Hello everybody,

I created a simple and a complex heatmap comparing gene expressions in two groups (control vs SAH) and noticed that the z-score color coding of the complex heatmap does not seem to fit the z-scores.

I picked one special molecule in the heatmap to display the differences in the simple and the complex heatmaps. Here you can find the log fold change values:

And the corresponding z-scores:

lcpm.df.scaled <- t(apply(lcpm.df, 1, scale))

Simple heatmap:

library(gplots)
Control.vs.SAH.topgenes <- Control.vs.SAH$ENTREZID[1:100]
i <- which(v$genes$ENTREZID %in% Control.vs.SAH.topgenes)
mycol <- colorpanel(1000,"blue","white","red")
heatmap.2(lcpm[i,], scale="row",
          labRow=v$genes$SYMBOL[i], labCol=group, 
          col=mycol, trace="none", density.info="none", 
          margin=c(8,6), lhei=c(2,10), dendrogram="column")
sessionInfo(R version 4.2.1 (2022-06-23))

This color-coding makes sense for me.

The problem: It seems as if something went wrong with the z-score color coding in the complex heatmap.

Complex Heatmap:

ha <- HeatmapAnnotation(summary = anno_summary(gp = gpar(fill=2),
                                               height = unit(2, "cm")))
h1 <- Heatmap(lcpm.df.scaled[rows_keep,], cluster_rows = F,
              column_labels = colnames(lcpm.df.scaled), name = "Z-Score",
              cluster_columns = T)
h2 <- Heatmap(l2_val, row_labels = df.top$SYMBOL[rows_keep],
              cluster_rows = F, name = "logFC", top_annotation = ha, col = col_logFC,
              cell_fun = function (j, i, x, y, w, h, col)
                grid.text(round(l2_val[i, j], 2), x, y))
h3 <- Heatmap(mean, row_labels = df.top$SYMBOL[rows_keep],
              cluster_rows = F, name = "AveExpr", col=col_AveExpr,
              cell_fun = function (j, i, x, y, w, h, col)
                grid.text(round(mean[i,j],2), x, y))
h <- h1+h2+h3

h

On the left you can see the z-score color coding, followed by the log-FC(6.98) and on the right you can see the AveExpr (12.55).

Do you have any idea why the complex heatmap does not really fit the z-scores?

Thank you a lot for your help.

Have you checked the matrix lcpm[i,] for heatmap.2() and lcpm.df.scaled[rows_keep,] for Heatmap() are the same?

Also heatmap.2() requires the matrix having rnow > 1 and ncol > 1. I guess lcpm[i,] should have at least two rows?

The following plot is based on the data you provided:

When posting what you might think is a bug, it's important to provide a self-contained example that others can use to replicate your results. Your code is not complete, we don't have access to the data, and it's not clear that you are using the data you say you are using.

Put another way, the heatmap using ComplexHeatmap has as the highest value the Cntrl_5 sample, which is one of the lower values. It is highly unlikely that a package that is used as regularly as ComplexHeatmap would be doing something so obviously incorrect and nobody but you has noticed.