# How can I improve very low assigned rate in featureCounts?

**URL:** <https://help.galaxyproject.org/t/how-can-i-improve-very-low-assigned-rate-in-featurecounts/758>\
**Category:** usegalaxy.org support\
**Created:** [March 7, 2019, 7:36pm UTC](https://help.galaxyproject.org/t/how-can-i-improve-very-low-assigned-rate-in-featurecounts/758 "2019-03-07T19:36:55Z")\
**Posts on this page:** 1\
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**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\
**Post date:** [March 7, 2019, 9:19pm UTC](https://help.galaxyproject.org/t/how-can-i-improve-very-low-assigned-rate-in-featurecounts/758/2 "2019-03-07T21:19:32Z")

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Hi,

> [@Satsea](#):
>
> HISAT2 (with default settings except for strandedness, R)

The strand for HISAT2 paired-end inputs should be `FR`, `RF`, or `Unstranded`, so this might be a typo and you meant `RF`?

`Featurecounts` also requires strandedness to match what was used for mapping. That is an `F`, `R`, or `Unstranded` toggle.

The data is failing for three primary reasons:

- **Unmapped** :

- **Mapping quality** :

- **NoFeatures** :

I would suggest comparing your methods to those in this Galaxy Training Network (GTN) tutorial. QA/QC, strandedness assessment, and usage for these tools are all covered.

- RNA-seq: [Discovering and quantifying new transcripts](https://galaxyproject.org/tutorials/nt_rnaseq) - an in-depth transcriptome analysis example.

Using the built-in annotation for mm10 is usually a very good choice for RNA-seq data. There are other sources but I don’t think the result will be much different if using a basic transcript reference annotation dataset from any source.

But if you want to try, Gencode and iGenomes are good alternative sources, with Gencode a bit simpler to get into Galaxy. This prior Q&A is about human (hg38), but both sources also have data for mouse (mm10): [RNA-STAR and hg38 GTF reference annotation - #2 by jennaj](https://help.galaxyproject.org/t/rna-star-and-hg38-gtf-reference-annotation/749/2)

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