Hi Jim, Seth, Reddy, Paul, Thank you very much for your suggestion. I may be make cdf environment. Could you please help me how to confirm the env is OK or No? Next I tried cel file reading and normalize from our custom affy array. If my working flow are useful for affy beginner like me, could you please help me? At first, I typed below... >source("http://www.bioconductor.org/getBioC.R") >getBioC("all") >library(makecdfenv) >Library(affy) >make.cdf.package ("arabidopsistlgF.cdf") And move to Terminal on my Mac, >R CMD INSTALL arabidopsistlgFcdf Return to R, >arabidopsistlgF = make.cdf.env("arabidopsistlgF.cdf") And I shut down my Mac. Is these step correct for making cdf environment? And then I started again. > source("http://www.bioconductor.org/getBioC.R") > getBioC() > library(affy) >Data <- readAffy() >eset <- rma(data) I got Error below, *********** Note: You did not specify a download type. Using a default value of: Source This will be fine for almost all users Error in getCdfInfo(object) : Could not obtain CDF environment, problems encountered: Specified environment specified did not contain arabidopsis_tlgF_4x Library - package arabidopsistlgf4xcdf not installed Data for package affy did not contain arabidopsis_tlgF_4x Bioconductor - arabidopsistlgf4xcdf not available ********* Q1. I have question. Do I need typing below every time after restart? If I need the typing every time for making cdf env, I need lot of time for this step (cdf file is big). ********** >source("http://www.bioconductor.org/getBioC.R") >getBioC("all") >library(makecdfenv) >Library(affy) >make.cdf.package ("arabidopsistlgF.cdf") ********** And next, I tried makecdfenv again like below, > env = make.cdf.env("arabidopsistlgF.cdf") > library(makecdfenv) > env = make.cdf.env("arabidopsistlgF.cdf") > cel.files=list.files(pattern=".CEL$") > data=ReadAffy(filenames=cel.files) > pname<- cleancdfname(whatcdf("J_HpaII_Wt_10uM.CEL")) > temp=rma(data) I got Error below, ****** Note: You did not specify a download type. Using a default value of: Source This will be fine for almost all users Error in getCdfInfo(object) : Could not obtain CDF environment, problems encountered: Specified environment specified did not contain arabidopsis_tlgF_4x Library - package arabidopsistlgf4xcdf not installed Data for package affy did not contain arabidopsis_tlgF_4x Bioconductor - arabidopsistlgf4xcdf not available ********* So I made copy of "arabidopsistlgF.cdf", and change name "arabidopsistlgF4x". And continue, > env = make.cdf.env("arabidopsistlgF4x.cdf") > cel.files=list.files(pattern=".CEL$") > data=ReadAffy(filenames=cel.files) > > pname<- cleancdfname(whatcdf("J_HpaII_Wt_10uM.CEL")) I got Error again, ******** Error in whatcdf("J_HpaII_Wt_10uM.CEL") : Could not open file J_HpaII_Wt_10uM.CEL ******** I thought I may be need for cel file normalization, below, > library(gcrma) Loading required package: matchprobes > Data <- ReadAffy() > eset <- gcrma(Data) I got Error again, ******** Computing affinities[1] "Checking to see if your internet connection works..." Warning message: unable to connect to 'www.bioconductor.org' on port 80. Note: http://www.bioconductor.org/repository/devel/package/Source does not seem to have a valid repository, skipping Warning messages: 1: Failed to read replisting at http://www.bioconductor.org/repository/devel/package/Source in: getReplisting(repURL, repFile, method = method) 2: unable to connect to 'www.bioconductor.org' on port 80. Note: http://www.bioconductor.org/repository/devel/package/Win32 does not seem to have a valid repository, skipping Note: You did not specify a download type. Using a default value of: Source This will be fine for almost all users Error in getCDF(cdfpackagename) : Environment arabidopsistlgf4xcdf was not found in the Bioconductor repository. In addition: Warning message: Failed to read replisting at http://www.bioconductor.org/repository/devel/package/Win32 in: getReplisting(repURL, repFile, method = method) ******** Q2. I can not read my cel file now. Our cdf file name is "arabidopsistlgF.cdf" . But cif file name is "arabidopsistlgF_4x.cif". Do I need to use same name for cif and cdf? Because cel file include cif file name. And how can I start to read cel file? Q3. And also I would like to read cel file and normalization using a lot of cel files. Could you please suggest me what package is better for reading and normalization of affy custom array? and which is better rma (Robust Multi-Array Average expression measure) or gcrma (Background adjustment using sequence information)? Q4. If our array has over 3 million data, how long do I need for reading and normalization for 1 data (depend on machine power?)? Do you have some speculation for calculation efficiency? I need to read cdf file for about 20min. Thank you very much, Junshi -- *********************************************************** *********************************************************** Junshi Yazaki The Salk Institute for Biological Studies

