`pipeline:process_bams:stringtie (1)` terminated with an error exit status (1)

[sme229]

Operating System

Other Linux (please specify below)

Other Linux

Linux 5.14.21-150400.24.28-default

Workflow Version

wf-single-cell v2.1.0-ga25ec6c

Workflow Execution

Command line (Cluster)

Other workflow execution

No response

EPI2ME Version

No response

CLI command run

nextflow run epi2me-labs/wf-single-cell --expected_cells 3000 -profile singularity --fastq 'Elena-cDNA-2/20240624_1312_1D_PAW58377_76308a2c/fastq_pass' --kit_name '3prime' --kit_version 'v3' --ref_genome_dir 'OryCun_genome'

Workflow Execution - CLI Execution Profile

singularity

What happened?

I get this error after ~5 h of execution: Caused by: Process pipeline:process_bams:stringtie (1) terminated with an error exit status (1)

Command executed: Add chromosome label (-l) to generated transcripts so we don't get name collisions during file merge later samtools view -h align.bam NC_001913.1 | tee >( stringtie -L -c 2 -p 8 -G chr.gtf -l "NC_001913.1.stringtie" -o "stringtie.gff" - ) | samtools fastq | bgzip --threads 2 -c > reads.fastq.gz Get transcriptome sequence gffread -g ref_genome.fa -w "transcriptome.fa" "stringtie.gff"

Command exit status: 1 Command output: (empty) Command error: [M::bam2fq_mainloop] discarded 0 singletons [M::bam2fq_mainloop] processed 333717 reads GffObj::getSpliced() error: improper genomic coordinate 1026 on NC_001913.1 for NC_001913.1.stringtie.1.1

I generated the reference and gtf file with 10x as suggested. I then renamed the headers in the reference file to keep only the IDs with no description. I'm not sure why it says 'improper genomic coordinate 1026 on NC_001913.1 for NC_001913.1.stringtie.1.1' and how could I fix this?

Relevant log output

Jul-22 14:36:04.390 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 134; name: pipeline:process_bams:stringtie (1); status: COMPLETED; exit: 1; error: -; workDir: /datasets/work/hb-rabbit-gbc/work/Maria_Jenkel_ONT_Data_04-07-2024/work/f2/e1eeb8d9fe3e7f883fe255830c3c27]
Jul-22 14:36:04.393 [TaskFinalizer-10] DEBUG nextflow.processor.TaskProcessor - Handling unexpected condition for
  task: name=pipeline:process_bams:stringtie (1); work-dir=/datasets/work/hb-rabbit-gbc/work/Maria_Jenkel_ONT_Data_04-07-2024/work/f2/e1eeb8d9fe3e7f883fe255830c3c27
  error [nextflow.exception.ProcessFailedException]: Process `pipeline:process_bams:stringtie (1)` terminated with an error exit status (1)
Jul-22 14:36:04.411 [TaskFinalizer-10] ERROR nextflow.processor.TaskProcessor - Error executing process > 'pipeline:process_bams:stringtie (1)'

Caused by:
  Process `pipeline:process_bams:stringtie (1)` terminated with an error exit status (1)

Command executed:

  # Add chromosome label (-l) to generated transcripts
  # so we don't get name collisions during file merge later
  samtools view -h align.bam NC_001913.1          | tee >(
          stringtie -L -c 2 -p 8                 -G chr.gtf -l "NC_001913.1.stringtie" -o "stringtie.gff" - )         | samtools fastq         | bgzip --threads 2 -c > reads.fastq.gz
  # Get transcriptome sequence
  gffread -g ref_genome.fa -w "transcriptome.fa" "stringtie.gff"

Command exit status:
  1

Command output:
  (empty)

Command error:
  [M::bam2fq_mainloop] discarded 0 singletons
  [M::bam2fq_mainloop] processed 333717 reads
  GffObj::getSpliced() error: improper genomic coordinate 1026 on NC_001913.1 for NC_001913.1.stringtie.1.1

Work dir:
  /datasets/work/hb-rabbit-gbc/work/Maria_Jenkel_ONT_Data_04-07-2024/work/f2/e1eeb8d9fe3e7f883fe255830c3c27

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line
Jul-22 14:36:04.412 [Task submitter] DEBUG n.executor.local.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run
Jul-22 14:36:04.412 [Task submitter] INFO  nextflow.Session - [db/4937d4] Submitted process > pipeline:process_bams:stringtie (15)
Jul-22 14:36:04.421 [TaskFinalizer-10] DEBUG nextflow.Session - Session aborted -- Cause: Process `pipeline:process_bams:stringtie (1)` terminated with an error exit status (1)
Jul-22 14:36:04.438 [TaskFinalizer-10] DEBUG nextflow.Session - The following nodes are still active:
[process] pipeline:process_bams:align_to_transcriptome
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:assign_features
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:create_matrix
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:process_matrix
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:merge_transcriptome
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:combine_final_tag_files
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:tag_bam
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:umi_gene_saturation
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:process_bams:pack_images
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:prepare_report_data
  status=ACTIVE
  port 0: (queue) OPEN  ; channel: -
  port 1: (cntrl) -     ; channel: $

