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 132, in <module> raise ValueError("Same name found on two strands: %s, %s" % (str(l[i]), str(l[i + 1]))) ValueError: Same name found on two strands: <GenomicFeature: exonic_part 'Gm4301+Gm4312' at chr10: 100366498 -> 100367412 (strand '+')>, <GenomicFeature: exonic_part 'Gm4301+Gm4312' at chr10: 100382>

The warnings about `SINGULARITYENV_*`/`APPTAINERENV_*` can be ignored, they are expected when you run with a`singularity` profile on a machine with Apptainer installed. The problem is that your GTF seems ambiguous/malformed:

ValueError: Same name found on two strands: <GenomicFeature: exonic_part 'Gm4301+Gm4312' at chr10: 100366498 -> 100367412 (strand '+')>, <GenomicFeature: exonic_part 'Gm4301+Gm4312' at chr10: 100382>

Where does your GTF come from? Can you try a different one (Ensembl usually works for me)? Pro tips: If you click in your icon (bottom left) and select

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thank for you reply, however the GTF was previously using in similar pipeline few week ago and work perfectly.

Maybe it got corrupted, or maybe some filtering had been applied earlier. I would look at the positions in the error message and confirm if something is wrong or not, and you can also try another GTF just to see if that works.