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

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