Hi, I am running the pipeline for a small part of my data to test if the pipeline works for my data set. The data is from two flowcells of a PromethION for one sample. It seems to run through the analysis just fine until the final steps. I get an error message at the seurat transcript step, but it is not entirely clear to me why it fails. Thank you for your help! Best, Annbjørg Here is the error message: ERROR ~ Error executing process > ‘NFCORE_SCNANOSEQSCNANOSEQSEURAT_TRANSCRIPT (GIST-T1-670)’ Caused by: Process

NFCORE_SCNANOSEQ:SCNANOSEQ:SEURAT_TRANSCRIPT (GIST-T1-670)

terminated with an error exit status (137) Command executed: seurat_qc.R \ \ -i GIST-T1-670.transcript_counts.tsv \ -s GIST-T1-670.dedup.sorted.flagstat \ -d GIST-T1-670 \ -r GIST-T1-670 cat <<-END_VERSIONS > versions.yml “NFCORE_SCNANOSEQSCNANOSEQSEURAT_TRANSCRIPT”: r-base: $(echo $(R --version 2>&1) | sed ‘s/^.*R version //; s/ .*$//’) r-seurat: $(Rscript -e “library(Seurat); cat(as.character(packageVersion(‘Seurat’)))“) END_VERSIONS Command exit status: 137 Command output: (empty) Command error: Attaching SeuratObject .command.sh: line 7: 32 Killed seurat_qc.R -i GIST-T1-670.transcript_counts.tsv -s GIST-T1-670.dedup.sorted.flagstat -d GIST-T1-670 -r GIST-T1-670 Work dir: /media/datastore/Leonardo_GIST/nf-core-test/work/0f/9d817f383e251ec682110bd1f7c8bb Tip: you can try to figure out what’s wrong by changing to the process work dir and showing the script file named

.command.sh

-- 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 -[nf-core/scnanoseq] Pipeline completed with errors-

Thank you for using our pipeline! Would you mind providing the command you used to run the pipeline and the

.nextflow.log

file? That’ll help us in tracking down the error

Command exit status:

137

Could indicate out of memory IIRC

Here is the code i used to run the pipeline: nextflow run nf-core/scnanoseq \ --input ./samplesheet.csv \ --outdir ./results/GIST-T1-670 \ --fasta /media/datastore/single_cell_analysis/ONT_analysis/10X-reference-files/refdata-gex-GRCh38-2024-A/fasta/genome.fa \ --gtf /media/datastore/single_cell_analysis/ONT_analysis/10X-reference-files/refdata-gex-GRCh38-2024-A/genes/genes.gtf \ --barcode_format 10X_3v3 \ -profile docker

Looks indeed like an out-of-memory error. The

SEURAT_TRANSCRIPT

process is retried once (after getting exit code 137), after it exits again with 137. How much memory do you have available on your system?

Yep agreed with the above. In our upcoming 1.1.0 release we have increased the default resources for the Seurat step to avoid this error. But in the meantime you can get around this by increasing the memory with a custom config, e.g.: (the 60GB below is given as an example. Not sure if this is fully needed for your job or not enough, but you can adjust as needed).

process
{
    withName: '.*:SEURAT_TRANSCRIPT'
    {
        memory = '60.GB'
    }
}

The above should be added to a file that can be named for example

custom.conf

and you can then add it to the pipeline run with the

-c

flag (e.g.:

-c custom.conf

) (and

-resume

of course). Let us know if this works!

Thank you! I think memory should not be a problem, so I will try this.

Great, happy to provide advice on resource optimization if needed. But be aware we are very close to a 1.1.0 release which contains improvements on data scalability for isoquant and the ability to run

oarfish

(which is quite fast). You will be able to run both quantifiers or one of them. Just a something to keep in the radar.