Hello, I'm analyzing human exon arrays normalized using Affymetrix Power tools for normalization (using 'core' probes) and limma for significantly modulated genes (all at the gene level, of course). The limma2annaffy function produce tables, but with all annotation table cells empty. I'm doing: limma2annaffy(eSet.gene, fit2, design,cont.matrix, lib = "huex10stv2hsentrezg.db", interactive=F, pfilt=0.05, fldfilt=0.8) where huex10stv2entrezg.db is from : http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/12. 1.0/entrezg.asp Is it the right file to use? Using onechannelGUI produce the same tables but with annotations, so I know there's a way to do it. To complicate things further, limma2biomaRt, which is another option, fails with: "Request to BioMart web service failed. Verify if you are still connected to the internet. Alternatively the BioMart web service is temporarily down." which from the mailing list seem to be an RCurl problem. I tried updating it to the latest and older (0.92) versions, using --internet2 doesn't solve this and as far as I know i'm not using a proxy to connect to the net. I'm under Windows 7. Any help would be appreciated, Michael [[alternative HTML version deleted]]

Hi Michael, Michael Imbeault wrote: > Hello, > > I'm analyzing human exon arrays normalized using Affymetrix Power tools > for normalization (using 'core' probes) and limma for significantly > modulated genes (all at the gene level, of course). > > The limma2annaffy function produce tables, but with all annotation table > cells empty. I'm doing: > > limma2annaffy(eSet.gene, fit2, design,cont.matrix, lib = > "huex10stv2hsentrezg.db", interactive=F, pfilt=0.05, fldfilt=0.8) > > where huex10stv2entrezg.db is from : > http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/1 2.1.0/entrezg.asp > > Is it the right file to use? Most likely. However, the MBNI folks have an unfortunate habit of adding _at to the end of all their probesets, regardless the source. So for instance, if I look at the probesets in this package, I get something like this: > head(Lkeys(huex10stv2hsentrezgGENENAME)) [1] "10000_at" "10001_at" "10002_at" [4] "100033423_at" "100033424_at" "100033425_at" And I am betting if you do something like head(featureNames(eSet.gene)), you won't have any of those nasty _at extensions. A simple albeit kludgy fix would be for you to first do featureNames(eSet.gene) <- paste(featureNames(eSet.gene), "_at") and then run limma2annaffy(). > > Using onechannelGUI produce the same tables but with annotations, so I > know there's a way to do it. I am betting that the onechannelGUI folks know about the extra _at extensions and are silently stripping them. I could hypothetically do the same, but I rebel against the idea that I should have to put code in my package to protect people from infelicities in other people's packages. Best, Jim > > To complicate things further, limma2biomaRt, which is another option, > fails with: > > "Request to BioMart web service failed. Verify if you are still > connected to the internet. Alternatively the BioMart web service is > temporarily down." > > which from the mailing list seem to be an RCurl problem. I tried > updating it to the latest and older (0.92) versions, using --internet2 > doesn't solve this and as far as I know i'm not using a proxy to connect > to the net. I'm under Windows 7. > > Any help would be appreciated, > Michael > > [[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 Douglas Lab University of Michigan Department of Human Genetics 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

