ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER (1)'

[IanCodes]

Description of the bug

My aim was to run rnasplice to output results for SUPPA2. It is not clear whether the following is an error on my part, or there are no significant results. !No genes left after filtering! makes we think it is the latter, but your opinion would be welcome. I am happy to provide more information if it help.

Command used and terminal output

Command line:
nextflow run nf-core/rnasplice --input samplesheet_fastqs_minus.csv --contrasts contrastsheet_minus.csv --outdir RNA-seq_fastqs_minus_samples -profile singularity -r 1.0.2 --rmats --source fastq --fasta Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa --gtf Saccharomyces_cerevisiae.R64-1-1.111.gtf --miso_genes <redacted> --aligner star_salmon --rmats_read_len 139

Output:
ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER (1)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER (1)` terminated with an error exit status (1)

Command executed:

  run_drimseq_filter.R salmon.merged.txi.dtu.rds tximport.tx2gene.tsv samplesheet_fastqs_minus.csv \
      4 \
      2 \
      2 \
      10 \
      0.1 \
      10
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER":
      r-base: $(echo $(R --version 2>&1) | sed 's/^.*R version //; s/ .*$//')
      bioconductor-drimseq: $(Rscript -e "library(DRIMSeq); cat(as.character(packageVersion('DRIMSeq')))")
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  INFO:    Converting SIF file to temporary sandbox...
  WARNING: Skipping mount /usr/local/singularity-ce-3.11.2/var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
  
  Attaching package: 'DRIMSeq'
  
  The following object is masked from 'package:base':
  
      proportions
  
  Error in dmDS_filter(counts = x@counts, min_samps_gene_expr = min_samps_gene_expr,  : 
    !No genes left after filtering!
  Calls: <Anonymous> -> <Anonymous> -> .local -> dmDS_filter
  Execution halted
  INFO:    Cleaning up image...

Work dir:
  <redacted>/work/69/970b0a2cad7dab41066bb18623d068

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

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

Relevant files

No response

System information

nextflow version 23.04.1.5866, run locally on server
singularity container 3.11.2
Linux Ubuntu 22.04.3 LTS
nf-core/rnasplice 1.0.2

[jma1991]

Hello @IanCodes

Would you mind re-running the pipeline with the following configuration:

min_samps_gene_expr: 0
min_gene_expr: 0
min_samps_feature_expr: 0
min_feature_expr: 0
min_samps_feature_prop: 0
min_feature_prop: 0

These parameters should effectively stop any filtering. If you still encounter the same issue, let me know and I can take a deeper look.

[IanCodes]

Hello @jma1991,

Thank you for your reply and apologies for the delayed response.

I ran the following command line:
nextflow run nf-core/rnasplice --input samplesheet_fastqs_minus.csv --contrasts contrastsheet_minus.csv --outdir RNA-seq_fastqs_minus_samples_MINSIG -profile singularity -r 1.0.2 --source fastq --fasta Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa --gtf Saccharomyces_cerevisiae.R64-1-1.111.gtf --aligner star_salmon --rmats false --suppa true --dexseq_exon false --dexseq_dtu false --sashimi_plot false --edger_exon false --min_samps_gene_expr 0 --min_gene_expr 0 --min_samps_feature_expr 0 --min_feature_expr 0 --min_samps_feature_prop 0 --min_feature_prop 0

But I still got an error:

ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER (1)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER (1)` terminated with an error exit status (1)

Command executed:

  run_drimseq_filter.R salmon.merged.txi.dtu.rds tximport.tx2gene.tsv samplesheet_fastqs_minus.csv \
      0 \
      0 \
      0 \
      0 \
      0 \
      0
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:DRIMSEQ_DEXSEQ_DTU_SALMON:DRIMSEQ_FILTER":
      r-base: $(echo $(R --version 2>&1) | sed 's/^.*R version //; s/ .*$//')
      bioconductor-drimseq: $(Rscript -e "library(DRIMSeq); cat(as.character(packageVersion('DRIMSeq')))")
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  INFO:    Converting SIF file to temporary sandbox...
  WARNING: Skipping mount /usr/local/singularity-ce-3.11.2/var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
  
  Attaching package: 'DRIMSeq'
  
  The following object is masked from 'package:base':
  
      proportions
  
  Error in dmDS_filter(counts = x@counts, min_samps_gene_expr = min_samps_gene_expr,  : 
    !No genes left after filtering!
  Calls: <Anonymous> -> <Anonymous> -> .local -> dmDS_filter
  Execution halted
  INFO:    Cleaning up image...

