Adjusting featureCounts settings in Galaxy for miRNA overlaps
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tjgray4 • 0
@c365719b
Last seen 10 months ago
United States

When featureCounts is used to count miRNAs present in genome aligned bam files, it gives miRNA size outputs, as well as long fragments (ie 8kb). I think this is happening because overlapping miRNAs exist, and thus featureCounts cannot distinguish where an individual miRNAs starts and ends in this situation (when there is an overlap of miRNAs in the same region, as confirmed by IGV visualization). In our protocol, we align the miRNAseq trimmed data to the genome, and we then use the same genome and its gtf format for miRNA counts. We do not use known miRNAs because we are trying to find novel miRNAs. We cannot use mirdeep2 for novel miRNAs because there are very few known miRNAs (and for some genomes there are 0 known miRNAs).

Thus I have the following questions: a. How can we change featureCounts settings to make this tool count individual miRNAs, rather than a cluster of overlapping miRNAs and label them as one fragment? b. If this is not possible, is there another tool that can be used in order to quantitate and discover all miRNAs (including novel miRNAs) from our genome aligned bam files? c. is it possible to tweak featureCounts to achieve this goal?

featureCounts Rsubread • 878 views
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Note to other Bioc Support moderators: although this question relates to Galaxy rather than to Bioconductor software, the Subread-featureCounts authors have asked users to send questions to this forum (https://subread.sourceforge.net/), so I think it's a legitimate question here.

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I added a featureCounts tag and removed metaMSdata as this question does not relate to the metaMS package.

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Thank you for doing this. I could not add the featureCounts tab or any other related tab myself. The metaMSdata tab was one of the only tabs I could originally apply for some reason.

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@gordon-smyth
Last seen 41 minutes ago
WEHI, Melbourne, Australia

You seem be making some incorrect assumptions about what featureCounts does.

featureCounts simply assigns sequence reads to genes or exons defined in the GTF file, no more and no less. It treats each miRNA individually. It does not merge or cluster overlapping miRNAs and it does not do any relabelling. If overlapping miRNAs have been merged into one, then that must have been done when the GTF file was constructed. It was not done by featureCounts.

featureCounts does not return fragment sizes. It simply returns the total genomic length of exons in each feature that reads are assigned to.

I don't understand your remarks about known vs novel miRNAs. It is usual to include all potentially expressed features in the GTF file. featureCounts only uses those features that are in the GTF. It cannot discover novel miRNAs if they are not in the GTF file.

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