Dear Wolfgang, Thanks for positive reply. I am sceptic about the trend of MA plot, namely a slope to the left. Normalization to many spike-ins is still tricky, and from my experience it was really hard to simply get one size factor. BTW, is it better to estimate dispersion in paires (treatment1 / control; treatment2 / control, ...) or rather for a whole dataset in case of 1 control / treatment 1-6 (each sample in 2 biol rep). Best, Micheal On Sun, Oct 7, 2012 at 3:31 PM, Wolfgang Huber <whuber@embl.de> wrote: > 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@gmail.com> > 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]] > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@r-project.org > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > > [[alternative HTML version deleted]]