I am following the tutorial here to learn how to process raw RNA-seq data and get gene read counts. I've had no issues until I got to step 3.9, "Assignment of RNA-Seq Reads to Genes." Here, the authors run the command

fc0 <- featureCounts(sam.list, annot.ext = 
"Ara-port11_GFF3_genes_transposons.201606.gtf", allowMultiOverlap = T, isPairedEnd = F, 
nthreads = 2, strandSpecific = 0, isGTFAnnotationFile = T)

I ran the same command except I set isGTFAnnotationFile = F because I couldn't find the gtf file, so I used the gff file TAIR10_GFF3_genes_transposons.gff instead, downloaded from here (they mentioned that using gtf only speeds up the process but that you can still do this using gff). I'm not sure if this is what is causing the issue, but the error I get is:

Error: Line 1 contains a format error. The expected annotation format is SAF.
Error in featureCounts(sam.list, annot.ext = "TAIR10_GFF3_genes_transposons.gff",  : 
  No counts were generated.
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    – M__
    Sep 13, 2023 at 16:28

1 Answer 1


Answer from @an-ignorant-wanderer, converted from comment:

I ended up using the annotation file from https://support.illumina.com/sequencing/sequencing_software/igenome.html and it worked.

[Please update/improve this answer if possible]


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