MIRTOP_STATS IndexError

[anastasiaprime]

Description of the bug

Hello!

I'm trying to process my small rnaseq data using only R1 reads and always get the same error. What could it be? nextflow.log

I use smrnaseq v2.4.0, nextflow version 24.04.4 I tried dev version, but had the same error. Command line: nextflow run nf-core/smrnaseq -profile docker --input samplesheet_1.csv --outdir Results_R1_test --fasta /mnt/cephfs8_rw/oncology/refseqs/Homo_sapiens/NCBI/GRCh38/Sequence/WholeGenomeFasta/genome.fa --mirgenedb true --mirgenedb_species Hsa --mirgenedb_mature /mnt/cephfs8_rw/oncology/refseqs/Homo_sapiens/miRNA/hsa.fas --mirgenedb_hairpin /mnt/cephfs8_rw/oncology/refseqs/Homo_sapiens/miRNA/hsa-pre.fas --mirgenedb_gff /mnt/cephfs8_rw/oncology/refseqs/Homo_sapiens/miRNA/hsa.gff --mirtrace_species hsa -c config Config only for resources (max_cpus, max_memory) Error: `ERROR ~ Error executing process > 'NFCORE_SMRNASEQ:MIRNA_QUANT:BAM_STATS_MIRNA_MIRTOP:MIRTOP_STATS (770902000404_S5)'

Caused by:
Process NFCORE_SMRNASEQ:MIRNA_QUANT:BAM_STATS_MIRNA_MIRTOP:MIRTOP_STATS (770902000404_S5) terminated with an error exit status (1)

Command executed:

mirtop \ stats \ \ --out stats \ 770902000404_S5_mirtop.gff

cat <<-END_VERSIONS > versions.yml "NFCORE_SMRNASEQ:MIRNA_QUANT:BAM_STATS_MIRNA_MIRTOP:MIRTOP_STATS": mirtop: $(echo $(mirtop --version 2>&1) | sed 's/^.*mirtop //') END_VERSIONS

Command exit status: 1

Command output: ['stats', '--out', 'stats', '770902000404_S5_mirtop.gff'] Command error: WARNING: Your kernel does not support swap limit capabilities or the cgroup is not mounted. Memory limited without swap. /opt/conda/lib/python3.12/site-packages/Bio/pairwise2.py:278: BiopythonDeprecationWarning: Bio.pairwise2 has been deprecated, and we intend to remove it in a future release of Biopython. As an alternative, please consider using Bio.Align.PairwiseAligner as a replacement, and contact the Biopython developers if you still need the Bio.pairwise2 module. warnings.warn( 10/15/2024 09:29:02 INFO Run stats. 10/15/2024 09:29:02 INFO Reading: 770902000404_S5_mirtop.gff Traceback (most recent call last): File "/opt/conda/bin/mirtop", line 10, in sys.exit(main()) ^^^^^^ File "/opt/conda/lib/python3.12/site-packages/mirtop/command_line.py", line 34, in main ['stats', '--out', 'stats', '770902000404_S5_mirtop.gff'] stats(kwargs["args"]) File "/opt/conda/lib/python3.12/site-packages/mirtop/gff/stats.py", line 38, in stats out.append(_calc_stats(fn)) ^^^^^^^^^^^^^^^ File "/opt/conda/lib/python3.12/site-packages/mirtop/gff/stats.py", line 82, in _calc_stats df = _summary(lines) ^^^^^^^^^^^^^^^ File "/opt/conda/lib/python3.12/site-packages/mirtop/gff/stats.py", line 130, in _summary df_sum = _add_missing(df_sum) ^^^^^^^^^^^^^^^^^^^^ File "/opt/conda/lib/python3.12/site-packages/mirtop/gff/stats.py", line 110, in _add_missing df2 = pd.DataFrame({'category': category, 'sample': df['sample'].iat[0], 'counts': 0}, index=[0])

    File "/opt/conda/lib/python3.12/site-packages/pandas/core/indexing.py", line 2527, in __getitem__
      return self.obj._get_value(*key, takeable=self._takeable)
             ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
    File "/opt/conda/lib/python3.12/site-packages/pandas/core/series.py", line 1234, in _get_value
      return self._values[label]
             ~~~~~~~~~~~~^^^^^^^
  IndexError: index 0 is out of bounds for axis 0 with size 0
Work dir:
  /mnt/cephfs8_rw/oncology/miRNA/220118_VH00195_67_AAAHV32M5_fastq4/work/39/2e514730d2d4ad416afc2023156668

Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`

 -- Check '.nextflow.log' file for details
ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting

 -- Check '.nextflow.log' file for details

`

### Command used and terminal output

_No response_

### Relevant files

_No response_

### System information

_No response_

[atrigila]

Hi! Thank you for reporting this bug. I think I know where the issue is and I am working on a solution. Just to confirm, could you please run the same command but without the --mirtrace_species hsa flag? Thank you!

[anastasiaprime]

Hi @atrigila ! I run the command as you asked and the pipeline completed successfully, but Mirtrace and Mirtop didn't run

[atrigila]

The mirtop step requires both the mirtrace_species and its corresponding mirtrace gff file. If mirtrace_species is provided but the mirtrace gff is not supplied, the pipeline still attempts to run mirtop, which results in an error. This occurs because mirtop expects input data that depends on the presence of a valid GFF file for the specified mirtrace_species. Without this, the tool cannot properly process the data and fails.

mirtop was also not available for runs with using mirgenedb even in previous versions of the pipeline v2.3.1, which required --mirtrace_species to be present, for example: https://github.com/nf-core/smrnaseq/blob/5901bea438cb719c186f883bb6e6a11f1de70a5b/subworkflows/local/mirna_quant.nf#L98-L105

This same behavior can be reproduced in older versions of the pipeline (nextflow run smnrnaseq -profile test_mirgenedb,docker --outdir test_mirgenedb_old --mirtrace_species hsa, commit id f8fd872034e214fe922118275cdfdf6e498a7f5c)

I will update documentation to clearly state that mirtop supports mirtrace inputs only and emit warnings in the code. I also contacted mirtop developers to see if there is a workaround using mirgenedb inputs. I'll add in this issue if I have any updates.

[atrigila]

@anastasiaprime the issue should be solved now, but let us know if you have any additional questions. Just take into account that when using MirgeneDB inputs mirtop is hard-coded to use the pre sequences, which originate from the hairpin FASTA, rather than the pri sequences, which come from the mature FASTA. Users must provide pre files from the start to ensure consistency between the FASTA and GFF files, as the coordinates in the GFF file are referenced to pre sequences. This also ensures that names in the BAM file will match those in the GFF.