# Rat Featurecount Selection **URL:** \ **Category:** Uncategorized\ **Tags:** troubleshooting, reference-annotation, featurecounts\ **Created:** [January 28, 2026, 12:14am UTC](https://help.galaxyproject.org/t/rat-featurecount-selection/17377 "2026-01-28T00:14:34Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![Tapan\_Dey](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/tapan_dey/32/8339_2.png) [@Tapan\_Dey](https://help.galaxyproject.org/u/Tapan_Dey)\ **Post date:** [January 28, 2026, 12:14am UTC](https://help.galaxyproject.org/t/rat-featurecount-selection/17377/1 "2026-01-28T00:14:34Z")
I aligned rat RNA-seq data using **HISAT2 with the Galaxy built-in rn6 (Jul 2014 / RGSC6) genome**. Since **featureCounts does not allow selecting a genome assembly** , which **GTF annotation** should be used for gene counting to ensure compatibility with the rn6 alignment? Tapan ---
**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:** [January 28, 2026, 2:46am UTC](https://help.galaxyproject.org/t/rat-featurecount-selection/17377/2 "2026-01-28T02:46:58Z")
Welcome @Tapan_Dey Hopefully we can help! You are correct that everyone will need to supply reference annotation to tools, including **Featurecounts**! You will want to obtain annotation that is based on the **same reference genome assembly** coordinate and labeling system. The **rn6** genome was sourced from UCSC. They also host annotation. To learn where to source UCSC annotation files in their Downloads area, this FAQ can help. - [FAQ: Working with GFF GFT GTF2 GFF3 reference annotation](https://galaxyproject.github.io/training-material/faqs/galaxy/datasets_working_with_reference_annotation.html) **Steps** 1. Start at the **UCSC Genome Browser** website [https://genome.ucsc.edu/](https://genome.ucsc.edu/). Navigate to their Downloads area, then choose Other Mammals → Rat. The URL of your browser window should be this once you are in the right place. 2. Then go into the assembly you already mapped against (rn6), and navigate down into the `/genes` directory. The URL should now be this. 3. Any of the choices are usually fine but one may be a better choice for your goals or downstream analysis plans. Each of the files represents a **Gene and Gene Prediction** annotation track in the main **Genome Browser at UCSC** → [https://genome.ucsc.edu/](https://genome.ucsc.edu/). You can inspect these tracks in the main graphical browser or their **Table Browser** to learn what each represents and how/when/who curated the data. 4. To get the data into Galaxy: copy the URL for the file, paste it into the Upload tool in Galaxy, [leave all settings at default](https://help.galaxyproject.org/t/getting-data-into-galaxy/10868), and the file will uncompress with the correct datatype, ready to use! 5. On a tool form, choose your newly uploaded **GTF** dataset from your history. Please give that a try and let us know if it works or not for you! 🙂 * * * Notes: There are a few ways to label the genome and features, and using the same choice throughout an analysis tends to work best. So, later on, should you notice that counts are not quite what you expected, or an error comes up about “mismatched reference data”, you can inspect your reference data closer and usually find the problem. If you get stuck, you can share the error and the community here can try to help more! Related topics that can help with additional context about #reference-annotation - [FAQ: Extended Help for Differential Expression Analysis Tools](https://galaxyproject.github.io/training-material/faqs/galaxy/analysis_differential_expression_help.html) - [Help with RNA Star, Featurecounts, and reference annotation choices - #2 by jennaj](https://help.galaxyproject.org/t/help-with-rna-star-featurecounts-and-reference-annotation-choices/16190/2) - [Ensembl gene annotation gtf for rat problem with RNA STAR - #2 by jennaj](https://help.galaxyproject.org/t/ensembl-gene-annotation-gtf-for-rat-problem-with-rna-star/14818/2) - [How to find the reference transcriptome for analysis tools: GRCh38/hg38 example - #2 by jennaj](https://help.galaxyproject.org/t/how-to-find-the-reference-transcriptome-for-analysis-tools-grch38-hg38-example/12506/2) - [Help in How to generate a GFF file](https://help.galaxyproject.org/t/help-in-how-to-generate-a-gff-file/9718) ---
**Author:** ![Tapan\_Dey](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/tapan_dey/32/8339_2.png) [@Tapan\_Dey](https://help.galaxyproject.org/u/Tapan_Dey)\ **Post date:** [January 28, 2026, 6:45pm UTC](https://help.galaxyproject.org/t/rat-featurecount-selection/17377/3 "2026-01-28T18:45:37Z")
**@[jennaj](https://help.galaxyproject.org/u/jennaj)** BINGO! It worked. Thank you so much for your kind help!