RNAseq TPM heatmap

Question

[tanyabioinfo]

Hi

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

wt0hr wt6hr wt24hr kd0hr kd6hr kd24hr

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: cols <- c("","wt0","wt6","wt24","kd0", "kd6","kd24") 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 .

Tanya

[Friederike]

what makes you think that this was not the correct way?

You don't really need to scale the matrix manually as heatmap.2 can do that for you (by choosing a different value than scale = "none") and my personal preference is to look at the unscaled values, too.

Answer 1

[Kevin Blighe]

If you are going to scale manually outside of the heatmap.2 function, be sure to also set your own breaks:

require(RColorBrewer)
myCol <- colorRampPalette(c("dodgerblue", "black", "yellow"))(100)
myBreaks <- seq(-3, 3, length.out=101)

require("gplots")

heat.scaled <- t(scale(t(heat)))

heatmap.2(heat.scaled, col=myCol, breaks=myBreaks, scale="none", ...)

However, as Friederike says, heatmap.2 will automatically scale your data with scale="row", thus de-necessitating the manual scaling to Z-scores as you have done with z.mat <- t(scale(t(counts_filtered_df), scale=TRUE, center=TRUE))

At the end of the day, your statistical tests will have been performed on the unscaled TPM data, and thus the scaling to Z-scores (or logging or something else) is purely for visualisation purposes.