Junshi Yazaki wrote: > Hi Jim, Seth, Reddy, Paul, > > Thank you very much for your suggestion. I may be make cdf > environment. Could you please help me how to confirm the env is OK or > No? Next I tried cel file reading and normalize from our custom affy > array. If my working flow are useful for affy beginner like me, could > you please help me? > > At first, I typed below... > >> source("http://www.bioconductor.org/getBioC.R") >> getBioC("all") >> library(makecdfenv) >> Library(affy) >> make.cdf.package ("arabidopsistlgF.cdf") > > > And move to Terminal on my Mac, > >> R CMD INSTALL arabidopsistlgFcdf It should actually be arabidopsistlgfcdf. Note that the F is lower case. > > Return to R, > >> arabidopsistlgF = make.cdf.env("arabidopsistlgF.cdf") > This is an unnecessary step - you already made and installed the package. > > And I shut down my Mac. Is these step correct for making cdf > environment? And then I started again. > >> source("http://www.bioconductor.org/getBioC.R") >> getBioC() >> library(affy) >> Data <- readAffy() At this point, try cleancdfname(cdfName(Data)) if the result is not arabidopsistlgfcdf, then you need to make your cdfenv again, using the cleancdfname(). I am betting the cleancdfname will be arabidopsistlgf4xcdf, so you will need to do make.cdf.package("arabidopsistlgF.cdf", packagename="arabidopsistlgf4xcdf") And then install using R CMD INSTALL >> eset <- rma(data) > > > I got Error below, > *********** > Note: You did not specify a download type. Using a default value of: > Source > This will be fine for almost all users > > Error in getCdfInfo(object) : Could not obtain CDF environment, problems > encountered: > Specified environment specified did not contain arabidopsis_tlgF_4x > Library - package arabidopsistlgf4xcdf not installed > Data for package affy did not contain arabidopsis_tlgF_4x > Bioconductor - arabidopsistlgf4xcdf not available > ********* > Q1. I have question. Do I need typing below every time after restart? If > I need the typing every time for making cdf env, I need lot of time for > this step (cdf file is big). No. If you install correctly, it should be there for you every time you run R. > ********** > >> source("http://www.bioconductor.org/getBioC.R") >> getBioC("all") >> library(makecdfenv) >> Library(affy) >> make.cdf.package ("arabidopsistlgF.cdf") > > ********** > And next, I tried makecdfenv again like below, > >> env = make.cdf.env("arabidopsistlgF.cdf") >> library(makecdfenv) >> env = make.cdf.env("arabidopsistlgF.cdf") >> cel.files=list.files(pattern=".CEL$") >> data=ReadAffy(filenames=cel.files) >> pname<- cleancdfname(whatcdf("J_HpaII_Wt_10uM.CEL")) >> temp=rma(data) > > > I got Error below, > ****** > Note: You did not specify a download type. Using a default value of: > Source > This will be fine for almost all users > > Error in getCdfInfo(object) : Could not obtain CDF environment, problems > encountered: > Specified environment specified did not contain arabidopsis_tlgF_4x > Library - package arabidopsistlgf4xcdf not installed > Data for package affy did not contain arabidopsis_tlgF_4x > Bioconductor - arabidopsistlgf4xcdf not available > ********* > So I made copy of "arabidopsistlgF.cdf", and change name > "arabidopsistlgF4x". And continue, > >> env = make.cdf.env("arabidopsistlgF4x.cdf") >> cel.files=list.files(pattern=".CEL$") >> data=ReadAffy(filenames=cel.files) >> >> pname<- cleancdfname(whatcdf("J_HpaII_Wt_10uM.CEL")) > > > I got Error again, > ******** > Error in whatcdf("J_HpaII_Wt_10uM.CEL") : Could not open file > J_HpaII_Wt_10uM.CEL > ******** > > I thought I may be need for cel file normalization, below, > >> library(gcrma) > > Loading required package: matchprobes > >> Data <- ReadAffy() >> eset <- gcrma(Data) > > > I got Error again, > ******** > Computing affinities[1] "Checking to see if your internet connection > works..." > Warning message: > unable to connect to 'www.bioconductor.org' on port 80. > Note: http://www.bioconductor.org/repository/devel/package/Source does > not seem to have a valid repository, skipping > Warning messages: > 1: Failed to read replisting at > http://www.bioconductor.org/repository/devel/package/Source in: > getReplisting(repURL, repFile, method = method) > 2: unable to connect to 'www.bioconductor.org' on port 80. > Note: http://www.bioconductor.org/repository/devel/package/Win32 does > not seem to have a valid repository, skipping > Note: You did not specify a download type. Using a default value of: > Source > This will be fine for almost all users > > Error in getCDF(cdfpackagename) : Environment arabidopsistlgf4xcdf was > not found in the Bioconductor repository. > In addition: Warning message: > Failed to read replisting at > http://www.bioconductor.org/repository/devel/package/Win32 in: > getReplisting(repURL, repFile, method = method) > ******** > Q2. I can not read my cel file now. Our cdf file name is > "arabidopsistlgF.cdf" . But cif file name is "arabidopsistlgF_4x.cif". > Do I need to use same name for cif and cdf? Because cel file include cif > file name. And how can I start to read cel file? Once you have the cdfenv installed correctly, you can read celfiles using ReadAffy(). > > Q3. And also I would like to read cel file and normalization using a lot > of cel files. Could you please suggest me what package is better for > reading and normalization of affy custom array? and which is better rma > (Robust Multi-Array Average expression measure) or gcrma (Background > adjustment using sequence information)? Which are better, apples or oranges? I guess it all depends on who you ask. > > Q4. If our array has over 3 million data, how long do I need for reading > and normalization for 1 data (depend on machine power?)? Do you have > some speculation for calculation efficiency? I need to read cdf file for > about 20min. If it takes that long to read in the cdf I am betting you are using virtual memory. In that case, you really need to get more RAM or things will be crushingly slow. HTH, Jim > > Thank you very much, > Junshi -- James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623