RA2111030010201

LAKKAVARAM S M SHRIYA VARNITA

COMPUTATIONAL GENOMICS ASSIGNMENT

MICROARRAY ANALYSIS

## ---- setup,
include=FALSE-------------------------------------------------------

library(knitr)
knitr::opts_chunk$set(echo = TRUE, message = FALSE, warning = FALSE,
comment = NA, prompt = TRUE, tidy = FALSE,
fig.width = 7, fig.height = 7, fig_caption =
TRUE,
cache=FALSE)
Sys.setlocale("LC_TIME", "C")

##
echo=FALSE----------------------------------------------------------

if(!(require(printr))) {
install.packages(
'printr',
type = 'source',
repos = c('http://yihui.name/xran', 'http://cran.rstudio.com')
)
}

## ----dataset, fig.cap="A simplified view of a gene expression

matrix", echo=FALSE----------------------------------------------
knitr::include_graphics("figures/Figure1.jpg")

## ----MDAProcess, fig.cap="The microarray data analysis process",

echo=FALSE----------------------------------------------------
knitr::include_graphics("figures/Figure2.png")

## ----CreateFolders, warning=FALSE,
eval=FALSE----------------------------------------------------------

## setwd(".")
## dir.create("data")
## dir.create("results")

##
ReadTargets---------------------------------------------------------

targets <- read.csv2("./data/targets.csv", header = TRUE, sep = ";")

knitr::kable(
targets, booktabs = TRUE,
caption = 'Content of the targets file used for the current
analysis')
## ----installBioC, message=FALSE, warning=FALSE,
eval=FALSE----------------------------------------------------------
## if (!requireNamespace("BiocManager", quietly = TRUE))
## install.packages("BiocManager")
## BiocManager::install()

## ----installPackages, message=FALSE, warning=FALSE,

eval=FALSE----------------------------------------------------------

## install.packages("knitr")
## install.packages("colorspace")
## install.packages("gplots")
## install.packages("ggplot2")
## install.packages("ggrepel")
## install.packages("htmlTable")
## install.packages("prettydoc")
## install.packages("devtools")
## install.packages("BiocManager")
## BiocManager::install("oligo")
## BiocManager::install("pd.mogene.2.1.st")
## BiocManager::install("arrayQualityMetrics")
## BiocManager::install("pvca")
## # NOT NEEDED UNTIL ANALYSES ARE PERFORMED
## BiocManager::install("limma")
## BiocManager::install("genefilter")
## BiocManager::install("mogene21sttranscriptcluster.db")
## BiocManager::install("annotate")
## BiocManager::install("org.Mm.eg.db")
## BiocManager::install("ReactomePA")
## BiocManager::install("reactome.db")

## ----ReadCELfiles, message=FALSE, results='hide',

warning=FALSE-------------------------------------------------------

library(oligo)
celFiles <- list.celfiles("./data", full.names = TRUE)
library(Biobase)
my.targets <-read.AnnotatedDataFrame(file.path("./
data","targets.csv"),
header = TRUE, row.names = 1,
sep=";")
rawData <- read.celfiles(celFiles, phenoData = my.targets)

##
ChangeName----------------------------------------------------------

my.targets@data$ShortName->rownames(pData(rawData))
colnames(rawData) <-rownames(pData(rawData))

head(rawData)
## ----QCRaw, message=FALSE, warning=FALSE,
eval=FALSE----------------------------------------------------------

## library(arrayQualityMetrics)
## arrayQualityMetrics(rawData)

## ----QCRawDataRes, fig.cap="Aspect of the summary table, in the

index.html file, produced by the arrayQualityMetrics package on the
raw data", echo=FALSE----
knitr::include_graphics("figures/Figure3.png")

