I have several issues using the version 3.26.0 of the rnaseq. 1-Do not download the genome reference. 2-do not generate the index give the fa. gtf files. 3.- i pass all the index and them they generate another folder with the star/index. 4- the most complicate issue is this:Last login: GNU nano 5.6.1 rnaAd36_job_error.out ===== / ____| | | | _ \| __ \| ____| /\ | __ \ ===== | (_ | | | | |_) | |__) | |__ / \ | | | | ==== \___ \| | | | _ <| _ /| __| / /\ \ | | | | ==== __) | |__| | |_) | | \ \| |____ / __ \| |__| | ========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/ v2.0.6 //========================== featureCounts setting ===========================\\ || || || Input files : 1 BAM file || || || || wt_2d.markdup.sorted.bam || || || || Output file : wt_2d.featureCounts.tsv || || Summary : wt_2d.featureCounts.tsv.summary || || Paired-end : no || || 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_biotype' An example of attributes included in your GTF annotation is 'gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id> Work dir: /mnt/lustre/koa/scratch/torres91/adeno/work/a2/77cc6dd2c604a4cb59db06735b33a2 For all the sample.. so looklike there is not conection with R??? somebody can help me to solve this. thank you in advance

The last issue (#4) is that your GTF does not contain any

gene_biotype

fields. You can either use a different GTF that has those fields, or use the parameter `--skip_biotype_qc`. I don't know what in #3 you consider a problem, and for #1 and #2 it would be helpful if you could show some error messages (e.g. from the

.command.{err,out,log}

files in the work directory of the process, or the

.nextflow.log

).

Hi thomas thank you for your reply.

Can you tell us the command you are using to start the pipeline, if there are any errors when running it, and what exactly it produces and you were expecting ("index" has many meanings, e.g. FASTA index [

*.fai

], BAM index [

*.bai

], the STAR index [a directory], etc.)? Also, note that RSEM uses its own parameters for STAR, which cannot be changed, as far as I know.

Chipping in here, as we're seeing a similar problem. It looks to us that the

featCounts -g $gene_id

variable is getting set to the once set in the parameters from the "`feature_group_type`" field: The error we see is:

The exit status of the task that caused the workflow execution to fail was: 255

The failure reason of the task that caused the workflow execution to fail was: ========== _____ _ _ ____ _____ ______ _____ ===== / ____| | | | _ \| __ \| ____| /\ | __ \ ===== | (___ | | | | |_) | |__) | |__ / \ | | | | ==== \___ \| | |

Error executing process > 'NFCORE_RNASEQ:RNASEQ:BAM_QC_RNASEQ:SUBREAD_FEATURECOUNTS (testes_4)'

Caused by:
  Process `NFCORE_RNASEQ:RNASEQ:BAM_QC_RNASEQ:SUBREAD_FEATURECOUNTS (testes_4)` terminated with an error exit status (255)
Command executed:

  featureCounts \
      -B -C -g ebi_biotype -t exon \
      -p \
      -T 18 \
      -a VectorBase-68_AaegyptiLVP_AGWG.filtered.gtf \
      -s 0 \
      -o testes_4.featureCounts.tsv \
      testes_4.markdup.sorted.bam

However, the only place in which we set the "ebi_biotype" value (that is getting passed to

featuireCounts -g

is in the

feature_group_type

parameter. I hope this helps diagnose!