RiboGalaxy: A Galaxy-based Web Platform for Ribosome Profiling Data Processing - 2023 Update.

Ribosome profiling (Ribo-Seq) captures a "snapshot" of ribosomes' locations at the entire transcriptome of a cell at sub-codon resolution providing insights into gene expression and enabling the discovery of novel translated regions. RiboGalaxy (https://ribogalaxy.genomicsdatascience.ie/), a Galaxy-based platform for processing Ribo-Seq data is a RiboSeq.Org (https://riboseq.org/) resource. RiboSeq.Org is an online gateway to a set of integrated tools for the processing and analysis of Ribo-Seq data. In this RiboGalaxy update we introduce changes to both the tools available on RiboGalaxy and to how the resource is managed on the backend. For example, in order to improve interoperability between Riboseq.Org resources, we added tools that link RiboGalaxy outputs with Trips-Viz and GWIPS-viz browsers for downstream analysis and visualisation. RiboGalaxy's backend now utilises Ansible configuration management which enhances its stability and jobs are executed within Singularity containers and are managed by Slurm, strengthening reproducibility and performance respectively.

Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.

A rapid protocol for ribosome profiling of low input samples.

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.