Hi

I have a count matrix (TPM) for my samples. I have 4 replicate for each sample. My samples are as follows :

wt0hr wt6hr wt24hr  mut0hr mut6hr  mut24hr

I have a TPM matrix with geneids as rows and the columns as the 6 samples above. I am using follwoing R code to generate the heatmap:

dat <- read.csv("TPM.csv", row.names = 1,stringsAsFactors = FALSE, col.names = cols, header = TRUE)

counts_filtered_df <- dat[apply(dat, MARGIN = 1, FUN = function(x) sd(x) != 0),]

z.mat <- t(scale(t(counts_filtered_df), scale=TRUE, center=TRUE))

heatmap.2(z.mat, dendrogram="both", scale="none", trace="none")

Can anyone suggest me if this is the correct way to generate the heatmaps from TPM rather than using log2 FC.

Tanya

Your saying rather than using log2FC, but are you sure that is what you mean?

You are currently running your heatmap function on z score normalised TPMs.

I would first take log(TPM+1) before doing the z score. That should be approx normally distributed which is required for doing z scores.

Other than that, this looks fine - to me at least.

Thanks Chris

I did that and it works fine for me.

So now the R script looks like this:

logTransformed.dat = log2(counts_filtered_df + 1)
z.mat <- t(scale(t(logTransformed.dat), scale=TRUE, center=TRUE))
heatmap.2(z.mat, dendrogram="both", scale="none", trace="none")

I have one more question. I have four replicates for each sample. How can I plot all the replicates into one heatmap.

Regards

Tanya