[BioC] Limma - data preparation problem

[Joanna Goscik]

Hello everyone,
 
My name is Joanna Goscik and I work as an assistant at the Bialystok Technical University, Poland.
 
I am quite new to limma package and a little bit confused about reading data into the R environment. 
 
The data I am trying to analyze has the following form:
- six excel files converted co text files - six different arrays - two channel data
 
The commands I use to read data is:
 
> library("limma")
> setwd("P:\\dane");
> targets=readTargets("targets.txt", row.names="Name");
> scolumns=list(G="Ch1 Median",Gb="Ch1 B Mean",R="Ch2 Median",Rb="Ch2 B Mean");
> sannotation= c("Name","ID");
> RG=read.maimages(targets,columns=scolumns,annotation=sannotation);
 
My targets file (tab delimited) has the following form:
 
Name FileName Cy3 Cy5
023_647 023_647.txt ciaza cykl
024_555 024_555.txt cykl ciaza
025_647 025_647.txt ciaza cykl
037_555 037_555.txt cykl ciaza
038_555 038_555.txt cykl ciaza
041_647 041_647.txt ciaza cykl
 
After reading the data I execute the following commands to obtain a list of genes with significantly differenf expression classes "ciaza" and "cykl":
 
> design=modelMatrix(targets,ref="cykl");
> RGb=backgroundCorrect(RG,method="normexp");
> MA=normalizeWithinArrays(RGb, method="loess",bc.method="none");
> MA=normalizeBetweenArrays(MA);
> fit=lmFit(MA,design);
 
In my input data - in ALL excel files - I have data from class "cykl" in column Ch1 and data from class "ciaza" in column Ch2. The difference between those files is, that in files containing "647" in the name of the file Channel1 is dyed Alexa 647 and Channel2 is dyed with Alexa 555. In files containing "555" in the name of the file Channel1 is dyed Alexa 555 and Channel2 is dyed with Alexa 647.
 
The question is - do I have to prepare my input files (rearrange colums for Channel1 and Channel2) so that they match the schema from targets file? I mean: after the rearrangement - in files containing "647" I woud have in column Ch1 data from class "ciaza" and in files containing "555" in the name I would have in column Ch1 data from class "cykl".
 
I would really appreciate Your help, my kindest regards,
Joanna Goscik
 
Bialystok Technical University
Faculty of Computer Science
Department of Software Engeneering