How to run miRNA analysis to identify miRNAs and for differential expression?

Currently using this tutorial as I am working with Arabidopsis Thaliana. I would assume the uploaded files of mature_miRNA , star_miRNA , and miRNA_stem-loop are all specific to A.Thaliana so no change is needed there. I ran my uploaded original sequencing up to the mapping and quantification using the tutorial’s reference datasets. However, how could I proceed with differential expression when I don’t have a control sequence to use?

Hi @alio

Yes, the reference data is in full as far as I know.

The DE analysis could compare the conditions you did generate. Two or more conditions, each with two or more replicates, are the minimum technical requirements. Three replicates per condition is a common minimum scientific requirement.

Thank you for the response @jennaj

The problem is I’m not sure what conditions were set when the sequencing of our samples was done. What I’ve tried so far is taking reference transcript FASTAs of A.Thaliana from databases, converting them into FastQ using Combine FASTA and QUAL, and proceeding with the workflow for it to serve as a control in DeSeq. However, when doing this the error attached appears when I try to run MiRDeep2 Quantifier on the collapsed reads of my “control” using the workflow’s attached reference data. I’m not sure if this is the right approach and how to move forward.

If this is true, then the experiment won’t have any meaningful scientific results to interpret.

If you are instead running the analysis just to learn how to use these tools, please consider using the training data instead.

Finished transcripts are not an appropriate substitute for actual controls matched with your other existing samples.

I’m sorry, but I don’t think we can help more here. This kind of input would fail the tool or produce unusable results even if not run inside Galaxy.