<p><em>Answer from @an-ignorant-wanderer, converted from <a href="https://bioinformatics.stackexchange.com/help/privileges/comment">comment</a>:</em></p>
<p>I ended up using the annotation file from <a href="https://support.illumina.com/sequencing/sequencing_software/igenome.html" rel="nofollow noreferrer">https://support.illumina.com/sequencing/sequencing_software/igenome.html</a> and it worked.</p>
<p><em>[Please update/improve this answer if possible]</em></p>

CC BY-SA 4.0
2022-09-02T11:13:03.143Z
2022-09-02T11:13:03.143Z
gringer
<p>I am following the tutorial <a href="https://pubmed.ncbi.nlm.nih.gov/29525965/" rel="nofollow noreferrer">here</a> 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, &quot;Assignment of RNA-Seq Reads to Genes.&quot; Here, the authors run the command</p>
<pre><code>fc0 &lt;- featureCounts(sam.list, annot.ext = 
&quot;Ara-port11_GFF3_genes_transposons.201606.gtf&quot;, allowMultiOverlap = T, isPairedEnd = F, 
nthreads = 2, strandSpecific = 0, isGTFAnnotationFile = T)
</code></pre>
<p>I ran the same command except I set isGTFAnnotationFile = F because I couldn't find the gtf file, so I used the gff file <code>TAIR10_GFF3_genes_transposons.gff</code> instead, downloaded from <a href="https://www.arabidopsis.org/download/index-auto.jsp?dir=%2Fdownload_files%2FGenes%2FTAIR10_genome_release%2FTAIR10_gff3" rel="nofollow noreferrer">here</a> (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:</p>
<pre><code>Error: Line 1 contains a format error. The expected annotation format is SAF.
Error in featureCounts(sam.list, annot.ext = &quot;TAIR10_GFF3_genes_transposons.gff&quot;,  : 
  No counts were generated.
</code></pre>

CC BY-SA 4.0
2023-09-13T16:28:46.783Z
Thanks for answering the question @AnIgnorantWanderer please consider "Accepting" your answer below because it helps with site statistics.
M__
<r><rna-seq>
Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data
# Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data

(Asked by: username_0 on 2020-06-05)

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.

**Tags:** r, rna-seq
> **Comments:**
> **username_1**: Thanks for answering the question @AnIgnorantWanderer please consider "Accepting" your answer below because it helps with site statistics.  

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## 1 answers

### Answer by username_2 on 2022-09-02. Score: 1 | Accepted answer

*Answer from @an-ignorant-wanderer, converted from [comment](https://bioinformatics.stackexchange.com/help/privileges/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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