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Tutorial On Microarray Analysis Using Bioconductor and R (Sample Study)

This document provides an introduction to analyzing microarray data using Bioconductor and R. It describes resveratrol's effect on lung cancer cells and the microarray data available from this study. The tasks involve importing and normalizing the data, comparing resveratrol-treated cells to controls, filtering differentially expressed genes, and clustering and viewing the analysis results. Code is provided to load packages, import the sample data and phenotype file, normalize the data using RMA, perform differential expression analysis with limma, and create a heatmap of differentially expressed genes.

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154 views

Tutorial On Microarray Analysis Using Bioconductor and R (Sample Study)

This document provides an introduction to analyzing microarray data using Bioconductor and R. It describes resveratrol's effect on lung cancer cells and the microarray data available from this study. The tasks involve importing and normalizing the data, comparing resveratrol-treated cells to controls, filtering differentially expressed genes, and clustering and viewing the analysis results. Code is provided to load packages, import the sample data and phenotype file, normalize the data using RMA, perform differential expression analysis with limma, and create a heatmap of differentially expressed genes.

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Coțovanu Iulian
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341: Introduction to Bioinformatics

Tutorial on Microarray Analysis using Bioconductor and R


(Sample Study)

February 11, 2011

Tools Used
• Bioconductor, http://www.bioconductor.org/, provides open source tools for the
analysis and comprehension of high-throughput genomic data
• R, http://www.r-project.org/, R is a free software environment for statistical computing
and graphics

Data Used
• Resveratrol effect on lung carcinoma cell line - Analysis of lung carcinoma A549 cells
treated with resveratrol. Resveratrol is a phytoestrogen found in red wine. Results
provide insight into protective effect of resveratrol against lung cancer. Available from
the Gene Expression http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS2966
• 4 CEL files (Micro-array output) Data {GSM228717.CEL, GSM228718.CEL,
GSM228719.CEL, GSM228720.CEL} available from above link
• 1 Sample Phenotype Mapping {GDS296-pheno.csv}

Tasks
1. Import and Normalise data
2. Compare tissue treated with Resveratrol with similar tissue treat with an Ethanol control
3. Filter most differentially expressed genes
4. Cluster and View Analysis
R Code
## Step 1- Load packages
library(affy)
library(limma)

## Step 2 - Import Sample Data


setwd("C:/Users/asrowe/Documents/Tutorial/celfiles")
phenoData <- read.AnnotatedDataFrame("GDS296-pheno.csv" ,header=TRUE, sep="\t")

## Step 3 - Import and Normalise Data using functions from the Bioconductor affy
package
eset <- justRMA(phenoData=phenoData)

## Step 4 - Differential expression filtering using Bioconductor limma package


design <- model.matrix(~substance, pData(eset))
fit <- lmFit(eset, design) # fit each probeset to model
efit <- eBayes(fit) # empirical Bayes adjustment
tt <-topTable(efit, coef=2) # table of differentially expressed probesets
fix(tt) #View

# Step 5 - H Cluster and Dendrogram from the R stats package


selected <- p.adjust(efit$p.value[, 2]) <0.05 #Select Adusted Points
esetSel <- eset [selected, ] #Filter Selected Points
heatmap(exprs(esetSel)) #Display Heatmap

GDS296-pheno.csv
Sample substance
GSM228717 Control
GSM228718 Wine
GSM228719 Wine
GSM228720 Wine

Notes:
For those interested in more information on Normalisation methods and why RMA (justRMA) is
used, start here:
Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003), A Comparison of
Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and
Variance. Bioinformatics 19(2):185-193

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