Wolfgang Huber | 7 Oct 15:31 2012
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Re: DESeq diagnostics

Dear Michael

the plots look fine, as far as it is possible to tell. What exactly are you worried about?

Normalising to the majority, as DESeq does, works well for experiments where the expression changes are
sparse, or symmetric. If these conditions are not met, spike-ins offer an alternative, but they can be
fiddly experimentally, and if there are only few, accuracy can be problematic. These problems are well
known, there is no simple cure, you just have to deal with them.

	Best wishes
	Wolfgang

Il giorno Oct 7, 2012, alle ore 1:28 AM, Michael
<mllmmllmmllmm@...> ha scritto:

> Hi,
> 
> 
> I'll try to ask once again :)
> 
> 
> My experiment is RNAseq of cells depletions from RNA decay factors, so one
> should expect to see more upregulations. The libraries are ribodepleted,
> paired end and stranded. After mapping with tophat I use HTSeq/DESeq combo
> to discover DE genes (among tophat genes.gtf, rRNA not included)
> 
> 
> I have problem with MA plots which are skewed (example attached), there is
> a clear slope suggesting that more upregulation of genes of lower
> expression. What should think about this?
> 
> Diagnostic scatter plots of log ratio also look weird (second one is match
> MA plot); PCA is ok, heat maps too.
> 
> I also tried to compare DESeq normalization with normalization to spike-ins
> present in the libraries, but the size factors assigned by DESeq seems
> much more accurate; although it's unclear why ?
> 
> 
> https://www.dropbox.com/s/0oykefjy1fvtq1i/1.pdf (MA plot)
> 
> https://www.dropbox.com/s/r95oydftyeoz9mu/scatter.pdf (scatter plots,
> second it for the MA plot)
> 
> 
> 
> Cheers,
> Michael
> 
> 	[[alternative HTML version deleted]]
> 
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