How do I get ribosomal RNA counts from featureCounts of my bam files?

Question: How do I get ribosomal RNA counts from featureCounts of my bam files?

7 months ago by

Qiagen Sciences - Frederick Maryland

daniel.kim • 30 wrote:

Hi,

I used HISAT2 to align more RNAseq fastq files and then featureCounts to count my features. All my mRNA counts look as expected but I want to count rRNA as well, however, it is saying 0 counts for rRNA even though I did not do rRNA depletion. I really want to know my rRNA counts. I used hg19 as my reference genome. Is there something I can do so that featureCounts will count rRNA?

Thanks

annotation rrna featurecounts de reference • 456 views

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modified 7 months ago by Jennifer Hillman Jackson ♦ 25k • written 7 months ago by daniel.kim • 30

7 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Please see the additional settings under Advanced Options on the tool form.

The feature type and attribute need to be a match for the input reference annotation data you want counts for.

Thanks! Jen, Galaxy team

ADD COMMENT • link written 7 months ago by Jennifer Hillman Jackson ♦ 25k

Update: Sorry, I think I misinterpreted your question earlier. You were using the built-in hg19 annotation, not one you provided from the history, correct?

The built-in annotation may or may not include rRNA features. Including reference annotation with tools is somewhat new and all the details are not worked out entirely. The annotation is a very good choice for gene/transcript/exon counts yet I don't know yet if it is appropriate for rRNA counts. I asked for more information from the tool author if you'd like to follow the feedback: https://gitter.im/galaxy-iuc/iuc?at=5aea702553ceca3604a9a04d

Should you want to move forward now (there is no guarantee the built-in annotation will meet your needs, eg: include rRNA features), locate an hg19 GTF reference annotation from a public source that includes rRNA annotation, upload that to Galaxy, use that from the history with the tool, and set the tool form parameters to capture those features/attributes.

ADD REPLY • link written 7 months ago by Jennifer Hillman Jackson ♦ 25k

Thank you Jennifer for the very helpful info and for replying back to me. I was able to use featureCounts to get my rRNA counts by getting the rRNA.gtf file from UCSC table browser and then adding that gtf file into the tool parameters. Galaxy is great and the community/forum is great! Thanks!

Daniel Kim

ADD REPLY • link written 7 months ago by daniel.kim • 30

Glad that worked! The tool author got back to us last night about the built-in reference data. It probably won't suit your needs (the reference annotation specifically for rRNA is better), but for others reading, this is the new Gitter thread: https://gitter.im/galaxy-iuc/iuc?at=5afbafcb5a1d895fae42cd8d

For anyone having trouble with the tool recognizing BAM/GFT inputs, please know that the Featurecounts tool now requires that the database metadata assignment is made to both the BAM and GTF inputs (when sourced from the history).

Please see this post for full details: https://biostar.usegalaxy.org/p/24154/#28027

ADD REPLY • link written 6 months ago by Jennifer Hillman Jackson ♦ 25k

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