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check_ruv.R
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check_ruv.R
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#########
# Preprocess StemCellDB in GEO
# Andrew Jaffe
# Updated 4/8/12
#########################
##### INSTRUCTIONS ######
# 1. PLACE "G4112F_annotation.txt" IN YOUR WORKING DIRECTORY
# 2. SOURCE FUNCTIONS AT THE TOP OF THIS SCRIPT (SEARCH TERM: 'FXN')
# 3. PREPROCESSING (SEARCH TERM: 'PREPROC')
# 4. MAKE PCA FIGURES (SEARCH TERM: 'FIG1')
# 5. MAKE D.E. FIGURES (SEARCH TERM: 'FIG2')
# 6. MAKE SEX AND GSTT1 FIGURES (SEARCH TERM: 'FIG3')
###### LOAD LIBRARIES ############
# if these aren't installed, here's the BioC install:
# source("http://bioconductor.org/biocLite.R")
## BIOC
library(limma)
library(GEOquery)
library(sva)
## CRAN
library(scales)
library(RColorBrewer)
########################################
#### FXN: SOME FUNCTIONS
# splits character strings, wrapper for strsplit
ss=function(x, pattern, slot=1,...) sapply(strsplit(x,pattern,...), function(y) y[slot])
# this function regresses SVs out of genomic data
cleaningP = function(y, mod, svaobj, P=ncol(mod)) {
X=cbind(mod,svaobj$sv)
Hat=solve(t(X)%*%X)%*%t(X)
beta=(Hat%*%t(y))
cleany=y-t(as.matrix(X[,-c(1:P)])%*%beta[-c(1:P),])
return(cleany)
}
# splits in factors
splitit=function(x) split(seq(along=x),x)
#####
# plotting code, expBySample()
# pp is a vector of expression values, or 1 row, of the expression matrix
# sampleIds is a vector of sample IDs
# conditions: colors the samples by treatment or condition, a vector
expBySample = function(pp, sampleIds, geneName, conditions=NULL,
pal="Dark2",legend=TRUE,cex=0.85,...) {
require(RColorBrewer)
sampleIds = factor(sampleIds)
N=length(levels(sampleIds))
if(!is.null(conditions)) {
conditions = factor(conditions)
cols = as.numeric(conditions)
Ncol = max(cols)
} else {
cols = rep(1,length(pp))
Ncol = 1
}
if(pal %in% rownames(brewer.pal.info)) {
palette(brewer.pal(max(Ncol,3), pal))
} else palette(pal)
xx = jitter(as.numeric(sampleIds),amount=0.25)
plot(pp~xx, pch = 21, bg=cols, cex=cex,
xaxt = "n", ylab = "Expression", xlab="",
main = geneName,...)
abline(v=seq(0.5,N+0.5, by=1),lty=2, lwd = 0.8)
axis(1, at = 1:N, labels = levels(sampleIds), las = 2,...)
if(legend) legend("topright", levels(conditions),
col = 1:Ncol,pch=15, nc =Ncol,bty="n",cex=0.9)
}
###############
## PREPROC: PREPROCESSING
tmp = getGEO("GSE32923")
pd = pData(tmp[[1]])
pd = pd[,c(1,2, grep("characteristics",names(pd)))]
# CLEAN UP PHENOTYPE DATA
for(i in 3:ncol(pd)) {
names(pd)[i] = ss(as.character(pd[,i]), ": ", 1)[1]
pd[,i] = ss(as.character(pd[,i]), ": ", 2)
}
names(pd) = c("Title", "GEO_Accession", "CellType",
"Treatment", "SampleID", "Passage", "Sex")
## only keep ESC and iPSC, drop last column
pd = pd[pd$CellType %in% c("ESC","iPSC"),-ncol(pd)]
## raw data
tmp = getGEOSuppFiles("GSE32923")
system("tar xvf GSE32923/GSE32923_RAW.tar -C GSE32923/")
system("gunzip GSE32923/*.gz GSE32923/")
### match to array data
id = ss(dir("GSE32923", pattern = "GSM"), "_",1)
fns = dir("GSE32923", pattern = "GSM",full.names=TRUE)
pd$FileName = fns[match(pd$GEO_Accession, id)]
