School of Evolution, University of Muenster, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany; Max Planck Research Group for Regulatory Genomics, Max Planck Institute for Molecular Biomedicine, Roentgenstrasse 20, 48149 Muenster, Germany.
authors.forename: Aarón
authors.initials: A
authors.lastname: Lecanda
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany.
authors.forename: Benedikt S
authors.initials: BS
authors.lastname: Nilges
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany.
authors.forename: Puneet
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany.
authors.forename: Danny D
authors.initials: DD
authors.lastname: Nedialkova
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Schwarz
authors.affiliation: Muenster Graduate School of Evolution, University of Muenster, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany; Max Planck Research Group for Regulatory Genomics, Max Planck Institute for Molecular Biomedicine, Roentgenstrasse 20, 48149 Muenster, Germany.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Vaquerizas
authors.affiliation: Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Strasse 54, 48149 Muenster, Germany; Muenster Graduate School of Evolution, University of Muenster, 48149 Muenster, Germany; Cells-in-Motion Cluster of Excellence, University of Muenster, 48149 Muenster, Germany. Electronic address: sebastian.leidel@mpi-muenster.mpg.de.
authors.forename: Sebastian A
authors.initials: SA
authors.lastname: Leidel
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics
chemical_list: D009841:Oligonucleotides
keywords: Codon-translation speed; RNA modification; Ribosome profiling; Sequencing bias; Translation; Translational control