Dataset Information

Small-scale sequencing enables quality assessment of Ribo-Seq data: an example from Arabidopsis cell culture.

ABSTRACT:

Background

Translation is a tightly regulated process, controlling the rate of protein synthesis in cells. Ribosome sequencing (Ribo-Seq) is a recently developed tool for studying actively translated mRNA and can thus directly address translational regulation. Ribo-Seq libraries need to be sequenced to a great depth due to high contamination by rRNA and other contaminating nucleic acid fragments. Deep sequencing is expensive, and it generates large volumes of data, making data analysis complicated and time consuming.

Methods and results

Here we developed a platform for Ribo-Seq library construction and data analysis to enable rapid quality assessment of Ribo-Seq libraries with the help of a small-scale sequencer. Our data show that several qualitative features of a Ribo-Seq library, such as read length distribution, P-site distribution, reading frame and triplet periodicity, can be effectively evaluated using only the data generated by a benchtop sequencer with a very limited number of reads.

Conclusion

Our pipeline enables rapid evaluation of Ribo-Seq libraries, opening up possibilities for optimization of Ribo-Seq library construction from difficult samples, and leading to better decision making prior to more costly deep sequencing.

SUBMITTER: Mahboubi A

PROVIDER: S-EPMC8386038 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, Ribo-Seq signal can be observed across RNAs that one would not expect to be bound by ribosomes. For example, Escherichia coli Ribo-Seq libraries also capture reads from most noncoding RNAs (ncRNAs). While some of these ncRNAs may overlap coding regions, this alone does not explain the majority of observed signal across ncRNAs. These fragments of ncRNAs in Ribo-Seq data pass all size selection steps of the Ribo-Seq protocol and survive hours of micrococcal nuclease (MNase) treatment. In this work, we specifically focus on Ribo-Seq signal across ncRNAs and provide evidence to suggest that RNA structure, as opposed to ribosome binding, protects them from degradation and allows them to persist in the Ribo-Seq sequencing library preparation. By inspecting these "contaminant reads" in bacterial Ribo-Seq, we show that data previously disregarded in bacterial Ribo-Seq experiments may, in fact, be used to gain partial information regarding the in vivo secondary structure of ncRNAs.IMPORTANCE Structured ncRNAs are pivotal mediators of bioregulation in bacteria, and their functions are often reliant on their specific structures. Here, we first inspect Ribo-Seq reads across noncoding regions, identifying contaminant reads in these libraries. We observe that contaminant reads in bacterial Ribo-Seq experiments that are often disregarded, in fact, strongly overlap with structured regions of ncRNAs. We then perform several bioinformatic analyses to determine why these contaminant reads may persist in Ribo-Seq libraries. Finally, we highlight some structured RNA contaminants in Ribo-Seq and support the hypothesis that structures in the RNA protect them from MNase digestion. We conclude that researchers should be cautious when interpreting Ribo-Seq signal as coding without considering signal distribution. These findings also may enable us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large scale and in vivo through the reanalysis of existing Ribo-Seq data sets.