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Hi team <!channel>, I have been using gencode as m...
# rnasplice
n
Hi team <!channel>, I have been using gencode as my reference and gtf file. I have used stated parameters like below to get gene_id conversion. I have been getting the following errors and it has not been performing dexdtu analysis as well any suggestions on id conversion to avoid sashimi plot error and other id conversion error if using gencode
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--fasta /gencode/GRCm38.primary_assembly.genome.fa \
--gtf /home/wangjh/paidi001/gencode/gencode.vM25.annotation.gtf \
--gtf_extra_attributes gene_id \
--egder_exon \
--dexseq_dtu \
--gencode
Error 
Workflow execution completed unsuccessfully!
The exit status of the task that caused the workflow execution to fail was: 1. The full error message was:
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Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (1)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (1)` terminated with an error exit status (1)

Command executed:

  sashimi_plot --plot-event ENSG00000004961 index miso_settings.txt --output-dir sashimi
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI":
      python: $(python --version | sed "s/Python //g")
      misopy: $(python -c "import pkg_resources; print(pkg_resources.get_distribution('misopy').version)")
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  INFO:    Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred
  /usr/local/lib/python2.7/site-packages/matplotlib/cbook/deprecation.py:107: MatplotlibDeprecationWarning: The mpl_toolkits.axes_grid module was deprecated in version 2.1. Use mpl_toolkits.axes_grid1 and mpl_toolkits.axisartist provies the same functionality instead.
    warnings.warn(message, mplDeprecation, stacklevel=1)
  Traceback (most recent call last):
    File "/usr/local/bin/sashimi_plot", line 11, in 
      sys.exit(main())
    File "/usr/local/lib/python2.7/site-packages/misopy/sashimi_plot/sashimi_plot.py", line 276, in main
      plot_label=plot_label)
    File "/usr/local/lib/python2.7/site-packages/misopy/sashimi_plot/sashimi_plot.py", line 142, in plot_event
      %(event_name, pickle_dir)
  Exception: Event ENSG00000004961 not found in pickled directory index. Are you sure this is the right directory for the event?

Work dir:
  /panfs/jay/groups/21/wangjh/paidi001/Nextflow/work/ec/3976cc86c876abf408a0284a24e229

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.s
t
Any particular reason you used 
--gtf_extra_attributes gene_id
? Not sure it is the source of your issue, but it might well be. (P.S.: Every time you type "@ channel", everyone gets an e-mail alert. We prefer to reserve that for special circumstances - thank you! 🙂)
this 1
n
Okay, will not use channel. I used the gtf attributes assuming it will convert ensemble id's to gene id. Should I remove that and run it 
--gtf_extra_attributes gene_id?
I just wanted to see if I get official gene names instead of ensemble id's.
t
Yes, I would try without 
--gtf_extra_attributes
. The problem with gene symbols is that they are not unique (you might have two or more IDs mapping to the same symbol) and they don't a exist for every gene, either (so you have a bunch of IDs mapping to symbol 
NA
, which is not helpful). For some tools the symbols are listed in the results table when available (e.g. rMATS), for others you can just make a table translating the IDs to symbols and use that to annotate your results. E.g. for exon-based DEXSeq, all the exons in the index have names derived from the gene IDs. Trying to use non-unique symbols would break this. And for the transcript-based DEXSeq, the important part are really the transcript names.
n
@Thomas Danhorn , its giving the same error without the 
gtf_extra_attributes gene_id .
t
I looked more closely at the parameters: The gene from 
Event ENSG00000004961 not found in pickled directory index
is from the default value of the `--miso_genes` parameter, and apparently that gene is not in your GTF, so that's an unfortunate choice of default value, I think I'll create a GitHub issue for that. You have two options here: • If you don't really care about Sashimi plots at this point, run with 
--sashimi_plot false
. • If you want to see the plots, use your favorite gene(s) with 
--miso_genes
(they must be in the GTF).
Since you seem to be using a Gencode GTF, you may also want to use the 
--gencode
option. This also seems to change the default for 
--miso_genes
, which would provide yet another option to fix your problem.
There was already a GitHub issue for that (https://github.com/nf-core/rnasplice/issues/111), but that was closed -- the user is apparently expected to provide a valid gene.
a
Hi there, I am trying to use the pipeline, but I'm encountering several errors. Could someone please help me understand what might be going wrong? Any assistance would be greatly appreciated. Thank you
-[nf-core/rnasplice] Pipeline completed with errors- ERROR ~ Error executing process > 'NFCORE_RNASPLICERNASPLICEDEXSEQ_DEU:DEXSEQ_ANNOTATION (genes.gtf)' Caused by: Process 
NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION (genes.gtf)
terminated with an error exit status (1) Command executed: dexseq_prepare_annotation.py genes.gtf DEXSeq.gff -r no cat <<-END_VERSIONS > versions.yml "NFCORE_RNASPLICERNASPLICEDEXSEQ_DEU:DEXSEQ_ANNOTATION": htseq: $(python -c "import pkg_resources; print(pkg_resources.get_distribution('htseq').version)") END_VERSIONS Command exit status: 1 Command output: (empty) Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred Traceback (most recent call last): File "/home/torres91/.nextflow/assets/nf-core/rnasplice/bin/dexseq_prepare_annotation.py", line 128, in <module> assert l[i].iv.end <= l[i + 1].iv.start, str(l[i + 1]) + " starts too early" AssertionError: <GenomicFeature: exonic_part 'Mir684-1' at chr11: 75065990 -> 75065905 (strand '-')> starts too early
t
This looks like a problem (inconsistency?) with your GTF -- the 
Mir684-1
entry seems to overlap whatever is before on that contig. Maybe try a different GTF ...
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