the permuted data is real data. just real data which has been subsampled with N samples for Y iterations to estimate population parameters for a random distribution of N samples. effectively that is the baseline distribution for a random process.

i recently wrote an R program that does just this. you'll have to tinker with it so it works for your data, but it's someplace to start.

```
library(rsgcc)
library(SNFtool)
args<-commandArgs(TRUE)
mrna_file <- args[1]
omics_file <- args[2]
group_name <- args[3]
query <- args[4]
try(mirna_file <- args[5])
ifis.na(mirna_file)){
try(n_permute <- args[5])
}else{
try(n_permute <- args[6])
}
ifis.na(n_permute)){n_permute=2000}
make_unique <- function(input.data) {
require(org.Hs.eg.db)
input.data <- data.frame(cbind(input.data,
entrez=as.numeric(mapIds(org.Hs.eg.db,
keys=gsub("\\..*","",rownames(input.data)),
column="ENTREZID", keytype="ENSEMBL",multiVals="first"))))
input.data <- input.data[!is.na(input.data$entrez),]
id.dup <- input.data[duplicated(input.data$entrez)
| duplicated(input.data$entrez,
fromLast = TRUE),ncol(input.data)]
data.dup <- as.matrix(input.data[duplicated(input.data$entrez) |
duplicated(input.data$entrez, fromLast = TRUE),])
ezid.dup <- unique(id.dup)
data.unique <- input.data[!input.data$entrez %in% id.dup,]
rownames(data.unique) <- data.unique$entrez
data.unique$entrez = NULL
data.dup2 <- lapply(ezid.dup, function(x) {
expr <- data.dup[id.dup == x,]
if(is.matrix(expr)){
sd.values <- apply(expr[,1:ncol(input.data)-1], 1, sd)
mean.values <- apply (expr[,1:ncol(input.data)-1], 1, mean)
CV.values <- sd.values/mean.values
CV.values <- gsub("NaN","0",CV.values)
expr <- expr[which(CV.values == max(CV.values))[[1]],]
} else {
expr
}
})
merger <- data.frame(do.call(rbind, data.dup2))
rownames(merger) <- merger$entrez
merger$entrez = NULL
eset <- as.matrix(rbind(data.unique, merger))
return(eset)
}
run_tests <- function(data, feature_type, n){
output = NULL
for (x in 2:dim(data)[[1]]){
#print(paste("current step",x-1,"out of",dim(data)[[1]]-1,sep=" "))
corpair <- cor.pair(c(1,x), GEMatrix = as.matrix(data),
rowORcol = "row", cormethod = "PCC", pernum = n,
sigmethod = "two.sided")
direction_corr="neg"
if(corpair$cor > 0){
direction_corr="pos"
}
output <- rbind(output, c(rownames(data)[[1]],
rownames(data)[[x]], feature_type, corpair$cor,
abs(corpair$cor), direction_corr, corpair$pvalue))
}
colnames(output) <- c("gene","feature","feature_type",
"correl", "abs_correl", "direction", "pvalue_2k_permutation")
write.table(output, file=paste(gene_name_query, "_", feature_type, "_",
group_name, ".csv",sep=""), row.names = FALSE, sep="\t")
}
mrna <- read.table(mrna_file, header=TRUE, row.names=1, sep="\t")
mrna <- make_unique(mrna)
genes <- mapIds(org.Hs.eg.db, keys=rownames(mrna),
column="SYMBOL", keytype="ENTREZID",multiVals="first")
rownames(mrna) <- genes
gene_name_query <- mapIds(org.Hs.eg.db, keys=gsub("\\..*","",query),
column="SYMBOL", keytype="ENSEMBL",multiVals="first")
query_row_num <- which(rownames(mrna) == gene_name_query)
group <- as.factor(unlist(read.table(omics_file, header=FALSE, sep="\t")))
if(group_name != "all"){
mrna <- mrna[,which(group == group_name)]
}
query_data <- data.frame(mrna[query_row_num,])
colnames(query_data) <- rownames(mrna)[[query_row_num]]
query_data <- t(query_data)
ifis.na(mirna_file)){
data <- rbind(query_data, mrna[-c(query_row_num),])
run_tests(data,"mrna",n_permute)
}else{
mirna <- read.table(mirna_file, header=TRUE, row.names=1, sep="\t")
if(group_name != "all"){
mirna <- mirna[,which(group == group_name)]
}
data <- rbind(query_data, mirna)
tdata <- t(data)
ntdata <- standardNormalization(tdata)
data <- t(ntdata)
run_tests(data,"mirna", as.numeric(n_permute))
}
```

If this isn't helpful, I can put a working example up on github. But that won't be until next week. Just let me know.

Thanks for you answer. Im not sure however this is answering my question. I'll try to be clearer. I do not have different conditions, i.e. control vs disease. My dataset is actually a time course of 20 samples and i don't intend to compare the correlation between different set of time points. If I generate a matrix of correlations coefficients calculated by correlating all the gene pairs (i.e. a matrix of ~20,000 cols x ~20,000 rows) i can see that the mean distribution of the correlations is 0.5. Now, i have been performing network analysis using this matrix to identify clusters of co-expressed genes. Prior to this i'd like to threshold the data to include only the 'significant' correlations.So, at which point should i consider a correlation 'significant' given that the majority of the genes seem to be correlated with an r of 0.5? I've been suggested to permute the data and compare the distribution of the permuted data vs the real one to identify an appropriate threshold. However, is this the way to go? Shouldnt i seek to calculate p-values based on the distribution of the real data? I hope i made myself clear. And thanks again for your answer.

Cheers

So if you wanted to know how to threshold your correlation matrix, why didn't you ask about this first ? You were asking about p-values for correlations instead. For clustering purposes, thresholding the matrix is meant to reinforce the cluster structure. In this case, what you want to do is remove correlations close to 0. What close to 0 means is up to you, one possibility is to keep those correlations with a low p-value. Permutations can tell you the likelihood of a given correlation value if the genes were randomly assigned profiles from your data. Here, p-values are of little help for assessing relevance. If you have large clusters, the probability of getting high correlation values in the permutations will be high. If you remove high correlations that have high p-value, you could end up destroying the cluster structure you're trying to identify. I think you should be guided by the biology behind your data. If you think there are too many genes that are highly correlated, try to understand why.

ok thanks so i guess permutation is the way to go. Cheers.