This repository is a code deposition of the performed Ribo-seq analyses and raw figures in the FUr readthrough manuscript. Code to generate the figures is found under figures. SessionInfo.RMD holds all required packages for the R code. Code to process Ribo-seq libraries to trimmed reads is located in the pipeline folder. Please note that our Ribo-seq pipeline is still in active development and that this is merely a historical snapshot to replicate our analyses. If your are interested in our current pipeline, please contact us.
- Data
- Data files for figure generation
- Figures
- Code to generate the figures
- Pipeline
- Code to process Ribo-seq libraries
Required files are in the Ribo-seq config located in the data folder. Our local computer cluster uses the SLURM workload manager. In addition, we use the LMOD system to load specific versions of packages. For the RNA-seq data, mrp_rna_trim.sh was used to pre-process the standard RNA-seq data.
- Quality control and 3' adapter trimming with TrimGalore
- Remove unwanted RNAs with bowtie2
- Map ribosome fragments with STAR
- Run RiboseQC for Ribo-seq QC
- Use FeatureCount from the subread package to quantify CDS counts
Before running the pipeline, make sure you have the following files:
- Filled in config file (pipeline/mrp.config)
- BOWTIE2 index of contaminant sequences that should be removed (rRNA, tRNA, snRNA, snoRNA, mtDNA)
- STAR index of your reference genome of choice (we used ensembl v102)
- Genome annotation file for your reference genome (we used ensembl v102)
- Create annotation files for RiboseQC (see https://github.com/ohlerlab/RiboseQC)
- cutadapt v3.4
- fastqc v0.11.9
- trimgalore v0.6.6
- bowtie2 v2.4.2
- STAR v2.7.8a
- samtools v1.12
- R v4.0.3 (cluster)
- R v4.0.4 (figures)
Contact us for more information regarding our analyses:
- Jip van Dinter ([email protected])
- Damon Hofman ([email protected])
- Sebastiaan van Heesch ([email protected])