Rp-Bp and the Ribotools box

Supplementary information 

This repository contains supplementary data for Rp-Bp and the Ribotools box for translatome profiling.

Description of files

• hiPSC-CMs.denovo.slim.gtf 
  A de novo assembled transcriptome annotation for human iPSCs differentiation into cardiomyocytes. Paired-end RNA-seq reads were first trimmed
  and filtered using Flexbar with the following options `flexbar -n 15 -z GZ -m 30 -u 0  -q TAIL -qt 28 -a adapters.fa -qf sanger -j`. Common contaminants
  were removed with Bowtie2 using a built-in index combining mitochondiral and rRNA sequences with  `bowtie2 --no-unal --omit-sec-seq --threads 20 --mm --seed 1337 --time --un-conc-gz`. Remaing reads were mapped using STAR with `STAR --runThreadN 40 --genomeLoad NoSharedMemory --readFilesCommand zcat --outReadsUnmapped Fastx --outSAMattributes NH HI AS nM NM MD jM jI XS --outSJfilterOverhangMin 15 15 15 15 --outFilterMultimapNmax 20 --chimMultimapNmax 20 --outFilterScoreMin 1 --outFilterMatchNminOverLread 0.7 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.05 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 15    
  --alignSJDBoverhangMin 10 --alignSoftClipAtReferenceEnds No --chimSegmentMin 15 --chimScoreMin 15 --chimScoreSeparation 10 --chimJunctionOverhangMin 15 --sjdbGTFfile GRCh38.102.gtf --quantMode GeneCounts --twopassMode Basic --chimOutType Junctions`. Soft clipping was removed and the output SAM file was passed to samtools for compression, sorting and indexing. To reconstruct de novo transcriptomes for each sample, guided by a reference transcriptome annotation (Ensembl release 102), stringtie was used `stringtie -G GRCh38.102.gtf -p 2 -v --rf --conservative`, followed by `stringtie --merge -G GRCh38.102.gtf -p 2 -v`. Finally, the annotations were merged into a non-redundant set of features and compared with the reference annotation using `gffcompare -r  GRCh38.102.gtf -V -R`.
• hiPSC-CMs.Ribo-seq-ORFs.bed.gz
  Ribo-seq ORFs predicted and annotated with Rp-Bp with `run-all-rpbp-instances --star-options "--quantMode TranscriptomeSAM GeneCounts" --use-slurm --num-cpus 6 --mem 250G --keep-intermediate-files --merge-replicates --run-replicates` (default options and parameters).  
• hiPSC-CMs.Ribo-seq-proteins.slim.fa 
  Translated Ribo-seq ORFs sequences. This file is short of 82 Ribo-seq ORFs that have duplicate protein sequences.
• rnaseq-results.zip
  Count tables (trimmed RNA-seq reads) obtained with Ribotools for both Ribo-seq ORFs and genes.
• riboseq-results.zip
  Count tables (periodic Ribo-seq reads) obtained with Ribotools for both Ribo-seq ORFs and genes.
• tea-results.zip
  Translation efficiency (TE) analyses obatine with Ribotools using the default LRT method for  both Ribo-seq ORFs and genes for selected contrasts (d1_vs_d0, d5_vs_d0, d5_vs_d1, d7_vs_d5, d17_vs_d5, d17_vs_d7, d31_vs_d17).
• adapters.fa
  General list of RNA-seq adapters.
• riboseq-adapters.fa
  List of Ribo-seq adapters.