Hi Benilton, If both of scripts do not generate errors, does it mean that both of them are correct? But the RMA results are very different, so one of them must be wrong? Regards, Peng On Mon, Aug 10, 2009 at 7:54 PM, Benilton Carvalho<bcarvalh at="" jhsph.edu=""> wrote: > For the Mouse Gene ST Array, 'oligo' needs the "pd.mogene.1.0.st.v1" > package. If it didn't complain about it when reading the CEL files, it's > because you already had it installed on your system. The next version of > 'oligo' will be able to identify the required package (as it does currently) > and automatically download/install the required packages, if available on > the BioC website. > > As a side note, please observe that the annotation packages used by 'affy' > are not compatible with those used by 'oligo', which are built via > 'pdInfoBuilder'. > > b > > On Aug 10, 2009, at 9:27 PM, Peng Yu wrote: > >> Hi Benilton, >> >> One thing I don't understand isf why 'oligo' didin't require me to >> install the .cdf file, but 'affy' required me to install the .cdf >> file. I am curious that how 'oligo' figure how where the correct .cdf >> file is and use it? >> >> Regards, >> Peng >> >> On Mon, Aug 10, 2009 at 5:25 PM, Benilton Carvalho<bcarvalh at="" jhsph.edu=""> >> wrote: >>> >>> Dear Peng, >>> >>> I can speak for oligo and the annotation package used by it. >>> >>> The current release of oligo summarizes to the probeset level. The next >>> release of oligo and annotation packages will allow you to summarize to >>> the >>> gene level. In your particular case, the count you'll get is roughly 35K. >>> >>> The updated packages have already been submitted to BioC and soon should >>> show up on the devel branch. >>> >>> Best wishes, >>> >>> b >>> >>> On Aug 10, 2009, at 6:39 PM, Peng Yu wrote: >>> >>>> Hi, >>>> >>>> I have run the two different R script to do RMA. Neither of them gives >>>> me any error messages. However, the RMA results are very >>>> different---they have very different number of lines. I don't know >>>> which one I should believe. Or neither of them is correct. It might be >>>> due to the difference in the cdf file used. Would you please point to >>>> me how to figure out the problem? >>>> >>>> $ Rscript probe2expr_affy.R >>>>> >>>>> library(affy) >>>> >>>> Loading required package: Biobase >>>> Loading required package: methods >>>> >>>> Welcome to Bioconductor >>>> >>>> Vignettes contain introductory material. To view, type >>>> 'openVignette()'. To cite Bioconductor, see >>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. >>>> >>>>> Data <- ReadAffy() >>>>> eset <- rma(Data) >>>> >>>> Background correcting >>>> Normalizing >>>> Calculating Expression >>>>> >>>>> write.exprs(eset, file="gene_expr_affy.txt", sep="\t") >>>>> >>>> >>>> $ Rscript probe2expr_oligo.R >>>>> >>>>> library(oligo) >>>> >>>> Loading required package: oligoClasses >>>> Loading required package: Biobase >>>> Loading required package: methods >>>> >>>> Welcome to Bioconductor >>>> >>>> Vignettes contain introductory material. To view, type >>>> 'openVignette()'. To cite Bioconductor, see >>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. >>>> >>>> Loading required package: preprocessCore >>>> Welcome to oligo version 1.8.1 >>>>> >>>>> data<-read.celfiles(list.celfiles()) >>>> >>>> Loading required package: pd.mogene.1.0.st.v1 >>>> Loading required package: RSQLite >>>> Loading required package: DBI >>>> Platform design info loaded. >>>> Reading in : koA-mth_HZ_5238_MST1_19389.cel >>>> Reading in : koB-mth_HZ_5238_MST1_19390.cel >>>> Reading in : koC-mth_HZ_5238_MST1_19391.cel >>>> Reading in : koD-mth_HZ_5238_MST1_19392.cel >>>> Reading in : wt1-mth_HZ_5238_MST1_19385.cel >>>> Reading in : wt2-mth_HZ_5238_MST1_19386.cel >>>> Reading in : wt3-mth_HZ_5238_MST1_19387.cel >>>> Reading in : wt4-mth_HZ_5238_MST1_19388.cel >>>>> >>>>> eset<-rma(data) >>>> >>>> Background correcting >>>> Normalizing >>>> Calculating Expression >>>>> >>>>> write.exprs(eset, file="gene_expr_oligo.txt", sep="\t") >>>>> >>>> >>>> $ wc gene_expr_affy.txt gene_expr_oligo.txt >>>> 34761 ? 312848 ?5002519 gene_expr_affy.txt >>>> 234591 ?2111318 33763075 gene_expr_oligo.txt >>>> 269352 ?2424166 38765594 total >>>> >>>> BTW, before I run 'Rscript probe2expr_affy.R'. I downloaded >>>> MoGene-1_0-st-v1.r3.unsupported-cdf and I run the following script. >>>> Then, I install the generated package 'mogene10stv1cdf'. >>>> $ cat make_cdf_package.R >>>> library(makecdfenv) >>>> make.cdf.package("MoGene-1_0-st-v1.r3.cdf", >>>> packagename="mogene10stv1cdf", species="Mus_musculus") >>>> >>>> Regards, >>>> Peng >>>> >>>> _______________________________________________ >>>> 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 >>> >>> >> >> _______________________________________________ >> 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 > >

