Hi #C0493FQQZ3P, I am currently running the

nf-core/rnasplice

pipeline with the

STAR_SALMON

aligner-quantification method, but I am encountering an issue where the job fails. My data consists of Illumina short-read RNA-Seq (150 bp paired-end), and my goal is to perform transcript quantification using Salmon, and additional transcript usage analysis. From my understanding, the nf-core/rnasplice pipeline automatically generates the transcript FASTA and Salmon index files from the provided GTF and genome FASTA by default. For isoform/transcript annotation, I used a merged GTF (custom) file that combines isoforms identified from PacBio long-read IsoSeq (via SQANTI3 filtering) data with the known GENCODE v48 annotation. Then, I basically created STAR Reference Index with the merged gtf file and fasta file from Gencode: GRCh38.primary_assembly.genome.fa.gz (renamed as hg38.fa) Ps: When I run with the standard GTF file directly from the Gencode, it runs successfully: https://www.gencodegenes.org/human/ I am attaching the logs for reference.

nextflow run /flashscratch/toufim/nfcore_rnasplice/1_0_4/rnasplice \
--input /flashscratch/toufim/sample_info_rnaplice.csv \
--contrasts /flashscratch/toufim/Contrastsheet.csv \
--outdir /flashscratch/toufim/ \
--star_index /flashscratch/toufim/ref_index/ \
--fasta /flashscratch/toufim/hg38.fa \
--gtf /flashscratch/toufim/merged.gtf \
--aligner star_salmon \
--gencode true \
--gtf_extra_attributes gene_id \
--skip_bigwig false \
--rmats false \
--sashimi_plot false \
--dexseq_dtu false \
--igenomes_ignore \
--suppa false \
-c /flashscratch/toufim/singularity_cache_nfcore/mod.config \
-c /flashscratch/toufim/singularity_cache_nfcore/rnasplice.config \
-resume

Logs

Command output:
  rsem-extract-reference-transcripts rsem/genome 0 hg38_genes.gtf None 0 rsem/hg38.fa
  Parsed 200000 lines
  Parsed 400000 lines
  Parsed 600000 lines
  Parsed 800000 lines
  Parsed 1000000 lines
  Parsed 1200000 lines
  Parsed 1400000 lines
  Parsed 1600000 lines
  Parsed 1800000 lines
  Parsed 2000000 lines
  Parsed 2200000 lines
  Parsed 2400000 lines
  Parsed 2600000 lines
  Parsed 2800000 lines
  Parsed 3000000 lines
  Parsed 3200000 lines
  Parsed 3400000 lines
  Parsed 3600000 lines
  Parsed 3800000 lines
  Parsed 4000000 lines
  Parsed 4200000 lines
  "rsem-extract-reference-transcripts rsem/genome 0 hg38_genes.gtf None 0 rsem/hg38.fa" failed! Plase check if you provide correct parameters/options for the pipeline!

Command error:
  INFO:    Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred
  Transcript ENST00000642072.1 is associated with multiple gene names!

Work dir:
  /flashscratch/toufim/RNAsplice_GT25_AnczukowO_SR/work/0b/5f7c17d8da51a23747422ac1c90ec9

Container:
  /projects/palucka-lab/toufim/singularity_cache_nfcore/depot.galaxyproject.org-singularity-mulled-v2-cf0123ef83b3c38c13e3b0696a3f285d3f20f15b-64aad4a4e144878400649e71f42105311be7ed87-0.img

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

executor >  slurm (29)
[7a/c356ea] NFC…:GTF_GENE_FILTER (hg38.fa) | 1 of 1 ✔
[0b/5f7c17] NFC…RIPTS_FASTA (rsem/hg38.fa) | 1 of 1, failed: 1 ✘
[61/e396f4] NFC…OM_GETCHROMSIZES (hg38.fa) | 1 of 1 ✔
[-        ] NFC…REPARE_GENOME:SALMON_INDEX -
[bd/7b45b8] NFC…_sample_info_rnaplice.csv) | 1 of 1 ✔
[0c/ddeb0b] NFC…czukowO_Contrastsheet.csv) | 1 of 1 ✔
[-        ] NFC…SPLICE:RNASPLICE:CAT_FASTQ -
[3d/292ca4] NFC…STQC (DD17_progenitors_B2) | 10 of 10 ✔
[c3/06b17a] NFC…LORE (DD46_progenitors_B2) | 10 of 10 ✔
[-        ] NFC…LICE:ALIGN_STAR:STAR_ALIGN -
[-        ] NFC…ATS_SAMTOOLS:SAMTOOLS_SORT -
[-        ] NFC…TS_SAMTOOLS:SAMTOOLS_INDEX -
[-        ] NFC…TS_SAMTOOLS:SAMTOOLS_STATS -
[-        ] NFC…SAMTOOLS:SAMTOOLS_FLAGSTAT -
[-        ] NFC…SAMTOOLS:SAMTOOLS_IDXSTATS -
[89/2cb4df] NFC…code_v48.merged_flair.gtf) | 1 of 1 ✔
[-        ] NFC…CE:DEXSEQ_DEU:DEXSEQ_COUNT -
[-        ] NFC…ICE:DEXSEQ_DEU:DEXSEQ_EXON -
[20/bb8663] NFC…code_v48.merged_flair.gtf) | 1 of 1 ✔
[-        ] NFC…_DEU:SUBREAD_FEATURECOUNTS -
[b5/5f713d] NFC…code_v48.merged_flair.gtf) | 1 of 1 ✔
[49/d403a7] NFC…code_v48.merged_flair.gtf) | 1 of 1 ✔
Plus 14 more processes waiting for tasks…
-[nf-core/rnasplice] Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_RNASPLICE:RNASPLICE:PREPARE_GENOME:MAKE_TRANSCRIPTS_FASTA (rsem/hg38.fa)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:PREPARE_GENOME:MAKE_TRANSCRIPTS_FASTA (rsem/hg38.fa)` terminated with an error exit status (255)


Command executed:

  rsem-prepare-reference \
      --gtf hg38_genes.gtf \
      --num-threads 12 \
       \
      rsem/hg38.fa \
      rsem/genome
  
  cp rsem/genome.transcripts.fa .
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:PREPARE_GENOME:MAKE_TRANSCRIPTS_FASTA":
      rsem: $(rsem-calculate-expression --version | sed -e "s/Current version: RSEM v//g")
      star: $(STAR --version | sed -e "s/STAR_//g")
  END_VERSIONS

Command exit status:
  255

Command output:
  rsem-extract-reference-transcripts rsem/genome 0 hg38_genes.gtf None 0 rsem/hg38.fa
  Parsed 200000 lines
  Parsed 400000 lines
  Parsed 600000 lines
  Parsed 800000 lines
  Parsed 1000000 lines
  Parsed 1200000 lines
  Parsed 1400000 lines
  Parsed 1600000 lines
  Parsed 1800000 lines
  Parsed 2000000 lines
  Parsed 2200000 lines
  Parsed 2400000 lines
  Parsed 2600000 lines
  Parsed 2800000 lines
  Parsed 3000000 lines
  Parsed 3200000 lines
  Parsed 3400000 lines
  Parsed 3600000 lines
  Parsed 3800000 lines
  Parsed 4000000 lines
  Parsed 4200000 lines
  "rsem-extract-reference-transcripts rsem/genome 0 hg38_genes.gtf None 0 rsem/hg38.fa" failed! Plase check if you provide correct parameters/options for the pipeline!

Command error:
  INFO:    Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred
  Transcript ENST00000642072.1 is associated with multiple gene names!

Thank you, Toufiq