Hi Florence,

I've copied the aroma.affymetrix mailing list, just in case others have extra 

On Jun 25, 2011, at 1:05 AM, Florence Jaffrezic wrote:

> Dear Professor Robinson,
> I am a French researcher working near Paris, and was trying to run an 
> analysis with Firma to detect alternative splicing.
> I wanted to re-analyze the colon cancer data available on the affymetrix 
> website for the Human exon chip, and used the
> R code provided in the Firma vignette. I ran the FirmaModel on the plmEx 
> object as shown below:
> plmEx <- ExonRmaPlm(csN,mergeGroups=FALSE)
> fit(plmEx, verbose=verbose)
> firma <- FirmaModel(plmEx)
> fit(firma, verbose=verbose)
> fs <- getFirmaScores(firma)
> scores=extractDataFrame(fs)
> I then obtain one score for each exon and each chip.
> I have a few questions:
> 1) First, there are 10 biological replicates in each condition. How should I 
> combine the Firma scores obtained for each replicate ?

You could take the average, or perhaps a more robust median.

> 2) For the detection of alternative splicing, I saw in the literature that we 
> should take the log2 value of the scores
> fsScores <- log2(extractDataFrame(fs)), and that "large negative values will 
> indicate exon skipping".
> Is this correct ? Is there a cut-off value that can be used for these scores 
> to detect alternative splicing ?

One thing: I see it more as detection of 'differential' splicing, i.e. 
different experimental conditions express transcripts differently.  But, yes, 
this is correct, you are looking for 'extreme values' and negative ones may 
indicate exon skipping.  As far as I know, we never set cutoffs because 
assigning estimated FDRs to them is non-trivial.


> Thank you very much in advance for your help,
> Florence
> -----------------------------------------------
> Dr Florence Jaffrezic
> Bat 211
> 78352 Jouy-en-Josas Cedex
> France
> Tel: (+33) 1 34 65 21 94
> Fax: (+33) 1 34 65 22 10
> -----------------------------------------------
> <Firma_Exon_question.r>

Mark Robinson, PhD (Melb)
Epigenetics Laboratory, Garvan
Bioinformatics Division, WEHI
e: mrobin...@wehi.edu.au
e: m.robin...@garvan.org.au
p: +61 (0)3 9345 2628
f: +61 (0)3 9347 0852

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