Dear conductors, we are currently trying to see if we can reproduce the results of GeneSpring analysis using Bioconductor/limma and we are wondering what the best interface is, i.e., at what point in the analysis to input the Agilent data (image, raw, Gene-Spring-normalized), and how to include the quality (present/absent) calls into the analysis. The package Agi4x44PreProcess does not seem to be being actively maintained (?). I wonder if anyone on this list would be willing to share scripts/experiences on this. THanks Peter -- Dr. med. Peter N. Robinson, MSc. Institut f?r Medizinische Genetik Universit?tsklinikum Charite Humboldt-Universit?t Augustenburger Platz 1 13353 Berlin Germany voice: 49-30-450569124 fax: 49-30-450569915 email: peter.robinson at charite.de http://compbio.charite.de/ http://www.human-phenotype-ontology.org

Hi Peter, Are you trying to just replicate the results exactly or you want to try another set of analysis method? It should be possible to replicate most of the results from GeneSpring using Bioconductor, although GeneSpring is not very good at describing all their methods (but maybe it's changed since). Unless you want to better understand the GeneSpring procedure (or if you don't trust their results), this maybe not what you want to do. If you want to try another analysis technique, then there is a whole world in front of you, both for the normalization and for the differential expression. If you just want to see what limma tells you, then you might just want to use the normalized values from GeneSpring, otherwise I would start from the raw values. Either way, I wouldn't start from the image level The Agilent scanner (if that's what you're using), can assign weights to each spot, which limma can use. I personally am not a big fan of present/absent calling methods, so maybe someone who use them more can chime in. The best advice is probably to read the limma user guide. you should see pretty much all the information that you need to get started (including normalization). HTH, Francois Peter Robinson wrote: > Dear conductors, > > we are currently trying to see if we can reproduce the results of > GeneSpring analysis using Bioconductor/limma and we are wondering what > the best interface is, i.e., at what point in the analysis to input the > Agilent data (image, raw, Gene-Spring-normalized), and how to include > the quality (present/absent) calls into the analysis. The package > Agi4x44PreProcess does not seem to be being actively maintained (?). I > wonder if anyone on this list would be willing to share > scripts/experiences on this. > > THanks Peter >

Hi Peter, I am probably in a similar situation. I have just been convinced by an agilent rep to test their platform. So far we used Affy arrays. Now, I am absolutely willing to share experiences. However, I guess that these also depend a bit on on the detail of applications and organisms. I am trying to do dual-color analysis comparing treated vs. untreated cells on 4x44 and we have Drosophila arrays. GeneSpring for whatever reason refuses to do proper analysis on dual color as far as i can see. So R/Bioconductor and manual processings seems to be the only way. As Agi4x44PreProcess does not appreciate dual-color approaches, I did not test this package and don't know its status. About absent/present calls: a simple solution would be to read the gIsPosAndSignif and/or rIsPosAndSignif column from your output files. for the rest, my analysis is mainly based on limma. I do loess normalization within arrays and Aquantile between. A big question for me is how to treat multiple probes mapping to a single gene in case I want to summarize expression per gene. This appears not to be straight forward and is even more complicated in dual color approach. best, Tobias On Jul 21, 2009, at 6:30 PM, Peter Robinson wrote: > Dear conductors, > > we are currently trying to see if we can reproduce the results of > GeneSpring analysis using Bioconductor/limma and we are wondering > what the best interface is, i.e., at what point in the analysis to > input the Agilent data (image, raw, Gene-Spring-normalized), and how > to include the quality (present/absent) calls into the analysis. The > package Agi4x44PreProcess does not seem to be being actively > maintained (?). I wonder if anyone on this list would be willing to > share scripts/experiences on this. > > THanks Peter > > -- > Dr. med. Peter N. Robinson, MSc. > Institut f?r Medizinische Genetik > Universit?tsklinikum Charite > Humboldt-Universit?t > Augustenburger Platz 1 > 13353 Berlin > Germany > voice: 49-30-450569124 > fax: 49-30-450569915 > email: peter.robinson at charite.de > http://compbio.charite.de/ > http://www.human-phenotype-ontology.org > > _______________________________________________ > 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 ---------------------------------------------------------------------- Tobias Straub ++4989218075439 Adolf-Butenandt-Institute, M?nchen D