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

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.="" pl=""> To Sent by: "bioconductor at stat.math.ethz.ch" bioconductor-boun <bioconductor at="" stat.math.ethz.ch=""> ces at stat.math.eth cc z.ch 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 _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor The information of this email and in any file transmitted with it is strictly confidential and may be legally privileged. It is intended solely for the addressee. If you are not the intended recipient, any copying, distribution or any other use of this email is prohibited and may be unlawful. In such case, you should please notify the sender immediately and destroy this email. The content of this email is not legally binding unless confirmed by letter. Any views expressed in this message are those of the individual sender, except where the message states otherwise and the sender is authorised to state them to be the views of the sender's company. For further information about Actelion please see our website at http://www.actelion.com