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Question: Limma paired sample analysis
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gravatar for anandprem1792
10 weeks ago by
anandprem17920 wrote:

I have an Affymetrix single channel DNA microarray dataset where normal and diseased tissue are taken from the same organ from 5 persons. So I have 10 CEL files with me. Now I want to analyse the differential gene expression between the normal and diseased tissues. I think I should use Limma Paired samples design matrix and compute paired moderated t test. Limma user guide. 9.4.1 pg no: 42 and 43. 

Can anyone please confirm whether my approach is correct or Should I follow different approach ?

My phenodata file looks like this 

FileName Subject Tissue
100.CEL 1 NORMAL
101.CEL 1 DISEASED
102.CEL 2 NORMAL
103.CEL 2 DISEASED
104.CEL 3 NORMAL
105.CEL 3 DISEASED
106.CEL 4 NORMAL
107.CEL 4 DISEASED
108.CEL 5 NORMAL
109.CEL 5 DISEASED

My code 

library(affy)

library(limma)

# Read all CEL FIles and put into an affybatch 
affy.data = ReadAffy()

#Importing the phenotype data
pData(affy.data) = read.table("phenodata.txt",  header=TRUE, row.names=1, as.is=TRUE)


#Visualise the phenotype data
pData(affy.data)

# Normalize the data
eset = rma(affy.data)

# DIFFERENTALLY GENE EXPRESSION ANALYSIS

library(limma)
pData(eset)
Subject <- factor(eset$Subject)

Tissue <- factor(eset$Tissue, levels = c("Normal", "Diseased"))

design <- model.matrix(~Subject+Tissue)
design

fit <- lmFit(eset, design)
eBayesfit <- eBayes(fit)

View(eBayesfit)
topTable(eBayesfit, coef="TissueDiseased")

ADD COMMENTlink modified 10 weeks ago • written 10 weeks ago by anandprem17920
1
gravatar for Aaron Lun
10 weeks ago by
Aaron Lun17k
Cambridge, United Kingdom
Aaron Lun17k wrote:

Looks fine to me. You should probably do some filtering on abundance, though. See the relevant section of the user's guide and case studies for examples. 

P.S. limma should be a separate tag, otherwise the maintainers don't get notified. 

ADD COMMENTlink modified 10 weeks ago • written 10 weeks ago by Aaron Lun17k

Thank you for the clarification. 

ADD REPLYlink written 10 weeks ago by anandprem17920
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