Dear all,
I am trying to find differentially expressed (DE) genes using EdgeR in a complicated experimental setting. There are two factors (each has different level of variation among replicates - the reason why the experimental setting is complicated to interpret) and each factor has a control and a treated group with two replicates for each.
I fit the GLM using a design matrix that describes my experimental setting. I get an unusual looking mean-variance plot (attached - mean-var-Plot.png). Here is my interpretation of the graph. The tagwise dispersion (light blue dots) is fit very well to the common dispersion (light blue line). However, since the raw variance (grey dots) is pretty far from the fitted tagwise dispersion, I am worried that the model might be overfitting my data and it could be bad for the analysis. Do you think this is the correct interpretation of this plot? I haven't seen this type of mean-variance plot before and therefore would like to know how would you interpret it.
Note: When I use each factor alone in the design matrix, the tagwise dispersion sits in the middle of the raw variance dots, which is how the mean-variance graphs typically look (attached -fac1.png, fac2.png).
Also, as I mentioned earlier that the level of variance (or dispersion) is different between my two factors (as the RNA levels were measured using different sequencing assays for both factors), I am wondering if EdgeR is the right method for these data, since EdgeR doesn't calculate the covariances separately for different factors.
I would highly appreciate any insights. Thanks in advance for your help.
For reference, this has been cross-posted to another site: http://seqanswers.com/forums/showthread.php?t=76023
I decided to post it on Biostars since Seqanswers didn't give me any confirmation message upon submission or didn't show my post up there. But thanks for the comment. I will keep this in mind for future.
Could you double check the pictures. The links point to one pic
See SeqAnswer's thread for images while OP fixes the links in this post.
Not sure why you don't see all three pics on that link. I do see all of them. Could you please try checking the pictures at http://seqanswers.com/forums/showthread.php?t=76023 as Genomax2 suggested! Thanks.
@ravi: If you edit the original post here you will see that both links are pointing to the same image for some reason.
@Genomax2, yes both of those links are pointing to an Imgur post where all three pictures are posted. I just noticed though that Biostars has a way of posting pictures directly. I am trying that to see if that fixes the confusion. Thanks.
P.S. I learned a way of posting pictures in Biostars using Imgur from one of the posts on Biostars.
UPDATE: Fixed the picture links. Thanks!