error running rmat2sashimiplot

[Miriam]

Hi I'm trying to run rmats2sashimiplot, and I've got an error printed on screen, but when I look at the log file it looks as if it has actually worked, althouth there's no sashimiplot to be found. Can anyone help? Thanks

This is my command:
rmats2sashimiplot -b1 data/T7.bam,data/T8.bam,data/T9.bam -b2 data/P0_1.bam,data/P0_2.bam,data/P0_3.bam -c  chr1:+:128056932:128057012:gtf/Rn5.gff3 -l1 Tissue -l2 P0 -exon_s 1 -intron_s 5 -o test_coordinate_output

And this is ERROR message:
Indexing GFF...
  - GFF: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/tmp.gff3
  - Outputting to: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index
ENST00000000000 has no exons
Through 0 genes...
Loaded 1 genes
  - Loading of genes from GFF took 0.00 seconds
Making directory: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/chr1
  - Chromosome serialization took 0.00 seconds
Outputting gene records in GFF format...
  - Output file: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/genes.gff
  - Serialization of genes from GFF took 0.13 seconds
Indexing of GFF took 0.13 seconds.
Traceback (most recent call last):
  File "/usr/local/bin/sashimi_plot", line 9, in <module>
    load_entry_point('misopy==0.5.3', 'console_scripts', 'sashimi_plot')()
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 276, in main
    plot_label=plot_label)
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 153, in plot_event
    plot_label=plot_label)
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/plot_utils/plot_gene.py", line 675, in plot_density_from_file
    parseGene(pickle_filename, event)
  File "/usr/local/lib/python2.7/dist-packages/misopy/parse_gene.py", line 48, in parseGene
    mRNAs, strand, chrom
UnboundLocalError: local variable 'strand' referenced before assignment
mv: cannot stat ‘/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_plot/chr1:128056932:128057013:+.pdf’: No such file or directory

And this is the log.file:

2015-12-09 10:11:17,325 convert sam to bam and build index..
2015-12-09 10:11:17,325 ################### folder names and associated input files #############
2015-12-09 10:13:46,428 SAMPLE_1\REP_1    data/T7.bam
2015-12-09 10:16:01,398 SAMPLE_1\REP_2    data/T8.bam
2015-12-09 10:18:47,496 SAMPLE_1\REP_3    data/T9.bam
2015-12-09 10:21:37,541 SAMPLE_2\REP_1    data/P0_1.bam
2015-12-09 10:24:28,778 SAMPLE_2\REP_2    data/P0_2.bam
2015-12-09 10:27:07,323 SAMPLE_2\REP_3    data/P0_3.bam
2015-12-09 10:27:07,323 #########################################################################

2015-12-09 10:27:07,323 get user input coordinates and convert to gff3 format file..
2015-12-09 10:27:07,323 coordinate is provided    chr1:+:128056932:128057012:gtf/Rn5.gff3
2015-12-09 10:27:07,323 drawPlotWithCoordinate()
2015-12-09 10:27:07,907 start preparing sashimi plot setting files
2015-12-09 10:27:07,907 prepare sashimi plot setting file..
2015-12-09 10:27:07,908 done prepare sashimi plot setting file/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/sashimi_plot_settings.txt
2015-12-09 10:27:07,908 done making setting file..
2015-12-09 10:27:07,908 use gff3 format file to run index_gff..
2015-12-09 10:27:08,676 output plot from events set..
2015-12-09 10:27:19,602 *** output plot chr1:128056932:128057013:+.pdf
2015-12-09 10:27:19,603 done drawPlotWithCoordinate()
2015-12-09 10:27:19,603 Program ended
2015-12-09 10:27:19,603 Program ran 00:16:02

[Ting Tseng]

Hello,

It seems that the length of genomic region you would like to draw is only 80 bp, and if the annotation you provided shows there is no alternatively-spliced transcripts be found between the region you input, you will get the message such as “ENST00000000000 has no exons”, please input a longer genomic region (recommended more than 10k bp) or check your annotation file to see is there any mRNA annotation located between the genomic region you input.

-Ting

[Miriam]

Thank you for your answer. Your help is greatly appreciated! I'll try a larger region.