##

library(ggplot2)
library(ggrepel)
plotPCA3 <- function (datos, labels, factor, title, scale,colores,
size = 1.5, glineas = 0.25) {
data <- prcomp(t(datos),scale=scale)
# plot adjustments
dataDf <- data.frame(data$x)
Group <- factor
loads <- round(data$sdev^2/sum(data$sdev^2)*100,1)
# main plot
p1 <- ggplot(dataDf,aes(x=PC1, y=PC2)) +
theme_classic() +
geom_hline(yintercept = 0, color = "gray70") +
geom_vline(xintercept = 0, color = "gray70") +
geom_point(aes(color = Group), alpha = 0.55, size = 3) +
coord_cartesian(xlim = c(min(data$x[,1])-5,max(data$x[,1])+5)) +
scale_fill_discrete(name = "Group")
# avoiding labels superposition
p1 + geom_text_repel(aes(y = PC2 + 0.25, label =
labels),segment.size = 0.25, size = size) +
labs(x =
c(paste("PC1",loads[1],"%")),y=c(paste("PC2",loads[2],"%"))) +
ggtitle(paste("Principal Component Analysis for: ",title,sep="
"))+
theme(plot.title = element_text(hjust = 0.5)) +
scale_color_manual(values=colores)
}

## ----PCARaw, message=FALSE, fig.cap="Visualization of the two

first Principal Components for raw data"-------------------------
plotPCA3(exprs(rawData), labels = targets$ShortName, factor =
targets$Group,
title="Raw data", scale = FALSE, size = 3,
colores = c("red", "blue", "green", "yellow"))

## ----BoxplotRaw, message=FALSE, fig.cap="Boxplot for arrays

intensities (Raw Data)"--------------------------------------------
boxplot(rawData, cex.axis=0.5, las=2, which="all",
col = c(rep("red", 3), rep("blue", 3), rep("green", 3),
rep("yellow", 3)),
main="Distribution of raw intensity values")

##
Normalization-------------------------------------------------------

eset_rma <- rma(rawData)

## ----QCNorm, message=FALSE, warning=FALSE,

eval=FALSE----------------------------------------------------------

## arrayQualityMetrics(eset_rma, outdir = file.path("./results",

"QCDir.Norm"), force=TRUE)

## ----QCNormDataRes, fig.cap="Aspect of the summary table, in the

index.html file, produced by the arrayQualityMetrics package on
normalized data", echo=FALSE----
knitr::include_graphics("figures/Figure6.png")

## ----fig:PCANorm, message=FALSE, fig.cap="Visualization of first

two principal components for normalized data"-----------------
plotPCA3(exprs(eset_rma), labels = targets$ShortName, factor =
targets$Group,
title="Normalized data", scale = FALSE, size = 3,
colores = c("red", "blue", "green", "yellow"))

## ----BoxplotNorm, message=FALSE, fig.cap="Distribution of

intensities for normalized data"------------------------------------
boxplot(eset_rma, cex.axis=0.5, las=2, which="all",
col = c(rep("red", 3), rep("blue", 3), rep("green", 3),
rep("yellow", 3)),
main="Boxplot for arrays intensity: Normalized Data")

## ----BatchDetection, message=FALSE,
warning=FALSE-------------------------------------------------------

#load the library

library(pvca)
pData(eset_rma) <- targets
#select the threshold
pct_threshold <- 0.6
#select the factors to analyze
batch.factors <- c("Genotype", "Temperature")
#run the analysis
pvcaObj <- pvcaBatchAssess (eset_rma, batch.factors, pct_threshold)
## ----plotPVCA, fig.cap="Relative importance of the different
factors -genotype, temperature and interaction- affecting gene
expression"----
#plot the results
bp <- barplot(pvcaObj$dat, xlab = "Effects",
ylab = "Weighted average proportion variance",
ylim= c(0,1.1),col = c("mediumorchid"), las=2,
main="PVCA estimation")
axis(1, at = bp, labels = pvcaObj$label, cex.axis = 0.75, las=2)
values = pvcaObj$dat
new_values = round(values , 3)
text(bp,pvcaObj$dat,labels = new_values, pos=3, cex = 0.7)

