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.

Transcriptome-wide measurement of translation by ribosome profiling

“…While this “ligation free” method is shown to have lower input requirements, it is yet to find wide application in ribosome profiling studies. Furthermore, in comparing circularization-based and dual-ligation libraries, there is evidence that dual-ligation libraries show can more variable coverage [15]. However, more than one dual-ligation strategy exists (e.g.…”

“…Furthermore, it can be used to avoid the variance that is associated with sequencing different samples on different lanes of a sequencer [14]. Secondly, the UMIs are likely to ameliorate the effect of sequence biases in ligation and circularization during library construction [15,16]. Thirdly, the UMIs will also allow for the computational removal of sequences likely to derive from selective amplification during the library construction PCR [15,16].…”

Mapping N6‐Methyladenosine (m6A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches

“…The click product did not only increase the steric demand of the modification but also allowed immobilization on streptavidin‐coated magnetic beads (Figure ). Thus, after clicking and immobilization, the N 6 ‐propargylated RNA caused 65 % termination in RT and the approach could be integrated into an existing NGS protocol . Interestingly, no mutations were observed opposite the N 6 ‐modified adenosine.…”

“…Thus, after clicking and immobilization, the N 6 -propargylatedR NA caused 65 %t ermination in RT and the approach couldb ei ntegrated into an existing NGS protocol. [62] Interestingly,n om utations were observed oppositethe N 6 -modified adenosine.…”

XPRESSyourself: Enhancing, Standardizing, and Automating Ribosome Profiling Computational Analyses Yields Improved Insight into Data

“…Additionally, standardized methods for handling this unique data type remain elusive. This has been problematic and evidenced by various studies using vague or opaque methods for data analysis (for examples, see [9][10][11][12][13]), or methods rely on outdated tools [5]. Very few labs have the tools necessary to separate the biological signals in ribosome profiling data from the inherent biases of the experimental measurements, and these tools are not readily accessible by the community.…”