library(RColorBrewer)
library(limma)
library(edgeR)
library(reshape)
library(scales)
library(ggplot2)
counttable_all <- read.table("~/Dropbox/rabba/P957_all_counts.tsv", header=T, row.names=1)
timepoints = rep(c(0,15,30,60,90),times=3)
t2 <- timepoints^2
design <- model.matrix(~timepoints)
counts <- counttable_all[,1:15]
dge <- DGEList(counts=counts)
dge <- calcNormFactors(dge)
dge <- dge[ rowSums(cpm(dge) > 1)>2, ]
v <- voom(dge,design,plot=TRUE)
fit <- lmFit(v, design)
fit <- eBayes(fit)
tt_genes_full<-topTable(fit,coef="timepoints",p.value=1,n=200000,sort="p")
table(pval=tt_genes_full$adj.P.Val < .95,beta=tt_genes_full$logFC < 0)
## beta
## pval FALSE TRUE
## FALSE 157 131
## TRUE 3049 2569
par(mfrow=c(3,3))
for (gene in rownames(tt_genes_full)[1:9]) {
boxplot(v$E[gene,]~timepoints,ylab=gene,col=timepoints,
main=paste("Adj. p-val:",prettyNum(tt_genes_full[gene,]$adj.P.Val)),cex=2)
}
#/////////////////////////////////////////////////////////////////////////////
# genklustring
#\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
cvpercent <- function(data)
{s <- apply(data,1,sd)
x <- apply(data,1,mean)
obj <- cbind(x,s/x)
colnames(obj) <- c("x","cv")
return(obj)
}
cvp <- cvpercent(v$E)
hist(cvp)
ClusterData <- scale(v$E[cvp[,"cv"] > .05,order(timepoints)])
dim(ClusterData)
## [1] 3524 15
heatmap.2(cor(ClusterData),col=redblue(256),Rowv=T,dendrogram="col",
scale="none",key=TRUE, keysize=1.5,
density.info="none", trace="none", cexRow=1,cexCol=1)
## Error: could not find function "heatmap.2"
#
# GeneCor <- cor(t(ClusterData))
#
# heatmap.2(GeneCor,col=redblue(256),Rowv=T,dendrogram="col",
# scale="none",key=TRUE, keysize=1.5,
# density.info="none", trace="none", cexRow=1,cexCol=1)
#
cl <- kmeans(ClusterData , 6)
#At this stage I also make a new variable indicating cluster membership as below.
# I have a good #idea of what my clusters will be called so
#I gave them those names in the second line of the code.
#Then I put it together and put the data in a form that is good for graphing.
cl.cluster<-as.factor(cl$cluster)
#cl.cluster<-as.vector(recode (cluster, "1='FC'; 2='FV'; 3='SO'; 4= 'OS' ",
# as.numeric.result=FALSE) )
clustT1WIN2<- data.frame (ClusterData , cl.cluster)
clustT1WIN2$.row=rownames(ClusterData)
molten2 <- melt(clustT1WIN2, id = c(".row", "cl.cluster") )
#OK set up the graph background.
#Following the ggplot book I also create a jit parameter cause it is
#much easier to alter this and type it in than the full code over and over again.
pcp_cl <- ggplot(molten2, aes(variable, value, group = .row, colour = cl.cluster) )
jit<- position_jitter (width = .08, height = .08)
#Ok first graph the cluster means.
#pcp_cl + stat_summary(aes(group = cl.cluster), fun.y = mean, geom = "line")
#Then we produce a colourful but uninformative parallel coordinates
#plot with a bit of alpha blending and jitter.
#pcp_cl + geom_line(position = jit, alpha = 1/5)
#All code up to this point is as per Wickham but
#I also add the cluster means graph that we
#first produced as well as changing the angle of the x axis text so it is readable.
pcp_cl + geom_line(position = jit, colour = alpha("black", 1/4))+
stat_summary(aes(group = cl.cluster), fun.y = mean, geom = "line", size = 1.5 )+
facet_wrap(~ cl.cluster,scales = "free_y")+ opts(axis.text.x=theme_text(angle=-45, hjust=0) )+
theme(axis.text.x=element_text(rep(c("red","blue"),each=18)))
## 'opts' is deprecated. Use 'theme' instead. (Deprecated; last used in version 0.9.1)
## theme_text is deprecated. Use 'element_text' instead. (Deprecated; last used in version 0.9.1)
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sessionInfo()
## R version 3.0.2 (2013-09-25)
## Platform: x86_64-redhat-linux-gnu (64-bit)
##
## locale:
## [1] LC_CTYPE=en_GB.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_GB.UTF-8 LC_COLLATE=en_GB.UTF-8
## [5] LC_MONETARY=en_GB.UTF-8 LC_MESSAGES=en_GB.UTF-8
## [7] LC_PAPER=en_GB.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] ggplot2_0.9.3.1 scales_0.2.4 reshape_0.8.5
## [4] edgeR_3.4.2 limma_3.18.13 RColorBrewer_1.0-5
## [7] knitr_1.5.21
##
## loaded via a namespace (and not attached):
## [1] colorspace_1.2-4 digest_0.6.4 evaluate_0.5.3 formatR_0.10.4
## [5] grid_3.0.2 gtable_0.1.2 labeling_0.2 MASS_7.3-32
## [9] munsell_0.4.2 plyr_1.8.1 proto_0.3-10 Rcpp_0.11.1
## [13] reshape2_1.4 stringr_0.6.2 tools_3.0.2