## ----SDplot, fig.cap="Values of standard deviations allong all

samples for all genes ordered from smallest to biggest"---------
sds <- apply (exprs(eset_rma), 1, sd)
sdsO<- sort(sds)
plot(1:length(sdsO), sdsO, main="Distribution of variability for all
genes",
sub="Vertical lines represent 90% and 95% percentiles",
xlab="Gene index (from least to most variable)", ylab="Standard
deviation")
abline(v=length(sds)*c(0.9,0.95))

## ----Filtering1, results='hide',
message=FALSE-------------------------------------------------------

library(genefilter)
library(mogene21sttranscriptcluster.db)
annotation(eset_rma) <- "mogene21sttranscriptcluster.db"
filtered <- nsFilter(eset_rma,
require.entrez = TRUE, remove.dupEntrez = TRUE,
var.filter=TRUE, var.func=IQR, var.cutoff=0.75,
filterByQuantile=TRUE, feature.exclude =
"^AFFX")

## ----FilterResults1, results='hide',
echo=FALSE----------------------------------------------------------

names(filtered)
class(filtered$eset)

##
FilterResults2------------------------------------------------------

print(filtered$filter.log)
eset_filtered <-filtered$eset
## ----SaveData1, results='hide',
message=FALSE-------------------------------------------------------

write.csv(exprs(eset_rma), file="./results/normalized.Data.csv")
write.csv(exprs(eset_filtered), file="./results/
normalized.Filtered.Data.csv")
save(eset_rma, eset_filtered, file="./results/normalized.Data.Rda")

##
LoadSavedData-------------------------------------------------------

if (!exists("eset_filtered")) load (file="./results/

normalized.Data.Rda")

## ---- DesignMatrix,
message=FALSE-------------------------------------------------------

library(limma)
designMat<- model.matrix(~0+Group, pData(eset_filtered))
colnames(designMat) <- c("KO.COLD", "KO.RT", "WT.COLD", "WT.RT")
print(designMat)

##
setContrasts--------------------------------------------------------

cont.matrix <- makeContrasts (KOvsWT.COLD = KO.COLD-WT.COLD,

KOvsWT.RT = KO.RT-WT.RT,
INT = (KO.COLD-WT.COLD) - (KO.RT-
WT.RT),
levels=designMat)
print(cont.matrix)

##
linearmodelfit------------------------------------------------------

library(limma)
fit<-lmFit(eset_filtered, designMat)
fit.main<-contrasts.fit(fit, cont.matrix)
fit.main<-eBayes(fit.main)
class(fit.main)

##
topTabs1------------------------------------------------------------

topTab_KOvsWT.COLD <- topTable (fit.main, number=nrow(fit.main),

coef="KOvsWT.COLD", adjust="fdr")
head(topTab_KOvsWT.COLD)
##
topTabs2------------------------------------------------------------

topTab_KOvsWT.RT <- topTable (fit.main, number=nrow(fit.main),

coef="KOvsWT.RT", adjust="fdr")
head(topTab_KOvsWT.RT)

##
topTabs3------------------------------------------------------------

topTab_INT <- topTable (fit.main, number=nrow(fit.main),

coef="INT", adjust="fdr")
head(topTab_INT)

## ----GeneAnnotation, message=FALSE,
warning=FALSE-------------------------------------------------------

annotatedTopTable <- function(topTab, anotPackage)

{
topTab <- cbind(PROBEID=rownames(topTab), topTab)
myProbes <- rownames(topTab)
thePackage <- eval(parse(text = anotPackage))
geneAnots <- select(thePackage, myProbes, c("SYMBOL", "ENTREZID",
"GENENAME"))
annotatedTopTab<- merge(x=geneAnots, y=topTab, by.x="PROBEID",
by.y="PROBEID")
return(annotatedTopTab)
}

