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Hi James, James Anderson wrote: > Hi, > > There are some commands in Affy package which can finish almost all the steps, however, I am trying to understand every single details of RMA and MAS5, > for RMA, I guess it has the following steps: > 1. Background subtraction > 2. quantile normalization > 3. median polish to convert probe level to probe set level. > > What's the command for doing median polish to convert probe level to probe set level? In addition, since there is memory problem, justRMA can solve the problem in probe set level, is there any similar method that can solve the problem in probe level, something like justRMAprobe? A justRMAprobe wouldn't make any sense, since the background and normalization steps are carried out on all the data from each chip. The median polish is done by C code, so there isn't a command per se. However, the implementation is identical to the R function medpolish(), so if you had a matrix of background corrected, normalized PM probes from one probeset you could do medpolish(probematrix) and you would get the same results. > > Regarding MAS5, I guess it also has background subtraction and normalization, which normalize method does MAS5 use? I typed > > >>data(affybatch.example) >>normalize.methods(affybatch.example) > > [1] "constant" "contrasts" "invariantset" "loess" > [5] "qspline" "quantiles" "quantiles.robust" > > Does it use "invariantset" or something else? Something else. MAS5 does the normalization _after_ computing expression values, so it uses affy.scalevalue.exprSet(). > In addition, I think it's the same as RMA in the sense that after background subtraction and normalization, probe level is obtained, then what's the command to convert the probe level to probe set level in MAS5? Again, how to deal with the memory problem? > No it is not the same. The background subtraction is based on subtracting IM values (not the same as MM values, see the affy documentation if interested), and then doing a Tukey bi-weight on the resulting background subtracted values. After which the data are normalized using the above mentioned function. There is no need for a memory-bounded algorithm for MAS5 because the expression value calculation is done on a chip by chip basis. You could simply do the first step of the mas5() function on reasonably sized subsets of your data, and after all have been processed, you could then combine the data and run affy.scalevalue.exprSet(). The combination step might be too memory intensive, so it might be necessary to hack affy.scalevalue.exprSet() to use a matrix instead, but it would be simple to do. Best, Jim > Thank you very much! Your reply will be highly appreciated! > > James > > > > --------------------------------- > > > [[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 -- James W. MacDonald, M.S. Biostatistician Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues.

On Wed, May 02, 2007 at 07:27:21AM -0700, James Anderson wrote: <james>Hi, <james> <james>There are some commands in Affy package which can finish almost all the steps, however, I am trying to understand every single details of RMA and MAS5, <james>for RMA, I guess it has the following steps: <james>1. Background subtraction <james>2. quantile normalization <james>3. median polish to convert probe level to probe set level. <james> <james>What's the command for doing median polish to convert probe level to probe set level? In addition, since there is memory problem, justRMA can solve the problem in probe set level, is there any similar method that can solve the problem in probe level, something like justRMAprobe? As far as I know the function rma() uses some C code for summarization (and normalization). Nevertheless you can have a look at ?medianpolish if you are interested in it <james>Regarding MAS5, I guess it also has background subtraction and normalization, which normalize method does MAS5 use? I typed <james> <james>> data(affybatch.example) <james>> normalize.methods(affybatch.example) <james>[1] "constant" "contrasts" "invariantset" "loess" <james>[5] "qspline" "quantiles" "quantiles.robust" <james> <james>Does it use "invariantset" or something else? <james>In addition, I think it's the same as RMA in the sense that after background subtraction and normalization, probe level is obtained, then what's the command to convert the probe level to probe set level in MAS5? Again, how to deal with the memory problem? For MAS5 expression value, background correction and summarization are performed *before* normalization. Normalization is perfomed by the function affy.scalevalue.exprSet, while summarization is ultimately done by generateExprVal.method.mas again, type mas5 affy.scalevalue.exprSet generateExprVa.method.mas to see what they actually do. HIH, Stefano <james> <james>Thank you very much! Your reply will be highly appreciated! <james> <james>James <james> <james> <james> <james>--------------------------------- <james> <james> <james> [[alternative HTML version deleted]] <james> <james>_______________________________________________ <james>Bioconductor mailing list <james>Bioconductor at stat.math.ethz.ch <james>https://stat.ethz.ch/mailman/listinfo/bioconductor <james>Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor