List and count splicing sites
-Put all the bam files (or links to them) in the same folder. The scripts need not be in that folder, but you should invoke them from it.
-Run run_RNASeq.sh. Warning: In its present form, this script runs the script on each file in parallel. If you want to run them one at a time, you can delete the & symbol in the bash file. This will take a while, so you may want to run it with nohup.
-When the program is done, you should have a ".flagstat" and a "_raw.hsh" file per input bam. Each hsh file contains a description of all the splice sites found in each bam file, with the number of reads supporting it.
-Run compile_table2.pl. There should be a new file called "retained.hsh". It contains information of the reads spanning each splicing site, but not supporting it, for each sample.
Assign splice sites to genes
-Download the latest gene definitions from Ensembl. At this point, it would be ftp://ftp.ensembl.org/pub/release-91/gtf/homo_sapiens/Homo_sapiens.GRCh38.91.gtf.gz
perl hsu_up.pl Homo_sapiens.GRCh37.91.gtf (With full paths if necessary)
-A file called hsu.hsh should appear.
Create tables:
-Run
perl write_tables.pl
-Several txt files should appear. The table containing the scores is ratio.txt