Source code to produce the PSI plots

# Source code to produce the PSI plots The scripts were used to generate the PSI plots to appear in Radke, M and Badillo-Lisakowski, V et al "Therapeutic inhibition of RBM20 improves diastolic function in a murine heart failure model and human engineered heart tissue". deposited for reproducibility purposes. The workflow is based on Snakemake, see `Snakefile` for more information. Workflow was called with ```bash snakemake --configfile config.yml --printshellcmds --cluster "sbatch --mem {cluster.mem} --out {cluster.out} --error {cluster.out} -c {cluster.c} --parsable " --cluster-config cluster.yaml --jobs 10 ``` ## Dependencies: - stringtie/1.3.5 - cufflinks/2.2.1 - python2 - R/3.5.1 - perl - bedtools Dependencies were handled with Environment Modules. ## Workflow steps: 1. Guided transcriptome assembly with StringTie: - Parameters: - min_isoform_fraction: 0.2 - min_anchor_length: 8 - min_junction_coverage: 3 - reference: /biodb/genomes/mus_musculus/GRCm38_85/GRCm38.85.gtf The modified GRCm38 84 annotation accopanies this repository. The version of Ensembl has been used to study Titin and match the exons to domains. 2. Merge annotation with cuffmerge: - Parameters: - reference: /biodb/genomes/mus_musculus/GRCm38_85/GRCm38.85.gtf - min_isoform_fraction: 0.2 3. Find 'containers', loci with that contains alternative splicing events, and calculate PSI from STAR `SJ.out.tab` intron counts with compute_PSI.pl PSI is define by the ratio between the number of reads including an exon and total reads for that event (reads including + reads excluding). 4. Produce the plots using PSI_plot_aso.R script: One plot per gene, only for gene with two or more exons, in addiiton: - Removde duplicated exons - Merged overlapping exons The accompanying test data reproduces figure 4.b of the paper: `Rscript testdata/PSI_plot_aso.R testdata/output/ testdata/ldb3.gtf testdata/` The subset of the data was prepared with: ```{fish} set gtf "/beegfs/prj/Mitch_Gotthardt/Thiago/psi_plots/Mus_musculus.GRCm38.84_cleaned.gtf" grep 'gene_name "Ldb3";' $gtf | sort -k1,1 -k4,4n | bedtools merge -d 1000000 -s -c 5,6,7 -o first > testdata/ldb3.bed intersectBed -b testdata/ldb3.bed -a $gtf -wa -s > testdata/ldb3.gtf for f in stringtie_guided/*/psi.txt; mkdir testdata/(basename (dirname $f)); intersectBed -b testdata/ldb3.bed -a $f -wa -s > testdata/(basename (dirname $f))/psi.txt; end ```