cannot plot with rmats2sashimiplot

Question

[thomas.heigl.ibk]

Hi all.

after struggling with the installation, then with the run in rMATS, I finally reached a point, at which I hoped to see some nice sashimi plots. Well, not yet. I am not able to get rid of some warnings I encounter wenn running the code below.

I saw some entry in another forum and apparently all should have been fixed, but I still encounter the same problems as others some years ago. Maybe I failed already at another step.

Anyway, I downloaded the gtf file from ensembl and then used "agat_convert_sp_gxf2gxf.pl" to convert to gff3 format. to create the bam files, I used Rsubread (subjunc function). The genome for aligning was also taken from ensembl. Below the code for the sashimi plotting, thank you:

 rmats2sashimiplot --b1 ./b1.txt --b2 ./b2.txt -o ./sashimi2 --l1 normoxia --l2 hypoxia --exon_s 1 --intron_s 5 -c chr19:+:46396896:46488804:../../ref_genomes/mouse_38/annotation/ensembl/Mus_musculus.GRCm38.102.gff3 
'../../bulkRNAseq_lynn/BAM/subjunc/normoxia_rep1_R1.fastq.gz.subjunc.BAM' is indexed already: '../../bulkRNAseq_lynn/BAM/subjunc/normoxia_rep1_R1.fastq.gz.subjunc.BAM.bai'
'../../bulkRNAseq_lynn/BAM/subjunc/07-06h-zh_rep1_R1.fastq.gz.subjunc.BAM' is indexed already: '../../bulkRNAseq_lynn/BAM/subjunc/07-06h-zh_rep1_R1.fastq.gz.subjunc.BAM.bai'
<module 'misopy' from '/home/chuddy/miniconda3/envs/rmats/lib/python3.10/site-packages/MISO/misopy/__init__.py'>
Indexing GFF...
  - GFF: /home/chuddy/bioinformatics/bulkRNAseq_lynn/rmats/sashimi2/Sashimi_index/tmp.gff3
  - Outputting to: /home/chuddy/bioinformatics/bulkRNAseq_lynn/rmats/sashimi2/Sashimi_index
WARNING: No entries found for gene chr19_46396896_46488805_+ in GFF /home/chuddy/bioinformatics/bulkRNAseq_lynn/rmats/sashimi2/Sashimi_index/tmp.gff3
Skipping gene chr19_46396896_46488805_+...
Loaded 0 genes
  - Loading of genes from GFF took 0.00 seconds
Outputting gene records in GFF format...
  - Output file: /home/chuddy/bioinformatics/bulkRNAseq_lynn/rmats/sashimi2/Sashimi_index/genes.gff
  - Serialization of genes from GFF took 0.00 seconds
Indexing of GFF took 0.00 seconds.
Traceback (most recent call last):
  File "/home/chuddy/miniconda3/envs/rmats/lib/python3.10/site-packages/MISO/misopy/sashimi_plot/sashimi_plot.py", line 293, in <module>
    main()
  File "/home/chuddy/miniconda3/envs/rmats/lib/python3.10/site-packages/MISO/misopy/sashimi_plot/sashimi_plot.py", line 285, in main
    plot_event(event_name, pickle_dir, settings_filename, output_dir,
  File "/home/chuddy/miniconda3/envs/rmats/lib/python3.10/site-packages/MISO/misopy/sashimi_plot/sashimi_plot.py", line 146, in plot_event
    raise Exception("Event %s not found in pickled directory %s. " \
Exception: Event chr19_46396896_464