> Thanks Wolfgang for your prompt reply and clarifications! > > Still one question, though, for you or Gordon: with respect to the base > of vsn2 and limma: > To be sure, since I cannot deduce myselves from limma's code; is it only > the statement that needs to be updated, or do you mean that the values > returned by vsn2 are still converted by limma (while there is no reason > to do so anymore)? > Dear Guido Only the man page needs to be updated. The code is correct. Best wishes Wolfgang > Thanks, > Guido > >> -----Original Message----- >> From: bioconductor-bounces at stat.math.ethz.ch >> [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of >> Wolfgang Huber >> Sent: 02 September 2009 22:09 >> To: Hooiveld, Guido >> Cc: Bioconductor >> Subject: Re: [BioC] limma's implementation of VSN >> >> >> Dear Guido >> >> You raise several interesting points. >> >> * 'subtract' is spelled without an s between the b and the t >> in the arguments to vsn2. >> >> * the 'normalizeBetweenArrays' function calls the 'vsnMatrix' >> function directly, which does not have the >> 'backgroundsubtract' argument. That argument only exists for >> the 'NChannelSet' method of 'vsn2'. A workaround for you >> would be something like: >> >> RG <- read.maimages(targets$FileName, source="agilent") >> RG$R = RG$R-RG$Rb; RG$G = RG$G-RG$Gb; >> MA.vsn <- normalizeBetweenArrays(RG, method="vsn") >> >> * the statement in the normalizeBetweenArrays man page >> regarding the base used for the log function (2 or e) is >> outdated. In vsn2 (which is now used here), the base is 2. >> I'll ask Gordon to fix this. Although e is a much cooler >> number than 2, this fact never seems have to caught on with >> non-mathematicians, and I bowed in. >> >> Best wishes >> Wolfgang >> >> >> >> Hooiveld, Guido wrote: >>> Dear list, >>> >>> I am analyzing my first 2-color array experiment (Agilent arrays). >>> After reading the manual of LIMMA and the book BioC case studies, I >>> decided to use LIMMA applying VSN normalization. However, >> to me it is >>> not completely clear which arguments are passed to VSN when >> executed >>> from LIMMA. >>> >>> >From ?normalizeBetweenArrays i learn: >>> "method="vsn" uses the vsn function from the vsn package. For this >>> option the input object should contain raw intensities, >> i.e., prior to >>> background correction, log-transformation or any >> normalization. Note >>> that the normalized intensities are on the log-2 scale, not >> the log-e >>> scale output by the vsn function in the vsn package." >>> >>> The code I ran: >>> targets <- readTargets("targets.txt", row.names="Name") RG <- >>> read.maimages(targets$FileName, source="agilent") MA.vsn <- >>> normalizeBetweenArrays(RG, method="vsn") >>> >>> >>> Questions: >>> - in the 3rd line code above, is VSN run with the argument >>> 'backgroundsubstract=TRUE'? I am asking because by default VSN does >>> NOT background subtraction, and when I explicitly state to subtract >>> background an error occurs: >>>> MA.vsn <- normalizeBetweenArrays(RG, method="vsn", >>> backgroundsubstract=TRUE) >>> Error in vsnMatrix(x = y, ...) : >>> unused argument(s) (backgroundsubstract = TRUE) Error in >>> exprs(vsnMatrix(x = y, ...)) : >>> error in evaluating the argument 'object' in selecting a >> method for >>> function 'exprs' >>> >>> - Regarding this statement from ?normalizeBetweenArrays ("Note that >>> the normalized intensities are on the log-2 scale, not the >> log-e scale >>> output by the vsn function in the vsn package"): does this >> mean that >>> the raw array data is processed fully as per VSN >> methodology, and that >>> only in the end it is converted into log2 scale? Thus that the raw >>> intensities present in RG are glog-transformed, which are then used >>> for background correction (?; see above) and variance stabilization >>> normalization, and finally the VSN intensities are >> converted from glog >>> to log2 scale? Or does this mean the VSN methodology is >> used on log2, >>> and NOT glog transformed data? I assume the former, but plz >> correct me >>> if I am wrong. >>> >>> TIA, >>> Guido >>> >>> >>> ------------------------------------------------ >>> Guido Hooiveld, PhD >>> Nutrition, Metabolism & Genomics Group Division of Human Nutrition >>> Wageningen University Biotechnion, Bomenweg 2 >>> NL-6703 HD Wageningen >>> the Netherlands >>> tel: (+)31 317 485788 >>> fax: (+)31 317 483342 >>> internet: http://nutrigene.4t.com <http: nutrigene.4t.com=""/> >>> email: guido.hooiveld at wur.nl >>> >>> >>> [[alternative HTML version deleted]] >>> >>> _______________________________________________ >>> 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 >> -- >> >> Best wishes >> Wolfgang >> >> ------------------------------------------------------- >> Wolfgang Huber >> EMBL >> http://www.embl.de/research/units/genome_biology/huber >>