pd$Passage = as.numeric(pd$Passage)
## read in data
theData = read.maimages(pd$FileName, source="agilent",
green.only=TRUE, columns = list(G = "gMedianSignal", Gb = "gBGMedianSignal"),
annotation = c("accessions","chr_coord", "ControlType",
"ProbeName", "GeneName", "SystematicName", "Description","Sequence"))
# get processing date
pd$ArrayDate = sapply(pd$FileName, function(x) {
tmp = read.delim(file=x,skip=1,nrows=1,header=T,as.is=T)
out = strsplit(tmp[1,"FeatureExtractor_ExtractionTime"], " ")[[1]][1]
return(out)
})
theData$targets = pd
##################
# for now, default background correct
theDataBack = backgroundCorrect(theData, offset = 50)
# lets normalize all cell types together for now
theDataNorm = normalizeBetweenArrays(theDataBack, method="quantile")
# save
save(theDataNorm, file = "normalizedES_GEO_withNegControl.rda",
compress=TRUE)
####################
library(ruv)
library(impute)
# load data
load("normalizedES_GEO_withNegControl.rda")
p = theDataNorm$E
p = impute.knn(p)$data # some missingness
pd = theDataNorm$targets
map = theDataNorm$genes
ctrlIndex=map$ControlType == 1
mod = model.matrix(~pd$Treatment)[,-1]
fitFull = lmFit(p, model.matrix(~pd$Treatment))
ebFull = ebayes(fitFull)
pdf("controlGene_suppfigure.pdf")
palette(brewer.pal(8,"Dark2"))
plot(density(abs(ebFull$t[-ctrlIndex,2])), col = 2, lty=2,lwd=2,
main = "Control Probes T-stat Distribution",xlab="|T-statistic|",
cex.axis=1.5, cex.lab=1.5,cex.main = 1.4)
lines(density(abs(ebFull$t[ctrlIndex,2])), col = 2, lwd=2)
lines(density(abs(ebFull$t[-hkIndex,3])), col = 3, lty=2,lwd=2)
lines(density(abs(ebFull$t[hkIndex,3])), col = 3, lty=1,lwd=2)
legend("topright", c("KSR vs FBS", "UNDIFF vs FBS"), col = 2:3,
pch = 15)
legend("right", c("Housekeeping", "Non Housekeeping"), col = "black",
lty = c(1,2))
dev.off()
ruvOut = RUV2(Y=t(p), X=mod, ctrlIndex, k=27)
outStats = data.frame(cbind(t(ruvOut$betahat), ruvOut$sigma2,
t(ruvOut$t),t(ruvOut$p)))
colnames(outStats) = c("logFC_TreatmentKSR","logFC_TreatmentUNDIFF",
"sigma2", "t_TreatmentKSR","t_TreatmentUNDIFF",
"p_TreatmentKSR","p_TreatmentUNDIFF")
load("sva_obj.rda")
mm = match(rownames(eb1$p), map$ProbeName)
outStats = outStats[mm,]
plot(outStats$logFC_TreatmentKSR, fit1$coef[,2])
plot(outStats$t_TreatmentKSR, eb1$t[,2])
abline(0,1,col="red")
hk = read.delim("http://www.stat.berkeley.edu/~johann/ruv/resources/hk.txt",
header=FALSE)$V1
# fit linear model without SVA
fit0 = lmFit(p, model.matrix(~pd$Treatment))
eb0 = ebayes(fit0) # empirical bayes for t-stats
### plot of density of t-stat by hk status
pdf("housekeeping_suppfigure.pdf")
palette(brewer.pal(8,"Dark2"))
hkIndex = which(map$GeneSymbol %in% hk)
plot(density(abs(eb0$t[-hkIndex,2])), col = 2, lty=2,lwd=2,
main = "Housekeeping Gene T-stat Distribution",xlab="|T-statistic|",
cex.axis=1.5, cex.lab=1.5,cex.main = 1.4)
lines(density(abs(eb0$t[hkIndex,2])), col = 2, lwd=2)
lines(density(abs(eb0$t[-hkIndex,3])), col = 3, lty=2,lwd=2)
lines(density(abs(eb0$t[hkIndex,3])), col = 3, lty=1,lwd=2)
legend("topright", c("KSR vs FBS", "UNDIFF vs FBS"), col = 2:3,
pch = 15)
legend("right", c("Housekeeping", "Non Housekeeping"), col = "black",
lty = c(1,2))
dev.off()