Hi Viritha, On 8/31/2011 9:36 AM, viritha kaza wrote: > Hi James, > Thanks for your quick reply. > > So my last step should be > exprs<-eset-median+median1. > Is it? If your data are not log transformed, yes. This of course assumes that this is what the original authors meant by 'median normalization'. > > In the paper they say > "RNA fluorescent labeling reaction and hybridization were performed > using the Affymetrix Gene Chips HG-U133A and HG-U133B according to the > manufacturer?s instructions (http://www.affymetrix.com/). The arrays > consist of 22,283 (HG-U133A) and 22,645 (HG-U133B) probe sets, which > together amount to 23,583 unique genes based on Unigene build 173. > Microarray analysis was performed using Affymetrix Microarray Suite 5.0 > and in-house Visual Basic software MATRIX 1.26. Array data were median > normalized, and replicate genes were combined by averaging. Samples (or > averages of samples) were then compared against each other by > calculating log ratios for each gene, and statistical significance was > presented as a p value calculated by Student?s t test. The microarray > data have been uploaded to GEO (Gene Expression Omnibus), and the > accession number is GSE-4824." > > In GSE4824 in geo: > "Data processing:Data were analyzed with Microarray Suite version 5.0 > (MAS 5.0) using Affymetrix default analysis settings and global scaling > as normalization method. The trimmed mean target intensity of each array > was arbitrarily set to 250". > Could you please suggest whether my steps are correct : > Since there are 3 platforms namely GPL570 which contains 6 arrays which > are referenece profiles and other 2 i.e HG-U133A and HG-U133B contains > 79 samples. > Steps as suggested by paper: > 1)combine both platforms with mas5 intensity of both the platform for > the 79samples from series matrix file(unlogged) > * what about the common probes between HG U133A and HG U133B(168)? > 2)Add annotation with Unigene and then combine the reference profile > which is HGU133 plus2 > *Do I ignore those probes which are unique to HGU133plus2 (9921) and > probes of HGU133A and HGU133B(6) > 3)Then perform median normalization or median centering. > 4)Then averaging the replicate genes. > 5)Log ratios for each genes(fold change) > 6)then perform statistical student t-test. > * During which step do I convert the expression to log2 ? I would assume somewhere before step 5. But this is your project, so you have to make that decision for yourself. Best, Jim > wiating for your suggestions. > Thanks, > Viritha > On Mon, Aug 29, 2011 at 1:27 PM, James W. MacDonald > <jmacdon at="" med.umich.edu="" <mailto:jmacdon="" at="" med.umich.edu="">> wrote: > > Hi Viritha, > > > On 8/29/2011 12:10 PM, viritha kaza wrote: > > Hi group, > I am trying to replicate a dataset GSE4824 from a paper. > There are actually 3 platforms in them. But right now I am > concentrating > only on one platform GPL570.This contains 6 arrays. > I have written the code to perform Microarray Suite version 5.0 > (MAS 5.0) > using Affymetrix default analysis settings and global scaling as > normalization method. The trimmed mean target intensity of each > array was > arbitrarily set to 250.After which median normalization. > > > source("http://bioconductor.__org/biocLite.R > <http: bioconductor.org="" bioclite.r="">") > > > biocLite("affy") > > > library(affy) > > > mydata<- ReadAffy() > > > eset.mas5 = mas5(mydata,sc=250,normalize=__TRUE) > > > write.exprs(eset.mas5,"__GSE4824_GPL570.txt",sep='\t') > > > eset=exprs(eset.mas5) > > > median = apply(eset, 2, median) > > > median1=median(median) > exprs<-eset/median*median1 > > > The output from mas5() isn't log transformed, so you should be > subtracting and adding, not dividing and multiplying. > > This assumes that by 'median normalization' the original authors > simply meant median centering. > > Best, > > Jim > > > write.table(exprs,"GSE4824___GPL570_Median.txt",sep='\t') > > > Please let me know if the my code performs corectly the above task, > especially if last few steps would perform median normalization > correctly or > not? Also let me know if this is the right way to do median > normalization. > Thanks, > Viritha > > [[alternative HTML version deleted]] > > _________________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org <mailto:bioconductor at="" r-project.org=""> > https://stat.ethz.ch/mailman/__listinfo/bioconductor > <https: stat.ethz.ch="" mailman="" listinfo="" bioconductor=""> > Search the archives: > http://news.gmane.org/gmane.__science.biology.informatics.__conductor > <http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> > > > -- > James W. MacDonald, M.S. > Biostatistician > Douglas Lab > University of Michigan > Department of Human Genetics > 5912 Buhl > 1241 E. Catherine St. > Ann Arbor MI 48109-5618 > 734-615-7826 <tel:734-615-7826> > ******************************__**************************** > Electronic Mail is not secure, may not be read every day, and should > not be used for urgent or sensitive issues > > -- James W. 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