One thing to consider when doing clever things with expresso is validating your approach. I have found that using the same data, different normalizations can come up with completely different lists of significantly differentially expressing genes. If you want to share your results with others (for example by publishing) then you need to justify your normalization method by e.g. running experiments with known outcomes such as spike-in genes, or extensive follow-up on another platform such as RT-PCR. The pre-packaged methods have their problems, but at least these problems are well-understood. --Naomi At 05:25 PM 1/21/2009, Michal Blazejczyk wrote: >Hi James, > >What I was wandering about more specifically was: if I run expresso >with a different summary.method parameter (say, medianpolish for RMA), >but select mas as the PM correction method or the background correction >method, should I be worrying about the additional normalization as well? >Or is it only applicable if I use summary.method=mas regardless of my >pmcorrect.method and bgcorrect.method? > >Best regards, > >Michal Blazejczyk >FlexArray Lead Developer >McGill University and Genome Quebec Innovation Centre >http://genomequebec.mcgill.ca/FlexArray > > >James W. MacDonald <jmacdon at="" med.umich.edu=""> wrote: > > Hi Michal, > > > Technically, the mas5 method is implemented by the function mas5(), > > so you would certainly need to normalize after that step. Note that > > mas5() is just a wrapper to expresso: > > > res <- expresso(object, bgcorrect.method = "mas", pmcorrect.method = > > "mas", normalize = FALSE, summary.method = "mas", ...) > > > As for expresso(), this is a bit of a 'roll your own' function, so > > it is incumbent upon the user to figure out whether or not the data > > need to be normalized, which would be dependent on the arguments > > 'normalize' and 'normalize.method'. > > > Best, > > > Jim > > > > > Michal Blazejczyk wrote: > >> Hello group, > >> > >> According to the Bioconductor Help for function expresso(): > >> > >> "For the mas 5.0 and 4.0 methods ones need to normalize after > >> obtaining expression. The function affy.scalevalue.exprSet > >> does this." > >> > >> What does it really mean, "for the mas 5.0 method" in this case? > >> When calling expresso, "mas" can be set as a method for background > >> correction, PM correction, and probe summarization. In which of > >> these cases should the custom normalization (affy.scalevalue.exprSet) > >> be used? > >> > >> Best regards, > >> > >> Michal Blazejczyk > >> FlexArray Lead Developer > >> McGill University and Genome Quebec Innovation Centre > >> http://genomequebec.mcgill.ca/FlexArray > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111

Hi Michal, The only things in expresso() that control normalization are the 'normalize' and 'normalize.method' arguments. If you tell expresso() to normalize, then your data will be normalized. If not, then you will have to do something additional if you want normalized data. So it doesn't matter what you choose for PM adjustment or summary methods, as these have nothing to do with normalization. Best, Jim Michal Blazejczyk wrote: > Hi James, > > What I was wandering about more specifically was: if I run expresso > with a different summary.method parameter (say, medianpolish for RMA), > but select mas as the PM correction method or the background correction > method, should I be worrying about the additional normalization as well? > Or is it only applicable if I use summary.method=mas regardless of my > pmcorrect.method and bgcorrect.method? > > Best regards, > > Michal Blazejczyk > FlexArray Lead Developer > McGill University and Genome Quebec Innovation Centre > http://genomequebec.mcgill.ca/FlexArray > > > James W. MacDonald <jmacdon at="" med.umich.edu=""> wrote: >> Hi Michal, > >> Technically, the mas5 method is implemented by the function mas5(), >> so you would certainly need to normalize after that step. Note that >> mas5() is just a wrapper to expresso: > >> res <- expresso(object, bgcorrect.method = "mas", pmcorrect.method = >> "mas", normalize = FALSE, summary.method = "mas", ...) > >> As for expresso(), this is a bit of a 'roll your own' function, so >> it is incumbent upon the user to figure out whether or not the data >> need to be normalized, which would be dependent on the arguments >> 'normalize' and 'normalize.method'. > >> Best, > >> Jim > > > >> Michal Blazejczyk wrote: >>> Hello group, >>> >>> According to the Bioconductor Help for function expresso(): >>> >>> "For the mas 5.0 and 4.0 methods ones need to normalize after >>> obtaining expression. The function affy.scalevalue.exprSet >>> does this." >>> >>> What does it really mean, "for the mas 5.0 method" in this case? >>> When calling expresso, "mas" can be set as a method for background >>> correction, PM correction, and probe summarization. In which of >>> these cases should the custom normalization (affy.scalevalue.exprSet) >>> be used? >>> >>> Best regards, >>> >>> Michal Blazejczyk >>> FlexArray Lead Developer >>> McGill University and Genome Quebec Innovation Centre >>> http://genomequebec.mcgill.ca/FlexArray > > -- James W. MacDonald, M.S. Biostatistician Hildebrandt Lab 8220D MSRB III 1150 W. Medical Center Drive Ann Arbor MI 48109-0646 734-936-8662