FeatureCounts No Features

I am running DESeq2 on different samples but with basically the same input. Everything works fine except for one sample.
Here, I have a good % assigned in RNAStar, but when I try to run featureCounts, I only get around 1 % assigned, most are Unassigned_NoFeatures.
I don’t really understand why that is the case, as I did exactly the same for the other samples and they work fine. I am also not sure what this means exactly, as I am new to Galaxy.
I use the mm10 reference genome (in gtf format).
If someone has any suggestions, I would be very greatful.
I apologize if this is repetitive, but I did not find a post with exactly the same problem.
Thank you :slight_smile:

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Hi @ko1013

Double-check the reference genome that you mapped against. Was it really mm10?

More help here:

Hi @jennaj,
Thank you for your reply.
Yes, I triple checked and it’s mm10, I used this exact same file for my other samples too and it worked fine.
I checked all the points of the list you send me, the input should be correct as it is.

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The problem then may be with the parameters used. Or, there is a content problem with that particular sample.

This GTN tutorial covers upstream QA/QC plus featureCounts usage:

The solution can vary for any unexpected results, but the resource links and discussion from prior Q&A may help.

  • Prior Q&A at this site can be found by clicking on the “featurecounts” tag added to this topic.

  • A web search also locates much prior Q&A, and what people needed to do to resolve the issue. Most of the options available for this tool line-command are also available in the Galaxy wrapped version of the tool.