Error when running on rMATS generated BAM files

[nicholas-moskwa]

Hi,

I am trying to generate sashimi plots using BAM files made from running rMATS on FASTQ files. I am using two files that hold my replicates.

My run:
python /Packages/rmats2sashimiplot/src/rmats2sashimiplot/rmats2sashimiplot.py --b1 10_129S.txt --b2 10_B6.txt -c chr7:+:27256438:27284145:/genomes/mm10/gencode.vM36.annotation.gff3 --l1 129S --l2 B6 --exon_s 1 --intron_s 5 -o ${dir_out}

My error:
File "/Packages/rmats2sashimiplot/src/MISO/misopy/index_gff.py", line 77
print "Making directory: %s" %(chrom_dir)
^
SyntaxError: invalid syntax
File "/Packages/rmats2sashimiplot/src/MISO/misopy/sashimi_plot/sashimi_plot.py", line 46
print "WARNING: %s does not end in .miso_bf, are you sure it is the "
^
SyntaxError: Missing parentheses in call to 'print'. Did you mean print("WARNING: %s does not end in .miso_bf, are you sure it is the " )?
'/mm10-tmp/2024-02-01-13_40_35_331660_bam1_1/Aligned.sortedByCoord.out.bam' is indexed already: '/mm10-tmp/2024-02-01-13_40_35_331660_bam1_1/Aligned.sortedByCoord.out.bam.bai'
'mm10-tmp/2024-02-01-13_40_35_331660_bam2_1/Aligned.sortedByCoord.out.bam' is indexed already: '/mm10-tmp/2024-02-01-13_40_35_331660_bam2_1/Aligned.sortedByCoord.out.bam.bai'

Thanks for your help!

[EricKutschera]

Here's a similar issue: #138 (comment)

You could try:

cd /Packages/rmats2sashimiplot
./2to3.sh

[nicholas-moskwa]

Okay, that changed the error, I think most of the script ran, but now I see this error:

Exception: Event 7_27256438_27284146_- not found in pickled directory /pod/2/ch-lee-lab/labdata/moskwn/RNA-seq/RMT0015/10_Rmats2sashimiplot/Sashimi_index. Are you sure this is the right directory for the event?

Does rmatsashimiplot only plot events that show up in rMATS?

[EricKutschera]

That error can happen if you use the same -o directory for multiple runs. The code will see files from a previous run (which may have had an error) and the old files can cause the error: #92 (comment)

[nicholas-moskwa]

I deleted the output directory and re-ran my scripts and saw this, should I use a gene name instead of a coordinate:

<module 'misopy' from '/pod/2/ch-lee-lab/labdata/Packages/rmats2sashimiplot/src/MISO/misopy/init.py'>
Indexing GFF...

• GFF: /10_Rmats2sashimiplot/Sashimi_index/tmp.gff3
• Outputting to: /10_Rmats2sashimiplot/Sashimi_index
  WARNING: No entries found for gene 7_27256438_27284146_+ in GFF /10_Rmats2sashimiplot/Sashimi_index/tmp.gff3
  Skipping gene 7_27256438_27284146_+...
  Loaded 0 genes
• Loading of genes from GFF took 0.00 seconds
  Outputting gene records in GFF format...
• Output file: /10_Rmats2sashimiplot/Sashimi_index/genes.gff
• Serialization of genes from GFF took 0.03 seconds
  Indexing of GFF took 0.04 seconds.
  Traceback (most recent call last):
  File "/rmats2sashimiplot/src/MISO/misopy/sashimi_plot/sashimi_plot.py", line 293, in
  main()
  File "/rmats2sashimiplot/src/MISO/misopy/sashimi_plot/sashimi_plot.py", line 289, in main
  plot_label=plot_label)
  File "/rmats2sashimiplot/src/MISO/misopy/sashimi_plot/sashimi_plot.py", line 148, in plot_event
  %(event_name, pickle_dir))
  Exception: Event 7_27256438_27284146_+ not found in pickled directory /10_Rmats2sashimiplot/Sashimi_index. Are you sure this is the right directory for the event?

[EricKutschera]

The issue might be that your bam files don't have chr but you're using a gff3 that does. It looks like you have gencode.vM36.annotation.gff3 which has chr7 and the error is about an event on just 7 which would be based on the coordinates from the -c argument. I think you could remove the chr prefix from your gff3 with:

sed s/^chr// gencode.vM36.annotation.gff3 > gencode.vM36.annotation_no_chr.gff3

Then you could use that updated gff3 file