Hello! I am trying to optimize my data processing based on the addition of ExFold ERCC controls. Ideally I would like to normalize with VSN using the procedure in chapter 7 of the vsn vignette. If I pull out the unprocessed intensities for the ERCC controls, order them by increasing concentration, and transform the concentrations and intensities by log base 2. I get a sigmoid curve which I can fit. Should I only use the probes which show a linear relationship as the spike-ins with lts.quantile=1? Should I order the "spikeins" by increasing concentration before passing to vsn2? I think that a significant source of error are intensities set as constant outside the dynamic range of the array. For example the intensity of the lowest 5 concentrations oscillate around log2(intensity)=5. We see a linear range in log base 2 intensity from 6.0-6.5 to 12. above 12 I see saturation. Also if it is only important that the "spike-in" probes are of similar intensities ("not expected to be different") should I use the hybridization controls or poly-A controls that are above log2(intensity) of six. Any advice or assistance is greatly appreciated. Thanks, Matt matthew.thornton at med.usc.edu

Dear Matthew it makes sense to subset the spike-in controls used for normalisation with vsn2 to those that respond about linearly to changes in the target mRNA abundance. lts.quantile=1 is reasonable choice for such a situation. The ordering of the features makes no difference to vsn2. Kind regards Wolfgang On 24 Jun 2014, at 21:36, Thornton, Matthew <matthew.thornton at="" med.usc.edu=""> wrote: > Hello! > > I am trying to optimize my data processing based on the addition of ExFold ERCC controls. Ideally I would like to normalize with VSN using the procedure in chapter 7 of the vsn vignette. If I pull out the unprocessed intensities for the ERCC controls, order them by increasing concentration, and transform the concentrations and intensities by log base 2. I get a sigmoid curve which I can fit. Should I only use the probes which show a linear relationship as the spike-ins with lts.quantile=1? Should I order the "spikeins" by increasing concentration before passing to vsn2? I think that a significant source of error are intensities set as constant outside the dynamic range of the array. For example the intensity of the lowest 5 concentrations oscillate around log2(intensity)=5. We see a linear range in log base 2 intensity from 6.0-6.5 to 12. above 12 I see saturation. Also if it is only important that the "spike-in" probes are of similar intensities ("not expected to be diffe! > rent") should I use the hybridization controls or poly-A controls that are above log2(intensity) of six. Any advice or assistance is greatly appreciated. > > Thanks, > > Matt > > > > matthew.thornton at med.usc.edu > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor