[BioC] Limma - data preparation problem

axel.klenk at actelion.com axel.klenk at actelion.com
Mon Nov 16 17:45:28 CET 2009


Dear Joanna,

if I understand your problem correctly, then yes, you'll have to rearrange
your columns
because normally channels correspond to dyes rather than sample classes,
such that
Ch1 <--> Cy3 <--> G/Gb and Ch2 <--> Cy5 <--> R/Rb and following this
convention,
limma will expect the sample class from the targets' Cy3 column in Ch1 as
specified
by the G = "Ch1 Median" in your scolumns -- whereas you're saying that Ch1
always
contains the same class, 'cykl'.

Therefore, you'd have to rename the columns in your XL files, e.g.
according to their
respective dyes, specify these dye columns in your call to read.maimages()
and their
sample classes in the targets.

Hope this makes sense :-),

 - axel


Axel Klenk
Research Informatician
Actelion Pharmaceuticals Ltd / Gewerbestrasse 16 / CH-4123 Allschwil /
Switzerland



                                                                           
             "Joanna Goscik"                                               
             <j.goscik at pb.edu.                                             
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                                                                   Subject 
                                       [BioC] Limma - data preparation     
             11/16/2009 04:41          problem                             
             PM                                                            
                                                                           
                                                                           
                                                                           
                                                                           
                                                                           





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

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