##
annotateTopTables---------------------------------------------------

topAnnotated_KOvsWT.COLD <- annotatedTopTable(topTab_KOvsWT.COLD,

anotPackage="mogene21sttranscriptcluster.db")
topAnnotated_KOvsWT.RT <- annotatedTopTable(topTab_KOvsWT.RT,
anotPackage="mogene21sttranscriptcluster.db")
topAnnotated_INT <- annotatedTopTable(topTab_INT,
anotPackage="mogene21sttranscriptcluster.db")
write.csv(topAnnotated_KOvsWT.COLD, file="./results/
topAnnotated_KOvsWT_COLD.csv")
write.csv(topAnnotated_KOvsWT.RT, file="./results/
topAnnotated_KOvsWT_RT.csv")
write.csv(topAnnotated_INT, file="./results/topAnnotated_INT.csv")

## ---- annotatedTop,
echo=FALSE----------------------------------------------------------

short<- head(topAnnotated_KOvsWT.COLD[1:5,1:4])
# library(kableExtra)
# knitr::kable(
# short, booktabs = TRUE,
# caption = 'Annotations added to results "topTable" for the
comparison "KOvsWT.COLD"'
# )
show(short)

## ----volcanoPlot, fig.cap="Volcano plot for the comparison between

KO and WT in COLD temperature. The names of the top 4 genes (i.e.
the first four genes in the topTable) are shown in the plot"----
library(mogene21sttranscriptcluster.db)
geneSymbols <- select(mogene21sttranscriptcluster.db,
rownames(fit.main), c("SYMBOL"))
SYMBOLS<- geneSymbols$SYMBOL
volcanoplot(fit.main, coef=1, highlight=4, names=SYMBOLS,
main=paste("Differentially expressed genes",
colnames(cont.matrix)[1], sep="\n"))
abline(v=c(-1,1))

## ----saveVolcanos, echo=FALSE,
results='hide'------------------------------------------------------

pdf("figures/Volcanos.pdf")
for (i in colnames(cont.matrix)){
volcanoplot(fit.main, coef=i, highlight=4, names=SYMBOLS,
main=paste("Differentially expressed genes",i,
sep="\n"))
abline(v=c(-1,1))
}
dev.off()

##
decideTests.1-------------------------------------------------------

library(limma)
res<-decideTests(fit.main, method="separate", adjust.method="fdr",
p.value=0.1, lfc=1)

##
resumeDecideTests---------------------------------------------------

sum.res.rows<-apply(abs(res),1,sum)
res.selected<-res[sum.res.rows!=0,]
print(summary(res))

## ---- vennDiagram, fig.cap="Venn diagram showing the genes in

common between the three comparisons performed"------------------
vennDiagram (res.selected[,1:3], cex=0.9)
title("Genes in common between the three comparisons\n Genes
selected with FDR < 0.1 and logFC > 1")

##
data4Heatmap--------------------------------------------------------

probesInHeatmap <- rownames(res.selected)

HMdata <- exprs(eset_filtered)[rownames(exprs(eset_filtered)) %in%
probesInHeatmap,]

geneSymbols <- select(mogene21sttranscriptcluster.db,

rownames(HMdata), c("SYMBOL"))
SYMBOLS<- geneSymbols$SYMBOL
rownames(HMdata) <- SYMBOLS
write.csv(HMdata, file = file.path("./results/data4Heatmap.csv"))

## ----heatmapNoclustering, fig.cap="Heatmap for expression data

without any grouping"-------------------------------------------
my_palette <- colorRampPalette(c("blue", "red"))(n = 299)
library(gplots)

heatmap.2(HMdata,
Rowv = FALSE,
Colv = FALSE,
main = "Differentially expressed genes \n FDR < 0,1, logFC
>=1",
scale = "row",
col = my_palette,
sepcolor = "white",
sepwidth = c(0.05,0.05),
cexRow = 0.5,
cexCol = 0.9,
key = TRUE,
keysize = 1.5,
density.info = "histogram",
ColSideColors = c(rep("red",3),rep("blue",3),
rep("green",3), rep("yellow",3)),
tracecol = NULL,
dendrogram = "none",
srtCol = 30)

