featureCounts parameters for RNA-Seq
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3.0 years ago

Dear Community,

I have been running featureCounts to count mapped reads in RNA-Seq paired-end data with the following parameters

featureCounts -T 10  -t exon -s 2 -p -g gene_id -a <path_to_annotation.gtf> -o <path_to_outoput_directory> mapped.bam


as mentioned in http://bioinf.wehi.edu.au/featureCounts/

However, I came across two posts where the parameters

and

have been used.

This got me a little confused and will like to to know if and when I should use these parameters as well as what will be the difference in the output from the default parameters?

Many thanks in advance

featurecounts RNA-Seq Subread ReadCounts • 3.8k views
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Hey do you get some answers for this? I am also using featurecounts now, paired ends RNAseq, and I am not sure if i have to use specify strandness? Could you please help me ?

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If your sequencing is stranded then you need to specify to featureCounts which strand has been sequenced. If your sequencing is not stranded then you don't. Pretty logical!

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Thanks for your answer. May I ask do you reccomand --primary to be set on?

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Generally, we recommend that featureCounts be run with default settings, which does not include --primary. Just follow the documentation. The specific use depends on your intended application.

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Thanks so much Gordon Smyth. In default setting -t exon is set on. So when is -t gene reccomanded? In my bacteria gff file, I do not see exon, but in my fungi gff file, I do. I wonder can I just set -t gene for both bacteria and fungi? I really appreciate your help. Best regards, Yanfang

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16 months ago

Honestly, if you aren't using something like Kallisto, which outputs counts more or less natively, I'd be using RSEM, not featureCounts RSEM is smarter about dealing with ambiguously assigned reads.