HuEx v2: Aroma - Firma scores NaN

Question

[Bioinformatics_NewComer]

I've Human Exon v2 microarray expression data for 223 people. I think I'm lost on my way to analyse data using different packages, normalization, etc. Would really appreciate your help. Here, I'm using aroma to get FIRMA scores but I get 0 or NULL value in output. I'm working with five .cel files to get some thing up and running.

Code and steps:

library("aroma.affymetrix")
cdf <- AffymetrixCdfFile$byChipType("HuEx-1_0-st-v2")
print(cdf)

cs <- AffymetrixCelSet$byName("celfiles", cdf=cdf) # rawdata in cwd. HuEx-1_0-st-v2 in celfiles # this should match cdf file names too
print(cs)
bc <- RmaBackgroundCorrection(cs) #backgroun
csBC <- process(bc,verbose=verbose)

qn <- QuantileNormalization(csBC, typesToUpdate="pm") #quantile norm
csN <- process(qn, verbose=verbose)

getCdf(csN) ##get CDF infor

plmTr <- ExonRmaPlm(csN, mergeGroups=TRUE) #entrire transcript #for exon-by-exon mergeGroup make FALSE
print(plmTr)

#----alternative splicing for transcript PLM - stage 1
firma <- FirmaModel(plmTr) #The FIRMA analysis only works from the PLM based on transcripts.
fit(firma, verbose=verbose)
fs <- getFirmaScores(firma)
cestrfs <- extractDataFrame(fs, units=1:3, addNames=TRUE) #extract the FIRMA scores -- but Nan

######-------------

#extract the estimates (transcript or probeset) use either - here transcript
#stage 2
cesTr <- getChipEffectSet(plmTr)
trFit <- extractDataFrame(cesTr, units=1:3, addNames=TRUE) # gives 0

I don't know what's wrong here.
For stage 1, FIRMA score per transcript is NAN and estimate for transcript is 0 in stage 2.
Aroma google groups is inactive. :(

Answer 1

[Kevin Blighe]

Please follow the tutorial posted by Henrik on the Aroma website: Vignette: FIRMA - Human exon array analysis

Note, in particular, the following warning:

Do not use the default CDF named HuEx-1_0-st-v2.cdf that is provided by Affymetrix.

You have to set-up a customised CDF file for this array type and version. In particular, execute the following as your first few commands:

chipType <- "HuEx-1_0-st-v2"
cdf <- AffymetrixCdfFile$byChipType(chipType, tags="coreR2,A20070914,EP")
print(cdf)

You are also missing the following command after you create you CEL-set:

setCdf(cs, cdf)

Essentially, just follow the tutorial line by line...

Kevin

[Bioinformatics_NewComer]

cdf <- AffymetrixCdfFile$byChipType(chipType,tags="coreR2,A20070914,EP")

This doesn't work because I don't have annotation file that ends with coreR2, A20070914,EP

I get error as:

Error: Failed to create AffymetrixCdfFile object. Could not locate an annotation data file for chip type 'HuEx-1_0-st-v2' with tags 'coreR2,A20070914,EP' and with filename extension 'cdf'.

[Kevin Blighe]

Oh, did you not see the statetment:

This custom CDF (and also more current versions) can be downloaded from subpage 'Affymetrix-Defined transcript clusters' on Page HuEx-1_0-st-v2.

When you go to the link in that statement on the ARoma website, you can then get the CDF and other necessary annotation files from here: http://www.aroma-project.org/chipTypes/HuEx-1_0-st-v2/ (see 'Resources')