Hi, I've got this error using

--genome GRCm38

pipeline version 3.26.0:

Caused by:
  Process `NFCORE_RNASEQ:RNASEQ:BAM_QC_RNASEQ:SUBREAD_FEATURECOUNTS (IIT_363_E5_B1_S4)` terminated with an error exit status (255)


Command executed:

  featureCounts \
      -B -C -g gene_type -t exon \
      -p \
      -T 6 \
      -a genes.filtered.gtf \
      -s 2 \
      -o IIT_363_E5_B1_S4.featureCounts.tsv \
      IIT_363_E5_B1_S4.markdup.sorted.bam

Command exit status:
  255

Command output:
  (empty)

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

          ==========     _____ _    _ ____  _____  ______          _____
          =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \
            =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
              ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
                ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
          ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
          v2.0.6

  //========================== featureCounts setting ===========================\\
  ||                                                                            ||
  ||             Input files : 1 BAM file                                       ||
  ||                                                                            ||
  ||                           IIT_363_E5_B1_S4.markdup.sorted.bam              ||
  ||                                                                            ||
  ||             Output file : IIT_363_E5_B1_S4.featureCounts.tsv               ||
  ||                 Summary : IIT_363_E5_B1_S4.featureCounts.tsv.summary       ||
  ||              Paired-end : yes                                              ||
  ||        Count read pairs : no                                               ||
  ||              Annotation : genes.filtered.gtf (GTF)                         ||
  ||      Dir for temp files : ./                                               ||
  ||                                                                            ||
  ||                 Threads : 6                                                ||
  ||                   Level : meta-feature level                               ||
  ||      Multimapping reads : not counted                                      ||
  || Multi-overlapping reads : not counted                                      ||
  ||   Min overlapping bases : 1                                                ||
  ||                                                                            ||
  \\============================================================================//

  //================================= Running ==================================\\
  ||                                                                            ||
  || Load annotation file genes.filtered.gtf ...                                ||

  ERROR: failed to find the gene identifier attribute in the 9th column of the provided GTF file.
  The specified gene identifier attribute is 'gene_type'
  An example of attributes included in your GTF annotation is 'exon_id "ENSMUSE00001343744"; exon_number "1"; exon_version "1"; gene_biotype "TEC"; gene_id "ENSMUSG00000102693"; gene_name "4933401J01Rik"; gene_source "havana"; gene_version "1"; havana_gene "OTTMUSG00000049935"; havana_gene_version "1"; havana_transcript "OTTMUST00000127109"; havana_transcript_version "1"; tag "basic"; transcript_biotype "TEC"; transcript_id "ENSMUST00000193812"; transcript_name "4933401J01Rik-001"; transcript_source "havana"; transcript_support_level "NA"; transcript_version "1"; tss_id "TSS81250";'.

It is an annotation-field mismatch. The GRCm38 iGenomes GTF is Ensembl and contains

gene_biotype

, but featureCounts is being run with

-g gene_type

. In rnaseq 3.26.0, that normally means either

--gencode

is enabled or

--featurecounts_group_type gene_type

is coming from a command/config. I’d suggest:

--gencode false \
--featurecounts_group_type gene_biotype \
-resume

Alternatively,

--skip_biotype_qc -resume

will bypass this process. It is only used for the MultiQC biotype QC plot, not the main expression quantification.

Ok I'll try that, thank you!