Featurecounts on Galaxy main

Question: Featurecounts on Galaxy main

5 months ago by

p.barnett • 50 wrote:

I have a general question/issue I wonder if anyone knows a solution to. Firstly, as I said in a previous message, i was pleased with the integration of built-in annotation files (mm10, Hg19 etc) in featureCounts on Galaxy main. I am now analyzing Rat genome data (rn6 build). The data looks good on conversion to BigWig and corresponds (on the whole) well with the FeatureCounts output. However, I did notice a couple of well annotated mRNAs that although giving clear signals looking at the BigWig genome alignments, fail to give representative counts under featurecounts. The annotation and exon locations etc. look fine in the GTF file I use for the analysis and as I said the BigWig clearly shows high signal counts for the exons, so the mapping is ok. Other genes, upon random selection, check out fine, so like I say its just the odd gene every now and again that seems to get miss-counted. I probably wouldn't have even noticed if it wasn't for the fact that one of the genes I checked was a key gene for our research. Ideas ?

Phil

rna-seq featurecounts annotation counts • 298 views

ADD COMMENT • link •

modified 5 months ago • written 5 months ago by p.barnett • 50

5 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Are you are certain now that the reference genome and reference annotation chromosome identifiers are an exact match? This is the type of incomplete result that can occur if one or more chroms are not a match - the mapped reads associated don't get counted (are ignored).

Another common item that comes up for missing counts is that the strand settings used for mapping are not the same as used with Featurecounts. So, double check those. Strand in the Galaxy wrapped version of Featurecounts is under Advanced Options.

If those check out, then review the results in the output "summary". It lists out why reads were not counted and how many were involved. There are many ways to tune settings under Advanced options & Options for pair-end reads. Read quality/content can be a factor. Docs and help for what these parameters do is covered here: http://subread.sourceforge.net/

If you do think there is a true bug after checking/testing the above, a Github issue against the tool's repository can be opened explaining the problem with a clear example provided for the developers to troubleshoot with. This is how: https://galaxyproject.org/issues/#bug-reporting and the dev repository for Featurecounts is linked from here: https://toolshed.g2.bx.psu.edu/view/iuc/featurecounts/85aaf50ad9dc

Thanks! Jen, Galaxy team

ADD COMMENT • link written 5 months ago by Jennifer Hillman Jackson ♦ 25k

5 months ago by

p.barnett • 50

p.barnett • 50 wrote:

Hi Jen,

Thanks for your help, again. After checking the output again I spotted (though why I didn't earlier) that those lacking counts had double registries in the GTF. So when I permitted multiple feature assignment of the same read.... hey presto!

Phil

ADD COMMENT • link written 5 months ago by p.barnett • 50

Please log in to add an answer.

Similar posts • Search »