Oh yeah, it finally worked its way through my dim bulb. All the IDs in that annotation package are based on Entrez Gene IDs, not Affymetrix IDs (this is true of all the MBNI chips - the IDs are based on the annotation database they used for re-mapping the probes). There is a package on BioC, the pd.huex.1.0.st.v2 package that has annotation information for the probes, but you would need to use oligo for the processing. I would be surprised if you would be able to use limma2annaffy for annotation however. Best, Jim James W. MacDonald, M.S. Biostatistician Douglas Lab 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 >>> Michael Imbeault <michael.imbeault at="" sympatico.ca=""> 12/21/09 6:05 PM >>> Hello James, "Error in .checkKeys(value, Lkeys(x), x at ifnotfound) : value for "3948543_at" not found" You're right, it's a mismatch problem; I just looked at all the "_at" probes in the db file and their id is not even right - some are 9994_at, which have no equivalent for the transcript clusters ids on my chip, which are of the format "3948543" - always 7 digits. I don't know why is it so in the db file, but it doesn't match anything on my chip, nor on NetAffx, so I don't know why the MBNI folks did it like so. My next options are to look at the source code of onechannelGUI (which doesn't use the MBNI file to accomplish its magic) or try to fix my RCurl / Biomart problem. I'll probably open a new mailing list topic for this one. Much frustration so far with the exon array chip workflow, it's so unlike the other chips; there really should be a wiki for this :) Thanks for the help, Michael On 21/12/2009 3:54 PM, James W. MacDonald wrote: > Hi Michael, > > Michael Imbeault wrote: >> Thanks for the help James, >> >> I did: >> >> featureNames(eSet.gene) <- paste(featureNames(eSet.gene), "_at" , >> sep="") >> >> (note the sep="", without it the probes were like "1000 _at"). >> Sadly, the end result is the same, except that as a side effect, the >> first column probe links don't work anymore (because of the added >> _at, they don't link to the right probe on the Affy site). >> >> I verified that the probes in eSet.gene contain _at after the >> operation. I build my eset with: >> >> eSet.gene <- new("ExpressionSet", exprs = rma.gene, phenoData = >> phenoData) >> >> Should I add annotation="huex10stv2" or "huex10stv2hsentrezg" or >> something similar? Do i need the cdf file in addition to the .db one? > > No. I think there is just a mismatch problem here. As you mentioned > below, onechannelGUI is able to create a table with annotation. > > All limma2annaffy is doing is passing the probeset IDs on to annaffy. > All the matching and link building are done there, but if the IDs > don't match to anything in the annotation package then annaffy will > just create an empty cell in the table. > > If you take the first 10 or so featureNames (with the _at appended) > and do e.g., > > mget(<thefeaturenames>, huex10stv2hsentrezgUNIGENE) > > do you get anything returned? > > Best, > > Jim >> >> Thanks, >> Michael >> >> On 21/12/2009 1:01 PM, James W. MacDonald wrote: >>> Hi Michael, >>> >>> Michael Imbeault wrote: >>>> Hello, >>>> >>>> I'm analyzing human exon arrays normalized using Affymetrix Power >>>> tools for normalization (using 'core' probes) and limma for >>>> significantly modulated genes (all at the gene level, of course). >>>> >>>> The limma2annaffy function produce tables, but with all annotation >>>> table cells empty. I'm doing: >>>> >>>> limma2annaffy(eSet.gene, fit2, design,cont.matrix, lib = >>>> "huex10stv2hsentrezg.db", interactive=F, pfilt=0.05, fldfilt=0.8) >>>> >>>> where huex10stv2entrezg.db is from : >>>> http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCD F/12.1.0/entrezg.asp >>>> >>>> >>>> Is it the right file to use? >>> >>> Most likely. However, the MBNI folks have an unfortunate habit of >>> adding _at to the end of all their probesets, regardless the source. >>> So for instance, if I look at the probesets in this package, I get >>> something like this: >>> >>> > head(Lkeys(huex10stv2hsentrezgGENENAME)) >>> [1] "10000_at" "10001_at" "10002_at" >>> [4] "100033423_at" "100033424_at" "100033425_at" >>> >>> And I am betting if you do something like >>> head(featureNames(eSet.gene)), you won't have any of those nasty _at >>> extensions. >>> >>> A simple albeit kludgy fix would be for you to first do >>> >>> featureNames(eSet.gene) <- paste(featureNames(eSet.gene), "_at") >>> >>> and then run limma2annaffy(). >>>> >>>> Using onechannelGUI produce the same tables but with annotations, >>>> so I know there's a way to do it. >>> >>> I am betting that the onechannelGUI folks know about the extra _at >>> extensions and are silently stripping them. I could hypothetically >>> do the same, but I rebel against the idea that I should have to put >>> code in my package to protect people from infelicities in other >>> people's packages. >>> >>> >>> Best, >>> >>> Jim >>> >>> >>>> >>>> To complicate things further, limma2biomaRt, which is another >>>> option, fails with: >>>> >>>> "Request to BioMart web service failed. Verify if you are still >>>> connected to the internet. Alternatively the BioMart web >>>> service is >>>> temporarily down." >>>> >>>> which from the mailing list seem to be an RCurl problem. I tried >>>> updating it to the latest and older (0.92) versions, using >>>> --internet2 doesn't solve this and as far as I know i'm not using a >>>> proxy to connect to the net. I'm under Windows 7. >>>> >>>> Any help would be appreciated, >>>> Michael >>>> >>>> [[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 >>> >> >> _______________________________________________ >> 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 > ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

