# Supplementary information for "RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls"
# This file describes the R/BioConductor commands used to analyze the raw data deposited in GEO with accession# GSE24084.

#Install package "Agi4x44PreProcess"
source("http://bioconductor.org/biocLite.R")
biocLite("Agi4x44PreProcess")
#Install package "vsn"
source("http://bioconductor.org/biocLite.R")
biocLite("vsn")

#################### QUARTILE NORMALIZATION
#Load library "Agi4x44PreProcess" (see this BioConductor package for details) 
library("Agi4x44PreProcess")

#read data filenames from file "targets.txt" listing the following raw data file names (available from GEO):
#FileName	Treatment	GErep	Array	SampleName	XT_Number
#251485046519_S01_GE1_105_Jan09_1_1.txt       	Ctrl	FALSE	519	Ctrl4	XT21M56
#251485046519_S01_GE1_105_Jan09_1_2.txt       	Ctrl	FALSE	519	Ctrl8	XT78F92
#251485046519_S01_GE1_105_Jan09_1_3.txt       	GBM	FALSE	519	GBM12	XT5672
#251485046519_S01_GE1_105_Jan09_1_4.txt       	GBM	FALSE	519	GBM15	XT5725
#251485047598_S01_GE1_105_Jan09_1_1.txt       	Ctrl	FALSE	598	Ctrl1	XT04N29
#251485047598_S01_GE1_105_Jan09_1_2.txt       	Ctrl	FALSE	598	Ctrl2	XT07J49
#251485047598_S01_GE1_105_Jan09_1_3.txt       	Ctrl	FALSE	598	Ctrl3	XT16E04
#251485047598_S01_GE1_105_Jan09_1_4.txt       	Ctrl	FALSE	598	Ctrl5	XT46B12
#251485047599_S01_GE1_105_Jan09_1_1.txt       	Ctrl	FALSE	599	Ctrl7	XT72C67
#251485047599_S01_GE1_105_Jan09_1_2.txt       	GBM	FALSE	599	GBM11	XT5650
#251485047599_S01_GE1_105_Jan09_1_3.txt       	GBM	FALSE	599	GBM13	XT5696
#251485047599_S01_GE1_105_Jan09_1_4.txt       	GBM	FALSE	599	GBM16	XT5737
#251485047600_S01_GE1_105_Jan09_1_1.txt       	GBM	FALSE	600	GBM17	XT5765
#251485047600_S01_GE1_105_Jan09_1_2.txt       	GBM	FALSE	600	GBM18	XT5767
#251485047600_S01_GE1_105_Jan09_1_3.txt       	GBM	FALSE	600	GBM19	XT5853
#251485047600_S01_GE1_105_Jan09_1_4.txt       	GBM	FALSE	600	GBM20	XT5865
targets=read.targets(infile="targets.txt")

#load data from raw data files into variable "dd"
dd=read.AgilentFE(targets, makePLOT=FALSE)

#Background subtract and Quartile Normalize data in dd using function BGandNorm. Load data into variable ddNORM 
ddNORM = BGandNorm(dd, BGmethod = "half", NORMmethod = "quantile", foreground = "MeanSignal", background = "BGMedianSignal", offset = 50, makePLOTpre = FALSE, makePLOTpost = FALSE)

#Alternative Quartile Normalization without background subtraction
#ddNORM = BGandNorm(dd, BGmethod = "none", NORMmethod = "quantile", foreground = "ProcessedSignal", background = "BGUsed", offset = 0, makePLOTpre = FALSE, makePLOTpost = FALSE)

#Write data to files
write.table(ddNORM$genes, file = "genenamesexport.txt")
write.table(ddNORM$R, file = "normdataexport.txt")

#################### VSN NORMALIZATION 
#Load libraries "limma" and "vsn" (se these BioConductor packages for details)
library(limma)
library(vsn)

#Load data from a file already combining the MeanSignal from all data files exported by the image analysis software Agilent Feature Extraction v 9.1
#The raw data files are available from GEO with accession# GSE24084, the coloumn of each file to be used is Column 26 ("Column Z" in Excel) with heading "gMeanSignal"
DataRawG=read.table("AgilentDataRawExport_MeanSignal.txt") 

# Normalize with VSN using the MeanSignal
AgilentVsnData_G <- justvsn(as.matrix(DataRawG))    
write.table(AgilentVsnData_G, file = "AgilentVsnNormalized_MeanSignal.txt", sep="\t")