Hi everyone, I'm trying to run the pipeline with my own reference and annotation file, but I'm getting the following error. I got it already for other genes that I have cancelled from the gtf file, but I'm not sure if I should continue to cancel more of them. Instead I would like to understand what is going on. The annotation and the reference are from uscs, I wonder if the problem is that the dexseq prefer the ensembl ones. I'm using those because they are the one used for the mapping (I'm starting the pipeline from the bam files). Can you help me? Thanks

ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION (genes_test.gtf)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_DEU:DEXSEQ_ANNOTATION (genes_test.gtf)` terminated with an error exit status (1)


Command executed:

  dexseq_prepare_annotation.py genes_test.gtf DEXSeq.gff

  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:DEXSEQ_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:
  Traceback (most recent call last):
    File "/.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 'Sumo2+Mir684-1' at chr11: 75065990 -> 75065905 (strand '-')> starts too early

I've run into similar issues trying to use UCSC/NCBI reference genomes as opposed to ensembl. I ultimately ended up running everything using the ensembl build instead. I just didn't have the time to dig in deeper as to why.

Thank you, I was thinking to do the same. My fastq file are single end, what shall I put in the samplesheet for fastq2 and strandness?

never ran single end in this pipeline, but for ChIP seq, you just leave the column empty, I presume it's the same here. For strand, that's dependent on your actual samples

The strandedness is determined by the library preparation kit (and independent of the way the library was sequenced, i.e. single- vs paired-end), so best to ask whoever sequenced it.

Thanks I fixed that but now I got another error, any idea about this? Thanks!

task: name=NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (2); work-dir=/igatech/rd/dev/nextflow-workflows/rnasplice/test_ID3314_Sanchez_fastq/work/b9/9280541cdf21bb8ecb28a34806b111
  error [nextflow.exception.ProcessFailedException]: Process `NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (2)` terminated with an error exit status (1)
Jul-10 21:53:27.466 [TaskFinalizer-8] ERROR nextflow.processor.TaskProcessor - Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (2)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:VISUALISE_MISO:MISO_SASHIMI (2)` terminated with an error exit status (1)


Command executed:

  sashimi_plot --plot-event ENSG00000005302 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:
  /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 <module>
      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 ENSG00000005302 not found in pickled directory index. Are you sure this is the right directory for the event?

Yes, I would probably run with

--sashimi_plot false

until you have figured out which genes you care about. The problem you ran into is that

--miso_genes

has a default value that is not compatible with your genome annotation (i.e. that gene ID does not exist), so if you want to use it, you'll have to set it to gene IDs that are actually in your annotation.

Thank you very much! The pipeline is actually running forward with the settings you suggested. I just wonder why it’s not compatible, since I run the whole pipeline starting with fastq and giving the gtf annotation file downloaded from ensembl to bypass the first problem I had.

It is a choice that is a bit unfortunate, in my opinion, to have a default setting for the

--miso_genes

parameter that mostly works with one gene annotation. You are not the first one to stumble over this, and there have been a couple of issues opened (https://github.com/nf-core/rnasplice/issues/119, https://github.com/nf-core/rnasplice/issues/78), but they were closed, apparently without addressing the root of the problem.

Thank you very much! In this way it completed the run successfully! When I have pinpoint the genes I’m interested in, how can I run the sashimi plot part without having to run again the whole pipeline? Thanks!

If you don't delete your work directory and use

-resume

, the pipeline should reuse any processes that have already been completed and are not affected by any changes (they show up as "cached" in the log). The pipeline can also be started from BAMs (by using a sample sheet that contains those), but that would still recalculate at least all the splicing-related tools.

great, thanks!