Work dir:
 <REDACTED>/work/44/6b45b957ccdafc9f5e93c4b0b7cc34

Tip: view the complete command output by changing to the process work dir and entering the command `cat .command.out`

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

This may or may not be related, but I cannot reproduce results obtained from the stand-alone rMATS-turbo (4.2) with rnasplice. The summary file of the stand-alone shows:

EventType       EventTypeDescription    TotalEventsJC   TotalEventsJCEC SignificantEventsJC     SigEventsJCSample1HigherInclusion       SigEventsJCSample2HigherInclusion       SignificantEventsJCEC   SigEventsJCECSample1HigherInclusion     SigEventsJCECSample2HigherInclusion
SE      skipped exon    22132   22465   756     385     371     841     436     405
A5SS    alternative 5' splice sites     10382   10438   168     94      74      185     109     76
A3SS    alternative 3' splice sites     13738   13777   332     190     142     346     191     155
MXE     mutually exclusive exons        2204    2243    74      24      50      82      26      56
RI      retained intron 6764    6926    364     199     165     407     233     174

Summary of rnasplice are all zeros.

The command line with the stand-alone was:
rmats.py --gtf Mus_musculus.GRCm39.106.gtf --b1 WT.txt --b2 mutAff.txt --libType fr-firststrand --readLength 90 --variable-read-length --novelSS --nthread 4 --od wt-mutRev_rMATsOUT --tmp wt-mutRev-rMATsTMP

For rnasplice I used:
nextflow run nf-core/rnasplice --input samplesheet.csv --contrasts contrastsheet.csv --outdir Rev_vs_Normal_Ens106_default_sig -profile singularity -r 1.0.2 --source genome_bam --fasta Mus_musculus.GRCm39.dna.primary_assembly.fa --gtf Mus_musculus.GRCm39.106.gtf --aligner star --rmats_read_len 90 --rmats --rmats_novel_splice_site true --dexseq_exon false --dexseq_dtu false --sashimi_plot false --edger_exon false

The samplesheet was:

sample,condition,genome_bam,strandedness
N1,Normal,1295_521_2_510_S31_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
N2,Normal,1395_721_1_510_S30_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
N3,Normal,711_2_3_1295_115_HT_S32_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
N4,Normal,711_6_1293_113_HT_S33_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
N5,Normal,7_129_115_HT_S34_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
N6,Normal,722_1_511_HT_S35_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
R1,Rev,3131_3_K27_511_HT_S39_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
R2,Rev,3150_2_512_HT_S40_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
R3,Rev,3158_11_514_HT_S42_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse
R4,Rev,R27_3139_4_512_HT3_S41_L005-vsHsap_Aligned.sortedByCoord.sorted.bam,reverse

Sorry to dump this one here. I can restart a separate thread if that helps.

Thank you.

[SergioManzano10]

H! I am having the same problem.

Did you finally solve it?

[IanCodes]

@SergioManzano10 I haven't had a response. I will use the standalone tool for the time being.

[SergioManzano10]

Ok, could you provide me a link or something where the standalone tool is explained? Thank you. El El dom, 12 may 2024 a las 15:52, IanCodes ***@***.***> escribió:

…

@SergioManzano10 <https://github.com/SergioManzano10> I haven't had a response. I will use the standalone tool for the time being. — Reply to this email directly, view it on GitHub <#126 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/BENHAIEYLM2JRYQ7X2MPCZ3ZB5X2PAVCNFSM6AAAAABD2QMTLWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDCMBWGI2TIMRUGM> . You are receiving this because you were mentioned.Message ID: ***@***.***>

[jma1991]

@SergioManzano10 I haven't had a response. I will use the standalone tool for the time being.

Apologies for the lack of progress on this issue. Is it possible to host your input files somewhere I can download and run locally? It's quite difficult for me to debug when the full test data seems to pass without issue.

[SergioManzano10]

Not me because are clinical/private data, but maybe @IanCodes can.

It would be so helpful!

• Maybe the problem is that all quantifications methods are set to true and it crashes, or that the default value for the aligner is star_salmon? These are just ideas...

[IanCodes]

@SergioManzano10 see https://github.com/Xinglab/rmats-turbo

[IanCodes]

@jma1991 I have an urgent deadline this week and a conference next week. When I get back i'll look into sourcing the files to you. I had to delete them last week to make space, so they need retrieving again. I'll also need permission to send them. I think Dropbox is probably the best route for me. If that works for you could you private message me, here, an email address to send the link.

EDIT:
I was easily able to get the files again, so if you can let me have contact details I can share them this week. Thanks.

[jma1991]

@jma1991 I have an urgent deadline this week and a conference next week. When I get back i'll look into sourcing the files to you. I had to delete them last week to make space, so they need retrieving again. I'll also need permission to send them. I think Dropbox is probably the best route for me. If that works for you could you private message me, here, an email address to send the link.

No problem, enjoy the conference. I'm not sure how to direct message on GitHub, but you can reach me on the nf-core Slack channel. Alternatively, if you have some public data that you used with the workflow and encountered the error, I can download it from SRA.

[jma1991]

EDIT: I was easily able to get the files again, so if you can let me have contact details I can share them this week. Thanks.

That's great, please send an email to tunics.scrums-0u@icloud.com when you have availability.