Pre-processing .CEL files in R

This post shows you how to compare data from two separate studies without the hassle of tackling batch effects, etc. By scaling and centring the data in both studies, you can look for trends in the data and look for gene expression changes that go in a similar direction.

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One of the most efficient ways to pre-process microarray data in R, is to use the oligo R/Bioconductor package. In a few lines of code you can go from raw .CEL files to a normalized data matrix you can work with for downstream analysis. It is particularly useful, if you wish to reanalyse a subset of .CEL files from a previously published dataset.

For example, say we are interested in comparing relative gene expression levels of Atf1, Atf3, Brca1 i in the lung, liver, and bone marrow of CB17 mice to previously published mouse tissue expression data on GEO. From our study, we have a data matrix of normalized expression values we obtained from our microarray study stored in CB17mat object.

First, we will scale all gene expression values by row to obtain center and scale these values to have an idea which organ expresses the highest levels of Atf1, Atf3, and Brca1 relative to the others in our study. To do this we will use the scale() with the default settings center=TRUE and scale=TRUE. Since the scale default function scales and centers columns we will need to transpose our matrix before proceeding.


# From your gene expression matrix stored in CB17mat
genes = c("Atf1", "Atf3", "Brca1")
CB17scal <- t(apply(CB17mat[genes, ], 1, scale))

# We will also add the missing column names to our scaled matrix
colnames(CB17scal) <- colnames(CB17mat)

# You can also plot a heatmap to look at the effects of the scaling on the expression levels across the tissues using the gplots package

# Clusters the rows (and potentially columns) by Pearson correlation as distance method
corrdist = function(x) as.dist(1-cor(t(x), method="pearson"))

# and Ward method as the agglomeration method
hclust.avl = function(x) hclust(x, method="ward.D2")

# with dendrogram="row" and Colv=NA, we are only clustering the rows i.e. genes
png(filename="example1.png", width=10, height=10, units = 'in', res = 300)
heatmap.2(CB17scal, dendrogram="row", Colv=NA, scale="none", key.title="", col=rev(redblue(250)), trace='none', cexCol=1.2, cexRow=1.5, hclustfun=hclust.avl, distfun=corrdist, margins=c(8, 12))
graphics.off()

example1

As you can see Atf1 and Brca1 are relatively higher in bone marrow, whereas Atf3 is relatively higher in the lung compared to the other organs.

Now let’s analyze the liver, lung and bone marrow data from the Large-scale analysis of the human and mouse transcriptomes study from Su et al, 2002, PNAS Apr 2;99(7):4465-70. The individual files from the liver, lung and bone marrow were downloaded from GSE97 and the data was normalized with the oligo package as follows:


# Move the CEL files to ~/MY_WORKING_DIRECTORY/filesToAnalyse
# Set the working directory to the folder you would like to save your results
setwd("~/MY_WORKING_DIRECTORY")

# Load the libraries needed for the analysis
library("oligo")
library("pd.mg.u74a")

# Load the packages needed for the analysis
# You can choose to save the CEL files for your tissues of interest
geneCELs <- list.celfiles("~/MY_WORKING_DIRECTORY/filesToAnalyse", full.names=TRUE)

affyGeneFS <- read.celfiles(geneCELs)
affyGeneFS

# RMA at the probet level
geneCore <- rma(affyGeneFS)

# Inspect the eset object
head(exprs(geneCore))
head(featureNames(geneCore))

# For the featureData info for the array used in this study
library("annotate")
#library("mouse4302.db")

source("http://bioconductor.org/biocLite.R")
library("mgu74a.db")
probeids <- featureNames(geneCore)
geneAnnotation <- select(mgu74a.db, probeids, c("SYMBOL", "ENTREZID", "GENENAME"), multiVals="first")

# Save the ESET data and annotation
save(geneCore, geneAnnotation, file="geneCoreTissueV2.RData")
saveRDS(geneCore, "geneCore.rds")
saveRDS(geneAnnotation, "geneAnnotation.rds")

# Convert the eset object to a matrix to get the gene expression values
# for Atf1, Atf3, and Brca1
M1 <- exprs(geneCore)
head(M1)

Gene_Symbols <- sapply(rownames(M1), getGeneSymbol, df=geneAnnotation)
DM1 <- data.frame(M1, Gene_Symbols=Gene_Symbols[rownames(M1)], stringsAsFactors=FALSE)

# Use can create a function to select the probe with the top interquantile range with IQR()
# to represent the gene expression value or use the TopIqrSymbolMatrix()
# in the "Useful Functions to Work with Microarrays" post (coming soon!)

tissueM1 <- TopIqrSymbolMatrix(DM1)
colnames(tissueM1)
tissueExprs <- tissueM1[, 1:ncol(M1)]
rownames(tissueExprs) <- tissueM1$Gene_Symbols
head(tissueExprs[, 1:4])

# To get the geo file ids
geoColnames <- gsub(".CEL", "", colnames(tissueExprs))

# Get the new colnames based on tissue
# Create an excel sheet with the GEO .CEL file id and tissue sample names you would like to use
# Load the excel sheet as a data frame using the gdata package
library("gdata")
TissueNames <- read.xls("GSE97_FileSampleIdentifier.xlsx", sheet=1, stringsAsFactors=FALSE, row.names=1)
TissueSamples <- TissueNames[geoColnames, 2]

# Replace the tissue exprs with the tissue names
colnames(tissueExprs) <- TissueSamples
saveRDS(tissueExprs, "tissueExprs.rds")
head(tissueExprs)

# Now create a matrix for our 3 genes of interest
genes = c("Atf1", "Atf3", "Brca1")
genesMat <- as.matrix(M1[genes, ])

# Let's scale and center the expression values
genesMatScal <- t(apply(genesMat, 1, scale))

# Add the column names
colnames(genesMatScal) <- rep(c("liver", "lung", "bone marrow"), each=2)

png(filename="example1b.png", width=10, height=10, units = 'in', res = 300)
heatmap.2(genesMatScal, dendrogram="row", Colv=NA, scale="none", key.title="", col=rev(redblue(250)), trace='none', cexCol=1.2, cexRow=1.5, hclustfun=hclust.avl, distfun=corrdist, margins=c(8, 12))
graphics.off()

example1b

By comparing both heatmaps, we can see that Atf1 and Brca1 have more similar expression patterns across the tissues than Atf3 in these mice compared to our CB17 mice tissue data.
Therefore, Brca1 and Atf1 tissue difference might be more consistent among the tissues from different mice.

Alternatively, it could just be an artifact of the small sample size and/or how the samples were processed before running the arrays. That being said, it’s a good thing to reanalyse studies and compare with your arrays to get an idea of sample variability and how the experimental design, pre- and post- processing affects the overall interpretation of the results.

Feel free to leave comments or email me at info@rjbioinformatics.com.

 

 

Author: radiaj

I am a Research Scientist & Bioinformatician who specializes in Immunology and Cancer Biology. I routinely use R and other programing languages to explore genomic data of cancer cells to identify molecular changes and risk factors that contributes to cancer development.

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