### --- BIOINFORMATICS TRAINING SCHOOL (EuGESMA) --- ###
### --- 23-24 Marzo 2010 - Barcelona - Spain --- ###

# 2nd SESSION: 
# Microarray data analysis: analysis of exon arrays and miRNA arrays; mapping of different RNAs to the genome landscape.  
# Dr. Javier DE LAS RIVAS (jrivas@usal.es)
# Centro de Investigacion del Cancer, CiC-IBMCC, CSIC/USAL, Salamanc, Spain

# See WEB SITE: GATExplorer
# http://bioinfow.dep.usal.es/xgate/




###  ANALYSIS OF EXON MICROARRAYS FROM Affymetrix (HUMAN_EXON_1.0) TO FIND GENES  ###

# Example of how to install a CDF package (in LINUX command line):
cd .../exon-arrays_protocol/Data_PackagesANNOT
R CMD INSTALL genemapperhumanexon1.0cdf_2.0.tar.gz


# Load libraries
library(affy)
library(siggenes)


# Set directory
setwd(".../exon-arrays_protocol")


# Load annotation package taken from GATExplorer 
load('Data_PackagesANNOT/genes-human-annotation.R')


# Read and normalization of samples
setwd('Data_CELs')
rma <- justRMA(cdfname="genemapperhumanexon1.0cdf")
exprs <- exprs(rma)


# Differential expression using SAM algorithm
diffExp <- sam(exprs,c(0,0,0,1,1,1),method=d.stat,var.equal=FALSE,na.method="median",rand=123)

diffExp
plot(diffExp)
plot(diffExp,2.4)
sigGenes <- summary(diffExp,2.4)@mat.sig


# Annotation
annotGenes <- genes.human.Annotation[rownames(sigGenes),]
annotGenes <- cbind(sigGenes[c("d.value","stdev","rawp","q.value","R.fold")],annotGenes)


# Export to file
setwd(".../exon-arrays_protocol")
write.table(annotGenes,quote=FALSE,sep='\t',row.names=FALSE,file='gene_list.tsv')




###  ANALYSIS OF EXON MICROARRAYS FROM Affymetrix (HUMAN_EXON_1.0) TO FIND ncRNAs  ###

# ncRNA
setwd('.../exon-arrays_protocol/Data_CELs')
rma.ncRNA <- justRMA(cdfname="ncrnamapperhumanexon1.0cdf")
exprs.ncRNA <- exprs(rma.ncRNA)


# differential expression using SAM algorithm
diffExp.ncRNA <- sam(exprs.ncRNA,c(0,0,0,1,1,1),method=d.stat,var.equal=FALSE,na.method="median",rand=123)

diffExp.ncRNA
plot(diffExp.ncRNA)
plot(diffExp.ncRNA,12)
sigGenes.ncRNA <- summary(diffExp.ncRNA,12)@mat.sig

sigGenes.ncRNA <- cbind(rownames(sigGenes.ncRNA),sigGenes.ncRNA)

# export to file
setwd(".../exon-arrays_protocol")
write.table(sigGenes.ncRNA,quote=FALSE,sep='\t',row.names=FALSE,file='ncRNA_list.tsv')