Hi, I am the maintainer of oneChannelGUI, concerning gene-level annotation for exon array, oneChannelGUI uses an internal annotation, builded parsing the netaffx annotation data via AffyCompatible package. The function buildingLocalAnnotation() produces a R object containing a data frame with the following columns for gene-level data: PROBESETID ACC SYMBOL DESCRIPTION CYTOBAND The internal annotation is updated when a new release of netaffx annotation is avilable. Users can build the annotation themselves with the previously described function. Cheers Raffaele P.S. oneChannleGUI also provides an internal annotation for exon-level probesets. This internal annotation will be replaced by three annotation packages, now present in the devel branch: http://www.bioconductor.org/packages/2.6/data/annotation/html/HuExExon ProbesetLocation.html http://www.bioconductor.org/packages/2.6/data/annotation/html/MoExExon ProbesetLocation.html http://www.bioconductor.org/packages/2.6/data/annotation/html/RaExExon ProbesetLocation.html >Hello James, >"Error in .checkKeys(value, Lkeys(x), x at ifnotfound) : value for "3948543_at" not found" >You're right, it's a mismatch problem; I just looked at all the "_at" >probes in the db file and their id is not even right - some are 9994_at, >which have no equivalent for the transcript clusters ids on my chip, >which are of the format "3948543" - always 7 digits. >I don't know why is it so in the db file, but it doesn't match anything >on my chip, nor on NetAffx, so I don't know why the MBNI folks did it >like so. >My next options are to look at the source code of onechannelGUI (which >doesn't use the MBNI file to accomplish its magic) or try to fix my >RCurl / Biomart problem. I'll probably open a new mailing list topic for >this one. Much frustration so far with the exon array chip workflow, >it's so unlike the other chips; there really should be a wiki for this :) >Thanks for the help, >Michael ---------------------------------------- Prof. Raffaele A. Calogero Bioinformatics and Genomics Unit Dipartimento di Scienze Cliniche e Biologiche Az. Ospedaliero-Universitaria S. Luigi Regione Gonzole 10, Orbassano 10043 Torino tel. ++39 0116705417 Lab. ++39 0116705408 Fax ++39 0119038639 Mobile ++39 3333827080 email: raffaele.calogero at unito.it raffaele[dot]calogero[at]gmail[dot]com www: http://www.bioinformatica.unito.it Info: http://publicationslist.org/raffaele.calogero

Dear Michael, Since we are entering in the winter holiday and I have some spare time, I will create a stand-alone function for gene level annotation for exon-arrays using as input a data frame, that can be generated by limma or other statistical tools. it will be present in oneChannelGUI and could be extracted from it without the need of installing oneChannelGUI. >On Wed, Dec 23, 2009 at 6:42 AM, Michael Imbeault <michael.imbeault at="" sympatico.ca=""> wrote: > Hello Raffaele, > Interesting; do you think you could provide a package to provide the same functionality that is >present in oneChannelGUI (for gene-level annotation, that is)? I looked at the code and it's a bit >interwined with the onChannelGUI environment; is there an easy way to extract the data frame and save >it as one external to the environment in the mean-time? In the mean time you can extract the anntoation data frame generated from netaffx data using: data("huex.annotation",package="oneChannelGUI") #for human data("moex.annotation",package="oneChannelGUI")#for mouse data("raex.annotation",package="oneChannelGUI")#for rat In the latest release of oneChannelGUI (1.12.2 ,22 December 2009) of stable Bioconductor 2.5 you can find the latest annotations released from Affymetrix in november. Cheers Raffaele -- ---------------------------------------- Prof. Raffaele A. Calogero Bioinformatics and Genomics Unit Dipartimento di Scienze Cliniche e Biologiche c/o Az. Ospedaliera S. Luigi Regione Gonzole 10, Orbassano 10043 Torino tel. ++39 0116705417 Lab. ++39 0116705408 Fax ++39 0119038639 Mobile ++39 3333827080 email: raffaele.calogero at unito.it raffaele[dot]calogero[at]gmail[dot]com www: www.bioinformatica.unito.it

>Michael wrote: >Hello Raffaele, >Interesting; do you think you could provide a package to provide the same functionality that is present in oneChannelGUI (for gene-level annotation, that is)? I looked >at the code and it's a bit interwined with the onChannelGUI environment; is there an easy way to extract the data frame and save it as one external to the environment >in the mean-time? >I can see from some mailing list topics that an easy to use gene- level annotation solution for exon arrays is really needed. > >Thanks, >Michael Dear Michael, I have created two stand-alone functions for gene level annotation for exon-arrays, which are available as part of oneChannelGUI package but can be used without the oneChannelGUI interface. The function standAloneBuildingLocalAnnotation creates a data frame with gene-level annotation data for exon arrays (human, mouse or rat) using the netaffx database informations. The function standAloneAddingAnnotation attaches gene-level annotation to a data frame. You will find more info on the functionalities of these functions in the vignette associated to oneChannelGUI release 1.12.4. I uploaded the package on 28th December and I am expecting that it will be available on the Bioconductor web in 24 hours. Cheers Raffaele P.S: let me know if these functions fulfill your needs, I will be happy to improve them upon suggestion -- ---------------------------------------- Prof. Raffaele A. Calogero Bioinformatics and Genomics Unit Dipartimento di Scienze Cliniche e Biologiche Az. Ospedaliero-Universitaria S. Luigi Regione Gonzole 10, Orbassano 10043 Torino tel. ++39 0116705417 Lab. ++39 0116705408 Fax ++39 0119038639 Mobile ++39 3333827080 email: raffaele.calogero at unito.it raffaele[dot]calogero[at]gmail[dot]com www: http://www.bioinformatica.unito.it Info: http://publicationslist.org/raffaele.calogero