[process] pipeline:makeReport
  status=ACTIVE
  port 0: (value) bound ; channel: metadata
  port 1: (value) bound ; channel: versions
  port 2: (value) bound ; channel: params.csv
  port 3: (value) bound ; channel: stats
  port 4: (queue) OPEN  ; channel: survival.tsv
  port 5: (value) OPEN  ; channel: umap_dirs
  port 6: (value) OPEN  ; channel: images
  port 7: (value) bound ; channel: umap_genes
  port 8: (value) bound ; channel: wf_version
  port 9: (cntrl) -     ; channel: $

Jul-22 14:36:04.470 [Task monitor] DEBUG n.processor.TaskPollingMonitor - <<< barrier arrives (monitor: local) - terminating tasks monitor poll loop
Jul-22 14:36:05.640 [main] DEBUG nextflow.Session - Session await > all processes finished
Jul-22 14:36:05.640 [main] DEBUG nextflow.Session - Session await > all barriers passed
Jul-22 14:36:05.649 [Actor Thread 20] DEBUG nextflow.processor.TaskProcessor - Handling unexpected condition for
  task: name=pipeline:process_bams:tag_bam; work-dir=null
  error [java.lang.InterruptedException]: java.lang.InterruptedException
Jul-22 14:36:05.649 [Actor Thread 33] DEBUG nextflow.file.SortFileCollector - FileCollector temp dir not removed: null
Jul-22 14:36:05.958 [main] WARN  n.processor.TaskPollingMonitor - Killing running tasks (1)
Jul-22 14:36:05.978 [main] DEBUG n.trace.WorkflowStatsObserver - Workflow completed > WorkflowStats[succeededCount=149; failedCount=1; ignoredCount=0; cachedCount=0; pendingCount=1685; submittedCount=1; runningCount=0; retriesCount=0; abortedCount=0; succeedDuration=1d 20h 31m 38s; failedDuration=31.4s; cachedDuration=0ms;loadCpus=0; loadMemory=0; peakRunning=8; peakCpus=12; peakMemory=18 GB; ]
Jul-22 14:36:05.978 [main] DEBUG nextflow.trace.TraceFileObserver - Workflow completed -- saving trace file
Jul-22 14:36:05.981 [main] DEBUG nextflow.trace.ReportObserver - Workflow completed -- rendering execution report
Jul-22 14:36:06.515 [main] DEBUG nextflow.trace.TimelineObserver - Workflow completed -- rendering execution timeline
Jul-22 14:36:06.620 [main] DEBUG nextflow.cache.CacheDB - Closing CacheDB done
Jul-22 14:36:06.671 [main] DEBUG nextflow.script.ScriptRunner - > Execution complete -- Goodbye

Application activity log entry

No response

Were you able to successfully run the latest version of the workflow with the demo data?

yes

Other demo data information

No response

[nrhorner]

Hi @sme229 Is it possible that a coordinate in your reference gtf file refers to a location that does not exist in your reference sequences?

The size of the mitochondrial genome NC_001913.1 is 17,245 bp. Are there any end coordinates for NC_001913.1 in your GTF file that extend further than this?

[sme229]

Hi @nrhorner Thanks for your response. No, there are no coordinates that extend beyond 17,245 in my gtf file:

[sme229]

@nrhorner I removed the NC_001913.1 entry from the GTF file to see if that helps Update: same type of error gffread -g ref_genome.fa -w "transcriptome.fa" "stringtie.gff"

Command exit status: 1

Command output: (empty)

Command error: [M::bam2fq_mainloop] discarded 0 singletons [M::bam2fq_mainloop] processed 3 reads GffObj::getSpliced() error: improper genomic coordinate 61536 on NW_026260071.1 for NW_026260071.1.stringtie.1.1

[sme229]

Hi @nrhorner Let me provide more information please. When I get this error:

GffObj::getSpliced() error: improper genomic coordinate 37565 on NW_026260069.1 for NW_026260069.1.stringtie.1.1

I can see that there is a duplication in the resulting stringtie.gff file:

I'm not sure if that's how it should be? upd. I can see that they are not exactly the same, sorry.

[nrhorner]

HI @sme229

Sorry for the late reply. Would it be possible to share the work directory with me? /datasets/work/hb-rabbit-gbc/work/Maria_Jenkel_ONT_Data_04-07-2024/work/f2/e1eeb8d9fe3e7f883fe255830c3c27

I can send a link for you to drop that into.

[sme229]

Hi @nrhorner

Sure, happy to do that. I have since re-run the pipeline but got the same error: Command error: [M::bam2fq_mainloop] discarded 0 singletons [M::bam2fq_mainloop] processed 1448 reads GffObj::getSpliced() error: improper genomic coordinate 37565 on NW_026260069.1 for NW_026260069.1.stringtie.1.1

Work dir: /datasets/work/hb-rabbit-gbc/work/Maria_Jenkel_ONT_Data_04-07-2024/work/ed/563d7d63a056ec563b717e48060582

So the directory has a different name.

[nrhorner]

Hi @sme229

I need an email address in order to grant access to a shared folder. Do you happen to have a linkedin profile or somewhere else I can message you to get your email address?

[sme229]

Hi @nrhorner Sure, here is my email address Elena.Smertina@csiro.au