Hi,

I am trying to plot heatmap on my counts from HTSeq. Here is my codes (partial).

dds<-DESeq(ddsHTSeq)
res<-results(dds)
res<-res[order(res$padj),]

rld <- rlogTransformation(dds, blind=TRUE)
vd <- varianceStabilizingTransformation(dds, blind=TRUE)

library("genefilter")
topVarGenes <- head(order(rowVars(assay(vd)),decreasing=TRUE),200)
mat <- assay(rld)[ topVarGenes, ]
mat <- mat - rowMeans(mat)
df <- as.data.frame(colData(rld)[,c("cond1","cond2, "cond3")])

pheatmap(mat, annotation_col=df, show_rownames=FALSE)

This is my summary of the results

out of 42075 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)     : 4674, 11%
LFC < 0 (down)   : 5123, 12%
outliers [1]     : 0, 0%
low counts [2]   : 11998, 29%
(mean count < 5.6)
 

This pheatmap results only for top 200 genes. My question is,

1) is it possible to heatmap based on threshold like above some p-value or fold change?

2) is it possible to use different colours for different conditions?

3) how can I force pheatmap to do the heatmap based on foldchange or adjusted p-value?

Thanks

J.

hi John

1) Yes, you would just change topVarGenes to a vector defined by res$padj or res$log2FoldChange. You can define this matrix you provide to pheatmap however you like.

2) Yes, please take a look at the pheatmap documentation for further details.

3) I guess, the point of the heatmap is to visualize the actual counts (normalized and transformed) across samples. There are other, perhaps better ways of visualizing fold changes and p-values (e.g. MA plots for fold changes, and histograms or "volcano" plots for p-values).

Hi Michael,

Thank you for the help.

>2) Yes, please take a look at the pheatmap documentation for further details.

I looked into that and the only colour change I could do was the following.

ann_colors=list(Condition=c(control="blue", health="yellow"))

pheatmap(mat, annotation_col=df,  cluster_rows=TRUE, cluster_cols=FALSE, legend=FALSE, show_rownames=FALSE, show_colnames=TRUE, annotation_colors = ann_colors)

This changes the label colours but not the actual heatmap pattern. I wanted each condition to have different colouring pattern on the heatmap. Do you think that is possible.

Thank you again

J.