What's going on with my sleuth output?

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Hello all,

I am using the Kallisto/Sleuth workflow to analyze some data following this tutorial (pachterlab.github.io/sleuth_walkthroughs/trapnell/analysis.html). I was able to successfully complete the tutorial and obtain outputs from Sleuth which I then observed through the R shiny web app interface. For now I'm just analyzing 12 samples (comparing 6 samples with condition A to 6 samples with condition B).

However, some of the outputs from Sleuth look bizarre and not what I expected, namely the mean-variance plot and volcano plot (I've attached images to this post). I'm also noticing that the qvalues for large groups of transcripts are exactly the same which is creating the horizontal or cutoff line in the volcano plot where all of the transcripts are congregating. I asked around about this before and was told that it is probably a processing or normalization error. How can I check for this? Is it something to do with the input data from Kallisto or is there something I can tweak in the Sleuth R script?

I'm pretty new to all this - I'm learning as I go so any feedback is much appreciated! Thanks in advance.

Volcano plot and Mean variance plot


RNA-seq


Sleuth

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