- Install pheatmap
- Load pheatmap library
- Load dataset
- Create heatmap
In bioinformatics, heatmaps are commonly used to visualize gene expression changes across multiple genes and conditions. This article describes how to create clustered and annotated heatmaps for visualization of gene expression data obtained from RNA-seq experiments using a pheatmap R package.
If you have not installed pheatmap package, you can install it using
# install pheatmap install.packages("pheatmap")
Load pheatmap library
You need to load the pheatmap library for creating the heatmaps. I am using the RStudio console with R version 4.2.1
# load pheatmap (v1.0.12) library(pheatmap)
For this pheatmap tuotrial, I will use the gene expressiion dataset generated from RNA-seq experiment in cotton plant
in response to pathogenic infection. For simplicity of understanding, I have put the conditions names as A to F. You
can import the CSV dataset using
read.csv() base R function. You can read my article on
different ways to import CSV datasets in R.
# load dataset df = read.csv("https://reneshbedre.github.io/assets/posts/heatmap/hm_data.csv", row.names="Gene") # convert to matrix df_mat = data.matrix(df) # view first 5 genes of data head(df, 5) # output A B C D E F B-CHI1 4.505700 3.260360 -1.249400 8.89807 8.05955 -0.842803 CTL2-1 3.508560 1.660790 -1.856680 -2.57336 -1.37370 1.196000 B-CHI2 2.160030 3.146520 0.982809 9.02430 6.05832 -2.967420 CTL2-2 1.884750 2.295690 0.408891 -3.91404 -2.28049 1.628820 CHIV1 0.255193 -0.761204 -1.022350 3.65059 2.46525 -1.188140
Tip: While importing a dataset, you should always make gene names columns as row names. Otherwise, you would encounter an error while creating a heatmap with pheatmap.
You can create a basic clustered heatmap using the pheatmap. You need to pass the data matrix to the
pheatmap(df_mat, main = "basic heatmap")
pheatmap provides a parameter
scale to rescale the default values in column or row direction. The rescaling is useful
when the values in the dataset are very different from each other. For example, some gene expression values are very high
and some are very low.
# row scaling pheatmap(df_mat, scale = "row", main = "row scaling") # column scaling pheatmap(df_mat, scale = "column", main = "column scaling")
Row and column clustering
By default, pheatmap applies hierarchical clustering to both rows and columns. You can choose if you want to display row/columns
clustering or not by using
# No row clustering pheatmap(df_mat, cluster_rows = FALSE, main = "no row clustering") # No column clustering pheatmap(df_mat, cluster_cols = FALSE, main = "no column clustering")
You can change the color palette of the heatmap using the
colorRampPalette() function, which returns a fixed number of
# green and red color palette color <- colorRampPalette((c("red", "black", "green")))(50) pheatmap(df_mat, color = color, main = "red-green color palette") # blue and green color palette color <- colorRampPalette((c("blue", "black", "red")))(50) pheatmap(df_mat, color = color, main = "blue-red color palette")
You can add annotations to the row and columns to enhance the informative visualization for the heatmap. For example, you can add an annotation to a group of genes involved in the same biological pathway or you can add an annotation to the samples based on their conditions.
Here, I will add an annotation to genes (row) and samples (column) based on their gene functions and conditions.
# create a data frame for column annotation smaple_group <- data.frame(sample = rep(c("infected", "control"), c(3, 3))) row.names(smaple_group) <- colnames(df_mat) # view data frame smaple_group sample A infected B infected C infected D control E control F control # create a data frame for row annotation gene_functions = read.csv("https://reneshbedre.github.io/assets/posts/heatmap/hmap_gene_functions.csv", row.names="Gene") # view data frame head(gene_functions) function. B-CHI1 antifungal CTL2-1 antifungal B-CHI2 antifungal CTL2-2 antifungal CHIV1 antifungal CHIA antifungal # heatmap with annotations pheatmap(df_mat, annotation_row = gene_functions, annotation_col = smaple_group, main = "heatmap with annotations")
Split heatmap clusters
pheatmap provides parameters
cutree_cols to split the rows and columns based on the number of
cluster. If you can visually see distinct clusters, you can provided that numbers to these paramaters. For example, for columns
I can see three distinct clusters.
# heatmap with cluster break pheatmap(df_mat, cutree_rows = 5, cutree_cols = 3, main = "heatmap with clusterized output")
Get the heatmap data belonging to each row cluster,
# get data that belong to row clusters hm <- pheatmap(df_mat) df_row_cluster = data.frame(cluster = cutree(hm$tree_row, k = 5)) # view the genes clsuters head(df_row_cluster) cluster B-CHI1 1 CTL2-1 2 B-CHI2 1 CTL2-2 2 CHIV1 1 CHIA 1
Get the heatmap data belonging to each column cluster,
# get data that belong to column clusters hm <- pheatmap(df_mat) df_col_cluster = data.frame(cluster = cutree(hm$tree_col, k = 3)) # view the genes clsuters head(df_col_cluster) cluster A 1 B 1 C 2 D 3 E 3 F 2
Add gaps in the heatmap
In pheatmap, you can add gaps in heatmap between specific columns or rows to separate the heatmap. For example, you can add gaps to differentiate the heatmaps by the functional significance of the genes.
# add gap between 2-3 and 4-5 columns pheatmap(df_mat, gaps_col = c(2, 4), gaps_row = c(5), cluster_cols = FALSE, cluster_row = FALSE, border_color = FALSE, main = "heatmap with row and column gap")
Tip: If you would like to enable gaps in heatmaps, you must off row and column clustering
In addition to the above function, you can also control other aesthetic parameters to control the fonts, border,
# heatmap with annotations pheatmap(df_mat, border_color = FALSE, # no border to cell fontsize_row = 5, # row label font size fontsize_col = 5, # column label font size angle_col = 90, # angle for column labels width = 5, # plot width in inches height = 7, # plot height in inches na_col = "black", # color of the cell with NA values legend = FALSE, # to draw legend or not (TRUE/FALSE) filename = "heatmap.png" # file name with path to save the heatmap )
Tip: If you save an image using the
dev.off()to close the plotting device
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