#C0493FQQZ3P For instance;

ENST00000642072.1

transcript ID is associated with multiple gene names. This turned out to be gene_fusion, hence I removed all the gene_fusion, and re-run, it still fail, now with a different error. Note: I just removed the gene_fusions, and did not create a new reference index: New logs:

executor >  slurm (30)
[95/692b0d] NFC…:GTF_GENE_FILTER (hg38.fa) | 1 of 1 ✔
[8a/71683a] NFC…RIPTS_FASTA (rsem/hg38.fa) | 1 of 1 ✔
[98/a76b44] NFC…OM_GETCHROMSIZES (hg38.fa) | 1 of 1 ✔
[a7/81983f] NFC…EX (genome.transcripts.fa) | 1 of 1, failed: 1 ✘
[c6/2cb944] NFC…_sample_info_rnaplice.csv) | 1 of 1 ✔
[66/b75205] NFC…czukowO_Contrastsheet.csv) | 1 of 1 ✔
[-        ] NFC…SPLICE:RNASPLICE:CAT_FASTQ -
[5e/09f6cd] NFC…STQC (DD58_progenitors_B2) | 10 of 10 ✔
[94/567fe7] 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 -
[ab/75f054] NFC…(Progenitors.noFUSION.gtf) | 1 of 1 ✔
[-        ] NFC…CE:DEXSEQ_DEU:DEXSEQ_COUNT -
[-        ] NFC…ICE:DEXSEQ_DEU:DEXSEQ_EXON -
[84/4e9ff4] NFC…(Progenitors.noFUSION.gtf) | 1 of 1 ✔
[-        ] NFC…_DEU:SUBREAD_FEATURECOUNTS -
[4f/99980c] NFC…(Progenitors.noFUSION.gtf) | 1 of 1 ✔
[33/d6b86e] NFC…(Progenitors.noFUSION.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:SALMON_INDEX (genome.transcripts.fa)'

Caused by:
  Process `NFCORE_RNASPLICE:RNASPLICE:PREPARE_GENOME:SALMON_INDEX (genome.transcripts.fa)` terminated with an error exit status (1)


Command executed:

  grep '^>' hg38.fa | cut -d ' ' -f 1 | cut -d $'\t' -f 1 > decoys.txt
  sed -i.bak -e 's/>//g' decoys.txt
  cat genome.transcripts.fa hg38.fa > gentrome.fa
  
  salmon \
      index \
      --threads 6 \
      -t gentrome.fa \
      -d decoys.txt \
      --gencode \
      -i salmon
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASPLICE:RNASPLICE:PREPARE_GENOME:SALMON_INDEX":
      salmon: $(echo $(salmon --version) | sed -e "s/salmon //g")
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390569.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390571.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390572.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390574.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390575.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390578.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390580.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390581.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390583.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390584.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390585.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390588.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390590.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.564] [puff::index::jointLog] [warning] Entry with header [ENST00000390591.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.809] [puff::index::jointLog] [warning] Entry with header [ENST00000415118.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:37.950] [puff::index::jointLog] [warning] Entry with header [ENST00000452198.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.062] [puff::index::jointLog] [warning] Entry with header [ENST00000451044.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.072] [puff::index::jointLog] [warning] Entry with header [ENST00000437320.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.104] [puff::index::jointLog] [warning] Entry with header [ENST00000431870.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.140] [puff::index::jointLog] [warning] Entry with header [ENST00000454691.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.217] [puff::index::jointLog] [warning] Entry with header [ENST00000448914.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.460] [puff::index::jointLog] [warning] Entry with header [ENST00000431440.2], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.536] [puff::index::jointLog] [warning] Entry with header [ENST00000414852an equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.217] [puff::index::jointLog] [warning] Entry with header [ENST00000448914.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.460] [puff::index::jointLog] [warning] Entry with header [ENST00000431440.2], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.536] [puff::index::jointLog] [warning] Entry with header [ENST00000414852.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.714] [puff::index::jointLog] [warning] Entry with header [ENST00000454908.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.745] [puff::index::jointLog] [warning] Entry with header [ENST00000439842.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.774] [puff::index::jointLog] [warning] Entry with header [ENST00000450276.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.789] [puff::index::jointLog] [warning] Entry with header [ENST00000430425.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.792] [puff::index::jointLog] [warning] Entry with header [ENST00000434970.2], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:38.870] [puff::index::jointLog] [warning] Entry with header [ENST00000473810.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:39.017] [puff::index::jointLog] [warning] Entry with header [ENST00000682202.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:39.696] [puff::index::jointLog] [warning] Entry with header [ENST00000518246.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:39.828] [puff::index::jointLog] [warning] Entry with header [ENST00000543745.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.296] [puff::index::jointLog] [warning] Entry with header [ENST00000579054.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.446] [puff::index::jointLog] [warning] Entry with header [ENST00000604838.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.453] [puff::index::jointLog] [warning] Entry with header [ENST00000603693.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.461] [puff::index::jointLog] [warning] Entry with header [ENST00000604446.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.463] [puff::index::jointLog] [warning] Entry with header [ENST00000604642.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.471] [puff::index::jointLog] [warning] Entry with header [ENST00000603326.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.472] [puff::index::jointLog] [warning] Entry with header [ENST00000605284.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.477] [puff::index::jointLog] [warning] Entry with header [ENST00000603775.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.795] [puff::index::jointLog] [warning] Entry with header [ENST00000603935.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.797] [puff::index::jointLog] [warning] Entry with header [ENST00000604102.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.798] [puff::index::jointLog] [warning] Entry with header [ENST00000632684.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.800] [puff::index::jointLog] [warning] Entry with header [ENST00000604950.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.802] [puff::index::jointLog] [warning] Entry with header [ENST00000603077.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:40.803] [puff::index::jointLog] [warning] Entry with header [ENST00000634174.1], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
  [2025-10-14 17:01:41.609] [puff::index::jointLog] [error] In FixFasta, two references with the same name but different sequences: ENST00000641182.1. We require that all input records have a unique name up to the first whitespace (or user-provided separator) character.

You can still use the gene fusions, if you want, you just have to call the fused gene something different from the original genes (e.g.

geneA_geneB_fusion

). The new error occurs because you are using the transcript ID

ENST00000641182.1

in your PacBio-derived annotation, but it already occurs in the Gencode GTF, so you now have a duplicate, but with different sequence. I would prefix every transcript (and any substantially different gene) derived from the PacBio data with something like

PacBio_

, so that such name clashes become impossible.