NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_INDEX (Mes_genes.gtf)` terminated with an error exit status (1

[sajjadasaf]

Description of the bug

I am facing the below issue Could you kindly help me to resolve this issue.
Input/output options
input : ./samplesheet.csv
outdir : ./results

Mandatory arguments
protocol : 10XV1

Reference genome options
fasta : Mes.fna
gtf : Mes.gtf
save_align_intermeds: true

Core Nextflow options
revision : dev
runName : focused_shannon
containerEngine : docker
launchDir : /media/omic/5a465bf0-239c-4f0f-ba19-5c5b3ed9baac/Singlecell
workDir : /media/omic/5a465bf0-239c-4f0f-ba19-5c5b3ed9baac/Singlecell/work
projectDir : /home/omic/.nextflow/assets/nf-core/scrnaseq
userName : omic
profile : docker
configFiles :

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------* The pipeline
https://doi.org/10.5281/zenodo.3568187

• The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

• Software dependencies
  https://github.com/nf-core/scrnaseq/blob/master/CITATIONS.md

WARN: The following invalid input values have been detected:

• --monochromeLogs: null

executor > local (3)
[d8/707115] NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (Control_stage1_Rep1) [ 0%] 0 of 1
[53/c4a8d0] NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (Mes.fna) [100%] 1 of 1 ✔
[b2/efd0f0] NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_INDEX (Mes_genes.gtf) [ 0%] 0 of 1
[- ] NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_QUANT -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:ALEVINQC -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:MTX_TO_H5AD -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:H5AD_CONVERSION:CONCAT_H5AD -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:H5AD_CONVERSION:ANNDATAR_CONVERT -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:EMPTY_DROPLET_REMOVAL:CELLBENDER_REMOVEBACKGROUND -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:EMPTY_DROPLET_REMOVAL:ADATA_BARCODES -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_H5AD_CONVERSION:CONCAT_H5AD -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_H5AD_CONVERSION:ANNDATAR_CONVERT -
[- ] NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC -
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_INDEX (Mes_genes.gtf)'

Caused by:
Process NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_INDEX (Mes_genes.gtf) terminated with an error exit status (1)

Command executed:

export required var

export ALEVIN_FRY_HOME=.
export NUMBA_CACHE_DIR=.

prep simpleaf

simpleaf set-paths

run simpleaf index

simpleaf
index
--threads 6
--fasta Mes.fna
--gtf Mes_genes.gtf
--rlen 91
-o salmon

cat <<-END_VERSIONS > versions.yml
"NFCORE_SCRNASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_INDEX":
simpleaf: $(simpleaf -V | tr -d '\n' | cut -d ' ' -f 2)
salmon: $(salmon --version | sed -e "s/salmon //g")
END_VERSIONS

Command exit status:
1

Command output:
2024-12-31T08:37:25.479747Z INFO simpleaf::utils::prog_utils: could not find piscem executable, so salmon will be required.
.nextflow.log

Command used and terminal output

Relevant files

No response

System information

No response

[sajjadasaf]

Kindly help how to solve this issue?

[kopichris]

Hi @sajjadasaf, I am not a maintainer, but happy to help troubleshoot. I reviewed a copy of the log file you attached and found the following error message:

[error] In FixFasta, two references with the same name but different sequences: unassigned_transcript_938. We require that all input records have a unique name up to the first whitespace (or user-provided separator) character.

Based on the error message, it looks like you might have duplicate header sequences in your FASTA file, which isn't allowed. I would recommend addressing this issue and then rerunning the pipeline.

Please let us know if this resolves your issue.

[sajjadasaf]

Thank you very much for your reply. Actually I am using genome downloaded from Ensemble and i am unable to find any duplicated sequences here. Should we make changes in GTF file before running this pipeline?

[kopichris]

Thank you very much for your reply. Actually I am using genome downloaded from Ensemble and i am unable to find any duplicated sequences here. Should we make changes in GTF file before running this pipeline?

If there are no duplicated sequences, I don't think changing the GTF file should be necessary. Happy to try reproducing the error using the same reference and gtf file. Would you mind sharing which reference genome and version was used from Ensembl?

[sajjadasaf]

Thank you very much for your help. The issue was caused by a mismatch between the GTF and FASTA files, but it has now been resolved. I successfully completed the pipeline; however, the output contains only two folders, and the quantification data is missing. This is my first time using this pipeline, and I would greatly appreciate your guidance.

[grst]

2.0.0 is a very old version and many things have been improved since then. Is there any reason why you use that version?

The results should be in the salmon folder though as results are organized by aligner.

[sajjadasaf]

Thank you very much for pointing this out. I have updated the version to 3.0.0 and re-ran the same command, but I am now encountering this error.

log.txt

[grst]

hmm, seems support for 10xv1 got removed from alevin at some point.

It is still possible to specify the chemistry using a description such as 1{b[16]u[10]x:}2{r:} (this is for 10xV2, we'd need to figure out the correct one for v1). The 'language' of these descriptions is specified here: https://hackmd.io/@PI7Og0l1ReeBZu_pjQGUQQ/rJMgmvr13