Hi Sundar, I've been leaving your messages to the FIRMA experts, because they can better answer you questions. However, I'll give a quick reply to the things I can answer.
On Mon, Aug 2, 2010 at 6:59 PM, Sundar <sundar...@gmail.com> wrote: > Hi, > I am trying to analyze Mouse Exon 1.0 st array data using > aroma.affymetrix and FIRMA model to find the splicing variants. > CDF file : MoEx-1_0-st-v1,coreR1,A20080718,MR.cdf ( downloaded from > the aroma.affymetrix website) > CEL files: 12 Mouse Exon 1.0 st arrays. ( 6 arrays for each strain A > and B) and Within A and B, i have 3 arrays each of two experimental > conditions) > > 1) I am not sure how the RMA normalization in aroma.affymetrix > performs, is the normalization performed within array or between the > array ? The aroma.affymetrix package implements a standard RMA normalization, that is, it reproduces it very well. I recommend that you read up on RMA model: Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003), A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics 19(2):185-193 Supplemental information Rafael. A. Irizarry, Benjamin M. Bolstad, Francois Collin, Leslie M. Cope, Bridget Hobbs and Terence P. Speed (2003), Summaries of Affymetrix GeneChip probe level data Nucleic Acids Research 31(4):e15 Irizarry, RA, Hobbs, B, Collin, F, Beazer-Barclay, YD, Antonellis, KJ, Scherf, U, Speed, TP (2002) Exploration, Normalization, and Summaries of High Density Oligonucleotide Array Probe Level Data. Accepted for publication in Biostatistics. Then you'll see that the RMA pipeline is a multi-array method. For more question on this, I recommend you to use the larger Bioconductor mailing list, because this one is used for aroma.* specific questions. > How do i pass arguments to the function to control the process > of normalization ? What do you want to control. > > 2) What are .js and .css files generated at the end of the analysis > described in vignette ( Human Exon array ). Are there any third party > software that could be used to analyze this out put ? Those are Javascript and CSS files used by the ArrayExplorer HTML reports. They do not contain any kind of data. They cannot be used by other software. > > 3) How do i convert the probe set ID or transcript ID into gene name ? > Can i calculate the fold change in aroma.affymetrix ? I leave this one to the FIRMA experts. Make sure to search/go through the aroma.affymetrix mailing archives - I think the question has been asked and answered before. More below.... > > > Thank you, > Sundar > > > > On Jul 27, 11:52 am, Sundar <sundar...@gmail.com> wrote: >> Hi, >> I am new to the Exon array concept. I am trying to analyze >> Mouse Exon 1.0 st array data using aroma.affymetrix and FIRMA model. >> Few questions i have are below. >> >> 1) To get a start i have just implemented the code described in >> vignette "FIRMA: Human exon array analysis. " There are certain .CEL >> files and other extension files generated. It is not clear which particular CEL files you are referring to. The ones under probeData/ contain normalized/calibrated probe signals in CEL files of the same format/layout as the once in rawData/. CEL files contains probe intensities, probe standard deviation, and number of pixels per probe. The ones in plmData/ are special aroma-specific CEL files that should be treated as internal file only, especially, they cannot be read by other software. These files contains chip effect estimates and standard deviation of those. There is also one CEL file containing probe affinity estimates. This is if you use the RMA-style probe-level modelling. >> How can i read them into >> R ? What do you want to do with the data that aroma doesn't do? >> How do i know what information they carry ? Is there any other >> software i can upload them to read these files ? Answered above. >> How do i get control >> of the analysis in applying extractMatrix() or extractDataFrame() , >> readUnits() functions ? Again, it is not clear what you want to do, but maybe the How-to page 'Extract probeset summaries (chip effects) as a data frame' [http://aroma-project.org/howtos/extractDataFrame] illustrates your options. I recommend that you use that instead of extractDataFrame(). Don't use readUnits(), unless you're a developer for aroma.affymetrix works. >> >> 2) I am unable to get a clear understanding of what the background >> calculation are taking place using the functions ( used to generate >> the files in Q.1) ? Except that these function performs QC, >> Normalization, Summarization etc. I am lacing clear understanding how >> those methods are implemented on Exon arrays , when compared to 3' >> prime arrays ? for instance,, In 3'-IVT arrays, I have the control of >> Normalizing within the group or across the group, where as in this >> exon array analysis I'm not sure what the function does ? I believe a detailed understand on the FIRMA paper will help here and help you be more precise what is unclear to you. >> >> 3) I am not sure what other applications i can use with the end result >> of summarization / FIRMA. can i get a list of differentially >> expressed genes ? splicing variant list ? upload the list of genes >> into pathway studio for further analysis ? What methods should i use ? >> can i use similar statistics that i use for 3' IVT arrays ? ( t-test, >> Bonferroni etc. ?) When you get to this level of analysis, you have to move your analysis to Bioconductor to use its package for further analysis and dealing with genomic annotations etc. Then extractDataFrame() is the best way to extract the data in a standard format. Hope this helps a bit. /Henrik >> >> Please enlighten me a bit on this exon array analysis >> >> Thank you, >> Sundar > > -- > When reporting problems on aroma.affymetrix, make sure 1) to run the latest > version of the package, 2) to report the output of sessionInfo() and > traceback(), and 3) to post a complete code example. > > > You received this message because you are subscribed to the Google Groups > "aroma.affymetrix" group with website http://www.aroma-project.org/. > To post to this group, send email to aroma-affymetrix@googlegroups.com > To unsubscribe and other options, go to http://www.aroma-project.org/forum/ > -- When reporting problems on aroma.affymetrix, make sure 1) to run the latest version of the package, 2) to report the output of sessionInfo() and traceback(), and 3) to post a complete code example. You received this message because you are subscribed to the Google Groups "aroma.affymetrix" group with website http://www.aroma-project.org/. 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