## ----heatmapClustering, fig.cap="Heatmap for expression data

grouping genes (rows) and samples (columns) by their similarity"----
heatmap.2(HMdata,
Rowv = TRUE,
Colv = TRUE,
dendrogram = "both",
main = "Differentially expressed genes \n FDR < 0,1, logFC
>=1",
scale = "row",
col = my_palette,
sepcolor = "white",
 sepwidth = c(0.05,0.05),
cexRow = 0.5,
cexCol = 0.9,
key = TRUE,
keysize = 1.5,
density.info = "histogram",
ColSideColors = c(rep("red",3),rep("blue",3),
rep("green",3), rep("yellow",3)),
tracecol = NULL,
srtCol = 30)

##
selectGenes---------------------------------------------------------

listOfTables <- list(KOvsWT.COLD = topTab_KOvsWT.COLD,

KOvsWT.RT = topTab_KOvsWT.RT,
INT = topTab_INT)
listOfSelected <- list()
for (i in 1:length(listOfTables)){
# select the toptable
topTab <- listOfTables[[i]]
# select the genes to be included in the analysis
whichGenes<-topTab["adj.P.Val"]<0.15
selectedIDs <- rownames(topTab)[whichGenes]
# convert the ID to Entrez
EntrezIDs<- select(mogene21sttranscriptcluster.db, selectedIDs,
c("ENTREZID"))
EntrezIDs <- EntrezIDs$ENTREZID
listOfSelected[[i]] <- EntrezIDs
names(listOfSelected)[i] <- names(listOfTables)[i]
}
sapply(listOfSelected, length)

##

mapped_genes2GO <- mappedkeys(org.Mm.egGO)

mapped_genes2KEGG <- mappedkeys(org.Mm.egPATH)
mapped_genes <- union(mapped_genes2GO , mapped_genes2KEGG)

##
BiologicalSig-------------------------------------------------------

library(ReactomePA)

listOfData <- listOfSelected[1:2]

comparisonsNames <- names(listOfData)
universe <- mapped_genes

for (i in 1:length(listOfData)){
 genesIn <- listOfData[[i]]
comparison <- comparisonsNames[i]
enrich.result <- enrichPathway(gene = genesIn,
pvalueCutoff = 0.05,
readable = T,
pAdjustMethod = "BH",
organism = "mouse",
universe = universe)

cat("##################################")
cat("\nComparison: ", comparison,"\n")
print(head(enrich.result))

if (length(rownames(enrich.result@result)) != 0) {
write.csv(as.data.frame(enrich.result),
file =paste0("./
results/","ReactomePA.Results.",comparison,".csv"),
row.names = FALSE)

pdf(file=paste0("./
results/","ReactomePABarplot.",comparison,".pdf"))
print(barplot(enrich.result, showCategory = 15, font.size = 4,
title = paste0("Reactome Pathway Analysis for ",
comparison,". Barplot")))
dev.off()

pdf(file = paste0("./
results/","ReactomePAcnetplot.",comparison,".pdf"))
print(cnetplot(enrich.result, categorySize = "geneNum",
schowCategory = 15,
vertex.label.cex = 0.75))
dev.off()
}
}

## ----network, fig.cap="Network obtained from the Reactome

enrichment analysis on the list obtained from the comparison between
KO and WT in RT"----
cnetplot(enrich.result, categorySize = "geneNum", schowCategory =
15,
vertex.label.cex = 0.75)

## ---- tableReacto,
echo=FALSE----------------------------------------------------------

Tab.react <- read.csv2(file.path("./results/

ReactomePA.Results.KOvsWT.RT.csv"),
sep = ",", header = TRUE, row.names = 1)

Tab.react <- Tab.react[1:4, 1:5]

knitr::kable(Tab.react, booktabs = TRUE, caption = "First rows and
columns for Reactome results on KOvsWT.RT.csv comparison")
## ---- listOfFiles,
echo=FALSE----------------------------------------------------------

listOfFiles <- dir("./results/")

knitr::kable(
listOfFiles, booktabs = TRUE,
caption = 'List of files generated in the analysis',
col.names="List_of_Files"
)