Question

I am trying to cluster a protein dna interaction dataset, and draw a heatmap using heatmap.2 from the R package gplots. My matrix is symmetrical.
Here is a copy of the data-set I am using after it is run through pearson:DataSet

Here is the complete process that I am following to generate these graphs: Generate a distance matrix using some correlation in my case pearson, then take that matrix and pass it to R and run the following code on it:

library(RColorBrewer);
library(gplots);
library(MASS);
args <- commandArgs(TRUE);
matrix_a <- read.table(args[1], sep='\t', header=T, row.names=1);
mtscaled <- as.matrix(scale(matrix_a))
# location <- args[2];
# setwd(args[2]);
pdf("result.pdf", pointsize = 15, width = 18, height = 18)
mycol <- c("blue","white","red")
my.breaks <- c(seq(-5, -.6, length.out=6),seq(-.5999999, .1, length.out=4),seq(.100009,5, length.out=7))
#colors <- colorpanel(75,"midnightblue","mediumseagreen","yellow") 
result <- heatmap.2(mtscaled, Rowv=T, scale='none', dendrogram="row", symm = T, col=bluered(16), breaks=my.breaks)
dev.off() 

The issue I am having is once I use breaks to help me control the color separation the heatmap no longer looks symmetrical.

Here is the heatmap before I use breaks, as you can see the heatmap looks symmetrical:

Here is the heatmap when breaks are used:

I have played with the cutoff’s for the sequences to make sure for instance one sequence does not end exactly where the other begins, but I am not able to solve this problem. I would like to use the breaks to help bring out the clusters more.

Here is an example of what it should look like, this image was made using cluster maker:

I don’t expect it to look identical to that, but I would like it if my heatmap is more symmetrical and I had better definition in terms of the clusters. The image was created using the same data.

Answer 1

After some investigating I noticed was that after running my matrix through heatmap, or heatmap.2 the values were changing, for example the interaction taken from the provided data set of

Pacdh-2
and
pegg-2

gave a value of 0.0250313 before the matrix was sent to heatmap.
After that I looked at the matrix values using result$carpet and the values were then

-0.224333135
-1.09805379

for the two interactions

So then I decided to reorder the original matrix based on the dendrogram from the clustered matrix so that I was sure that the values would be the same. I used the following stack overflow question for help:
Order of rows in heatmap?

Here is the code used for that:

rowInd <- rev(order.dendrogram(result$rowDendrogram))
colInd <- rowInd
data_ordered <- matrix_a[rowInd, colInd]

I then used another program “matrix2png” to draw the heatmap:

I still have to play around with the colors but at least now the heatmap is symmetrical and clustered.

Looking into it even more the issue seems to be that I was running scale(matrix_a) when I change my code to just be mtscaled <- as.matrix(matrix_a) the result now looks symmetrical.