Dear All, 
 
I have microRNA microaaray expression data from Agilent platform exported by
the Agilent Feature Extraction (AFE) image analysis software. Initially, we
performed quantile normalisation using GeneSpring (version 12.1) software
(demo version). Being an R user, I wanted to do rest of the analysis by
using R. So, I repeated quantile normalisation using "AgiMicroRna" library.
Before further analysis, I compared raw ( gTotalProbeSignal(raw) from
GeneSpring and ddTGS$TGS from AgiMicroRNA) and quantile normalised (
gTotalProbeSignal(normalized)  from GeneSpring and ddNORM$TGS from
AgiMicroRNA ) output of two softwares. Raw output of two softwares are the
same. However, there are some differences in the some of the quantile
normalised output, although majority of the output are very similar. I
suppose GeneSpring and  "AgiMicroRna" are using the same algorithm and they
should produce the same results. I was wondering whether I am doing
something wrong during the procees? Does any one faced similar situation? 
 
My sessionInfo() and steps I am following in AgiMicroRna are below. 
 
Any comment, help deeply appreciated.
 
Kind Regards
 
Seyit Ali
 
============================================================================
==============
 
> sessionInfo()
R version 2.15.1 (2012-06-22)
Platform: i386-pc-mingw32/i386 (32-bit)
 
locale:
[1] LC_COLLATE=English_Australia.1252  LC_CTYPE=English_Australia.1252
LC_MONETARY=English_Australia.1252
[4] LC_NUMERIC=C                       LC_TIME=English_Australia.1252    
 
attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     
 
other attached packages:
 [1] AgiMicroRna_2.6.0     affycoretools_1.28.0  KEGG.db_2.7.1
GO.db_2.7.1           RSQLite_0.11.1       
 [6] DBI_0.2-5             AnnotationDbi_1.18.1  preprocessCore_1.18.0
affy_1.34.0           limma_3.12.1         
[11] Biobase_2.16.0        BiocGenerics_0.2.0   
 
loaded via a namespace (and not attached):
 [1] affyio_1.24.0       annaffy_1.28.0      annotate_1.34.1
BiocInstaller_1.4.7 biomaRt_2.12.0      Biostrings_2.24.1  
 [7] Category_2.22.0     gcrma_2.28.0        genefilter_1.38.0
GOstats_2.22.0      graph_1.34.0        grid_2.15.1        
[13] GSEABase_1.18.0     IRanges_1.14.4      lattice_0.20-6      RBGL_1.32.1
RCurl_1.91-1.1      splines_2.15.1     
[19] stats4_2.15.1       survival_2.36-14    tools_2.15.1        XML_3.9-4.1
xtable_1.7-0        zlibbioc_1.2.0  
 
============================================================================
============
 
"AgiMicroRna" steps
 

library(AgiMicroRna)
 
sessionInfo()

AFE.TGS = TRUE

half=  FALSE  # ddTGS signal with 'half method'
offset=0    
makePLOT=FALSE  

 
# NORMALIZATION of ddTGS 
NORMmethod="quantile"
 makePLOTpre=TRUE
 makePLOTpost=TRUE 

# FILTERING PROBES 
control = TRUE 
IsGeneDetected = TRUE
wellaboveNEG = FALSE 
limIsGeneDetected = 50 
limNEG = 25
makePLOT = FALSE

#  READING THE Target File
targets=readTargets(infile="targets.txt",verbose=TRUE)
# READING THE DATA (RGList)
dd=readMicroRnaAFE(targets,verbose=TRUE)
names(dd)

 
# PRE-PROCESSING # USING AFE gTotalGeneSignal  &  # NORMALIZATION
#   tgsMicroRna: creates an uRNAList object that contains the Total Gene
Signal computed
#   by the Agilent Feature Extraction algorithms
#  tgsNormalization: creates an uRNAList object containing the Normalized
Total
#   Gene Signal in log 2 scale

if(AFE.TGS) {
message('pre-processing: AFE TGS')
      cat('\n')
ddTGS = tgsMicroRna(dd, half = FALSE, makePLOT = FALSE,verbose = FALSE)
ddNORM = tgsNormalization(ddTGS, "quantile", makePLOTpre = FALSE,
makePLOTpost = FALSE, targets, verbose = TRUE)
}

#  Obtaining raw data 
 
TGSTGS<-cbind(ddTGS$genes, ddTGS$TGS)
 
# Obtaining quantile normalised data
 
NormData<-cbind(ddNORM$genes, ddNORM$TGS)
 
============================================================================
=====
 
 
 
--------------------------------------------------------
Dr. Seyit Ali KAYIS 
Selcuk University, Faculty of Agriculture 
Kampus/Konya, Turkey 

skayis@selcuk.edu.tr, s_a_kayis@yahoo.com, s_a_kayis@hotmail.com

Tel: +90 332 223 2830 Mobile: +90 535 587 1139 

Greetings from Konya, Turkey 
http://www.ziraat.selcuk.edu.tr/skayis/ 
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