On Mon, Aug 10, 2009 at 9:30 PM, Peng Yu <pengyu.ut@gmail.com> wrote: > Hi Benilton, > > If both of scripts do not generate errors, does it mean that both of > them are correct? But the RMA results are very different, so one of > them must be wrong? > The probesets for the genest arrays are designed roughly against exons and as Benilton pointed out, the current behavior of oligo is to summarize the probesets. Given that there are >200k exons in the human genome, one should expect >200k rows in the rma-normalized data from oligo. Affy, on the other hand, uses the unofficial cdf that you downloaded and summarizes to transcripts. So, both are correct, but you are trying to compare apples to oranges. Sean > > Regards, > Peng > > On Mon, Aug 10, 2009 at 7:54 PM, Benilton Carvalho<bcarvalh@jhsph.edu> > wrote: > > For the Mouse Gene ST Array, 'oligo' needs the "pd.mogene.1.0.st.v1" > > package. If it didn't complain about it when reading the CEL files, it's > > because you already had it installed on your system. The next version of > > 'oligo' will be able to identify the required package (as it does > currently) > > and automatically download/install the required packages, if available on > > the BioC website. > > > > As a side note, please observe that the annotation packages used by > 'affy' > > are not compatible with those used by 'oligo', which are built via > > 'pdInfoBuilder'. > > > > b > > > > On Aug 10, 2009, at 9:27 PM, Peng Yu wrote: > > > >> Hi Benilton, > >> > >> One thing I don't understand isf why 'oligo' didin't require me to > >> install the .cdf file, but 'affy' required me to install the .cdf > >> file. I am curious that how 'oligo' figure how where the correct .cdf > >> file is and use it? > >> > >> Regards, > >> Peng > >> > >> On Mon, Aug 10, 2009 at 5:25 PM, Benilton Carvalho<bcarvalh@jhsph.edu> > >> wrote: > >>> > >>> Dear Peng, > >>> > >>> I can speak for oligo and the annotation package used by it. > >>> > >>> The current release of oligo summarizes to the probeset level. The next > >>> release of oligo and annotation packages will allow you to summarize to > >>> the > >>> gene level. In your particular case, the count you'll get is roughly > 35K. > >>> > >>> The updated packages have already been submitted to BioC and soon > should > >>> show up on the devel branch. > >>> > >>> Best wishes, > >>> > >>> b > >>> > >>> On Aug 10, 2009, at 6:39 PM, Peng Yu wrote: > >>> > >>>> Hi, > >>>> > >>>> I have run the two different R script to do RMA. Neither of them gives > >>>> me any error messages. However, the RMA results are very > >>>> different---they have very different number of lines. I don't know > >>>> which one I should believe. Or neither of them is correct. It might be > >>>> due to the difference in the cdf file used. Would you please point to > >>>> me how to figure out the problem? > >>>> > >>>> $ Rscript probe2expr_affy.R > >>>>> > >>>>> library(affy) > >>>> > >>>> Loading required package: Biobase > >>>> Loading required package: methods > >>>> > >>>> Welcome to Bioconductor > >>>> > >>>> Vignettes contain introductory material. To view, type > >>>> 'openVignette()'. To cite Bioconductor, see > >>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. > >>>> > >>>>> Data <- ReadAffy() > >>>>> eset <- rma(Data) > >>>> > >>>> Background correcting > >>>> Normalizing > >>>> Calculating Expression > >>>>> > >>>>> write.exprs(eset, file="gene_expr_affy.txt", sep="\t") > >>>>> > >>>> > >>>> $ Rscript probe2expr_oligo.R > >>>>> > >>>>> library(oligo) > >>>> > >>>> Loading required package: oligoClasses > >>>> Loading required package: Biobase > >>>> Loading required package: methods > >>>> > >>>> Welcome to Bioconductor > >>>> > >>>> Vignettes contain introductory material. To view, type > >>>> 'openVignette()'. To cite Bioconductor, see > >>>> 'citation("Biobase")' and for packages 'citation(pkgname)'. > >>>> > >>>> Loading required package: preprocessCore > >>>> Welcome to oligo version 1.8.1 > >>>>> > >>>>> data<-read.celfiles(list.celfiles()) > >>>> > >>>> Loading required package: pd.mogene.1.0.st.v1 > >>>> Loading required package: RSQLite > >>>> Loading required package: DBI > >>>> Platform design info loaded. > >>>> Reading in : koA-mth_HZ_5238_MST1_19389.cel > >>>> Reading in : koB-mth_HZ_5238_MST1_19390.cel > >>>> Reading in : koC-mth_HZ_5238_MST1_19391.cel > >>>> Reading in : koD-mth_HZ_5238_MST1_19392.cel > >>>> Reading in : wt1-mth_HZ_5238_MST1_19385.cel > >>>> Reading in : wt2-mth_HZ_5238_MST1_19386.cel > >>>> Reading in : wt3-mth_HZ_5238_MST1_19387.cel > >>>> Reading in : wt4-mth_HZ_5238_MST1_19388.cel > >>>>> > >>>>> eset<-rma(data) > >>>> > >>>> Background correcting > >>>> Normalizing > >>>> Calculating Expression > >>>>> > >>>>> write.exprs(eset, file="gene_expr_oligo.txt", sep="\t") > >>>>> > >>>> > >>>> $ wc gene_expr_affy.txt gene_expr_oligo.txt > >>>> 34761 312848 5002519 gene_expr_affy.txt > >>>> 234591 2111318 33763075 gene_expr_oligo.txt > >>>> 269352 2424166 38765594 total > >>>> > >>>> BTW, before I run 'Rscript probe2expr_affy.R'. I downloaded > >>>> MoGene-1_0-st-v1.r3.unsupported-cdf and I run the following script. > >>>> Then, I install the generated package 'mogene10stv1cdf'. > >>>> $ cat make_cdf_package.R > >>>> library(makecdfenv) > >>>> make.cdf.package("MoGene-1_0-st-v1.r3.cdf", > >>>> packagename="mogene10stv1cdf", species="Mus_musculus") > >>>> > >>>> Regards, > >>>> Peng > >>>> > >>>> _______________________________________________ > >>>> Bioconductor mailing list > >>>> Bioconductor@stat.math.ethz.ch > >>>> https://stat.ethz.ch/mailman/listinfo/bioconductor > >>>> Search the archives: > >>>> http://news.gmane.org/gmane.science.biology.informatics.conductor > >>> > >>> > >> > >> _______________________________________________ > >> Bioconductor mailing list > >> Bioconductor@stat.math.ethz.ch > >> https://stat.ethz.ch/mailman/listinfo/bioconductor > >> Search the archives: > >> http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]

Hi, On Aug 10, 2009, at 9:30 PM, Peng Yu wrote: > Hi Benilton, > > If both of scripts do not generate errors, does it mean that both of > them are correct? But the RMA results are very different, so one of > them must be wrong? Benilton, I think, has provided some hints which you can chew on for a bit. Sometimes it helps to digest some of the information and try to *really* figure out what it *means* instead of hoping to jump straight to an answer. Let's see if we can't do that with the information we have here: > On Mon, Aug 10, 2009 at 5:25 PM, Benilton Carvalho<bcarvalh at="" jhsph.edu=""> > wrote: >> >> Dear Peng, >> >> I can speak for oligo and the annotation package used by it. >> >> The current release of oligo summarizes to the probeset level. The >> next >> release of oligo and annotation packages will allow you to >> summarize to >> the >> gene level. In your particular case, the count you'll get is >> roughly 35K. He mentions summaries at "the probeset level," and then "the gene level." He also mentions that the upcoming release of oligo WILL allow you to summarize to THE GENE LEVEL, which I guess suggests that currently oligo is providing summaries at the probeset level. Once you can get GENE LEVEL summaries, you will get ROUGHLY 35K VALUES. Now, you say your files are very different, and that one (or both!) of these package must be wrong, since you've looked at this: $ wc gene_expr_affy.txt gene_expr_oligo.txt 34761 312848 5002519 gene_expr_affy.txt 234591 2111318 33763075 gene_expr_oligo.txt 269352 2424166 38765594 total It looks like your current oligo summary has many more numbers than your expr_affy file, which only has ROUGHLY 35K VALUES (oh wait, I think I've seen this number before). Now the question is: *what* are these two files telling you? Have you looked at their outputs, aside from the number of lines in each file? Do you see probes by the same names in each file? What is a probe set? How is it different from a gene? Since you have many more probeset level numbers than gene/transcript numbers, does that mean there are more than 1 probeset to a gene? Is that what this is saying here? http://www.affymetrix.com/support/help/faqs/mouse_430/faq_8.jsp Or this figure (and the rest of the publication), here?: http://www.biomedcentral.com/1471-2105/8/108/figure/F1 Has someone asked something like this on this mailing list before? https://stat.ethz.ch/pipermail/bioconductor/2009-January/025791.html I actually have no idea what your output files look like (I haven't used oligo before, actually), but I'm just trying to help put some pieces together. In the meantime, I see that Sean has provided you a direct answer, so kudos, but I'd just suggest taking a bit more of this work on yourself. I think it will help to make more sense of the answers you will inevitably get on this list. -steve -- Steve Lianoglou Graduate Student: Computational Systems Biology | Memorial Sloan-Kettering Cancer Center | Weill Medical College of Cornell University Contact Info: http://cbio.mskcc.org/~lianos/contact