Hi, I'm not a expert on exon analysis, but I'll try to answer some of your questions.
On Tue, Sep 13, 2011 at 5:45 AM, rene.rotterdam <rene.boettche...@googlemail.com> wrote: > Dear all, > > I fairly new to the field of Exon Arrays and my experiences with > Microarrays in general are quite limited, so I have a couple of > questions concerning the analysis of Exon Array data. > > First of all, I would like to do an RMA (or GCRMA) based on the > validated genes of the core probeset (so bg, qn + sum). The way I > understood the literature, this normalizes ALL probesets based on the > intensity values of the core probeset (which is also what I intend to > do). Actually, this depends on what settings/intermediate preprocessing methods you use. For instance, in vignette 'Human exon array analysis' [http://aroma-project.org/node/37] the quantile normalization step is: qn <- QuantileNormalization(csBC, typesToUpdate="pm") which says that it operates only on PM probes. The definition of what a PM probe is is defined by the CDF used. See also page 'Empirical probe-signal densities and rank-based quantile normalization' [http://aroma-project.org/node/141] for more information on how the settings for QuantileNormalization affect the outcome. > Afterwards, I want to extend my analysis on the full probeset, as > I will focus on some genes that are not included in 'core'. > I read about this procedure in a paper on APT and R by H. Lockstone > and wanted to implement it via aroma. The way the 'Human exon array analysis' vignette is outlined, changing to another CDF will affect what probes are considered PMs and hence also affect the preprocessing output. To me (my personal opinion) this is a bit unfortunate; ideally the early on preprocessing steps that are designed to normalize probe-level data (not summarized) should be invariant to the (CDF) annotation. An alternative approach is to use the "default" CDF throughout these steps, as done/suggested in thread 'Help Regarding GCRMA Human Exon' (Dec 2, 2010) [https://groups.google.com/forum/#!topic/aroma-affymetrix/QD6o-3XiifM/discussion]. > > Am I mislead in this case? > I tried to realize this by doing a GCRMA on 'core', but the resulting > probe intensities are to few (~285k) and the probes I'm looking for > are not included. > > Before that, I did a regular RMA on the full probeset and I got all > the probes /the intensities I was looking for, but I realized that > normalizing to not validated genes may not be the best idea. As I indicated above, from my experience with other chiptypes I *think* it is ok to probe-level normalization using the full CDF. However, I haven't investigated the impact of doing this, so I might be wrong. I leave it to others with more exon-array experience to comment on this/correct me. > > It would be a great help if you could set my thoughts straight and if > I'm correct tell me how to use aroma to achieve what I want. > > Note: I assembled my script as described in > http://aroma-project.org/node/37 > and > https://groups.google.com/forum/#!topic/aroma-affymetrix/QD6o-3XiifM/discussion FYI, it is always easier if you cut'n'paste you complete script to your message - avoids second guessing. Not much help, but at least some pointers and suggestions. /Henrik > > Thank you in advance for your help. > > With best regards, > Rene > > -- > 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/. 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/