Miriam

[Miriam]

Hi, I'm afraid I'm still getting errors. I have selected an event that is predicted by rMATS, the size should fit the recommended length. As region to plot I have selected the Upstream ES and the downstream EE, would that be acceptable?  what are the recommended

This is my command:

rmats2sashimiplot -b1 data/T7.bam,data/T8.bam,data/T9.bam -b2 data/P0_1.bam,data/P0_2.bam,data/P0_3.bam -c chr16:+:61720872:61762379:gtf/Stringtie_Rn5.gff3 -l1 Tissue -l2 P0 -exon_s 1 -intron_s 5 -o test_coordinate_output

And this is the error:
Indexing GFF...
/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/tmp.gff3 appears to already be indexed. Aborting.

Traceback (most recent call last):
  File "/usr/local/bin/sashimi_plot", line 9, in <module>
    load_entry_point('misopy==0.5.3', 'console_scripts', 'sashimi_plot')()
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 276, in main
    plot_label=plot_label)

  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 142, in plot_event
    %(event_name, pickle_dir)
Exception: Event chr16:61720872:61762380:+ not found in pickled directory /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index. Are you sure this is the right directory for the event?
mv: cannot stat ‘/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_plot/chr16:61720872:61762380:+.pdf’: No such file or directory

I have noticed that the error report has added a nt to my chr coordinates, why is that?

Thank you for your help,

Miriam

[Ting Tseng]

Hello,

It seems that you are trying to use same output folder name to execute again, but from your previous run, some index files have already been created in the same folder, so the error message shows "/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/tmp.gff3 appears to already be indexed. Aborting." Please delete the previous index files or you can just use another output folder name to run again.

-Ting

[Miriam]

Hi,

Thanks for your suggestion. Unfortunately, after deleting the output folder and re running the command, I got a different error. When I looked the error up in google, it says that there's a problem with Python and  variable definition, something about global variables, but since I don't know anything about python I don't understand. I would appreciate any help,

Thank you very much,

Miriam

This is the ERROR:

Indexing GFF...
  - GFF: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/tmp.gff3
  - Outputting to: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index
ENST00000000000 has no exons
Through 0 genes...
Loaded 1 genes
  - Loading of genes from GFF took 0.00 seconds

Making directory: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/chr16

  - Chromosome serialization took 0.00 seconds
Outputting gene records in GFF format...
  - Output file: /new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_index/genes.gff

  - Serialization of genes from GFF took 0.08 seconds
Indexing of GFF took 0.09 seconds.

Traceback (most recent call last):
  File "/usr/local/bin/sashimi_plot", line 9, in <module>
    load_entry_point('misopy==0.5.3', 'console_scripts', 'sashimi_plot')()
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 276, in main
    plot_label=plot_label)
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 153, in plot_event
    plot_label=plot_label)
  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/plot_utils/plot_gene.py", line 675, in plot_density_from_file
    parseGene(pickle_filename, event)
  File "/usr/local/lib/python2.7/dist-packages/misopy/parse_gene.py", line 48, in parseGene
    mRNAs, strand, chrom
UnboundLocalError: local variable 'strand' referenced before assignment

mv: cannot stat ‘/new-data/ml439/rMATS.3.0.9/test_coordinate_output/Sashimi_plot/chr16:61720872:61762380:+.pdf’: No such file or directory

[Gosia Komór]

Hi, 

I have the same error.

Traceback (most recent call last):

  File "/usr/local/bin/sashimi_plot", line 9, in <module>

    load_entry_point('misopy==0.5.3', 'console_scripts', 'sashimi_plot')()

  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 276, in main

    plot_label=plot_label)

  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/sashimi_plot.py", line 153, in plot_event

    plot_label=plot_label)

  File "/usr/local/lib/python2.7/dist-packages/misopy/sashimi_plot/plot_utils/plot_gene.py", line 675, in plot_density_from_file

    parseGene(pickle_filename, event)

  File "/usr/local/lib/python2.7/dist-packages/misopy/parse_gene.py", line 48, in parseGene

    mRNAs, strand, chrom

UnboundLocalError: local variable 'strand' referenced before assignment

Any solutions by now?

Greetings, 

Gosia