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---
title: "Publication-ready volcano plots with enhanced colouring and labeling"
author: "Kevin Blighe"
date: "`r Sys.Date()`"
package: "`r packageVersion('EnhancedVolcano')`"
output:
    html_document:
        highlight: pygments
        toc: true
            toc_depth: 2
            toc_float: true
    fig_width: 6
bibliography: library.bib
vignette: >
    %\VignetteIndexEntry{Publication-ready volcano plots with enhanced colouring and labeling}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
    %\usepackage[utf8]{inputenc}
---

Introduction.

Volcano plots represent a useful way to visualise the results of differential expression analyses. Here, we present a highly-configurable function that produces publication-ready volcano plots [@EnhancedVolcano]. EnhancedVolcano will attempt to fit as many transcript names in the plot window as possible, thus avoiding 'clogging' up the plot with labels that could not otherwise have been read.


    library(knitr)

    opts_chunk$set(tidy = TRUE, message = FALSE, warning = FALSE)

Installation.

1. Download the package from Bioconductor.


    if (!requireNamespace("BiocManager", quietly = TRUE))

        install.packages("BiocManager")

        BiocManager::install("EnhancedVolcano")

Note: to install development version:


    devtools::install_github("kevinblighe/EnhancedVolcano")

2. Load the package into R session.


    library(EnhancedVolcano)

Quick start.

Following data generated by tutorial of RNA-seq workflow: gene-level exploratory analysis and differential expression:

Load airway data:


    source("https://bioconductor.org/biocLite.R")

    biocLite("airway")

    library(airway)

    library(magrittr)


    data("airway")

    airway$dex %<>% relevel("untrt")

Conduct differential expression using DESeq2:


    library("DESeq2")

    dds <- DESeqDataSet(airway, design = ~ cell + dex)

    dds <- DESeq(dds, betaPrior=FALSE)

    res1 <- results(dds,

        contrast = c("dex","trt","untrt"))

    res1 <- lfcShrink(dds,

        contrast = c("dex","trt","untrt"), res=res1)

    res2 <- results(dds,

        contrast = c("cell", "N061011", "N61311"))

    res2 <- lfcShrink(dds,

        contrast = c("cell", "N061011", "N61311"), res=res2)

Example 1: plot the most basic volcano plot:


    EnhancedVolcano(res1,

        lab = rownames(res1),

        x = "log2FoldChange",

        y = "pvalue")

Advanced features

Virtually all aspects of an EnhancedVolcano plot can be configured for the purposes of accomodating all types of statistical distributions and labelling preferences. EnhancedVolcano will only attempt to label genes that pass the thresholds that you set for statistical significance, i.e., 'pCutoff' and 'FCcutoff'. In addition, it will only label as many of these that can reasonably fit in the plot space. The user can optionally supply a vector of transcript names (as 'selectLab') that s/he wishes to label in the plot.

Example 2: modify cut-offs for log2FC and pvalue; add title; adjust point and label size:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-12,

        FCcutoff = 1.5,

        transcriptPointSize = 1.5,

        transcriptLabSize = 3.0,

        title = "N061011 versus N61311")

Example 3: adjust colour and alpha for point shading:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-12,

        FCcutoff = 1.5,

        transcriptPointSize = 1.5,

        transcriptLabSize = 3.0,

        title = "N061011 versus N61311",

        col=c("black", "black", "black", "red3"),

        colAlpha = 1)

Example 4: adjust axis limits:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-12,

        FCcutoff = 1.5,

        transcriptPointSize = 1.5,

        transcriptLabSize = 3.0,

        title = "N061011 versus N61311",

        colAlpha = 1,

        xlim = c(-8, 8),

        ylim = c(0, -log10(10e-32)))

Example 5: adjust cut-off lines:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-12,

        FCcutoff = 1.5,

        transcriptPointSize = 1.5,

        transcriptLabSize = 3.0,

        title = "N061011 versus N61311",

        colAlpha = 1,

        xlim = c(-8, 8),

        ylim = c(0, -log10(10e-32)),

        cutoffLineType = "twodash",

        cutoffLineCol = "red3",

        cutoffLineWidth = 1.5)

Example 6: adjust legend position, size, and text:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-12,

        FCcutoff = 1.5,

        transcriptPointSize = 1.5,

        transcriptLabSize = 3.0,

        colAlpha = 1,

        cutoffLineType = "twodash",

        cutoffLineCol = "red4",

        cutoffLineWidth = 1.0,

        legend=c("NS","Log (base 2) fold-change","P value",

            "P value & Log (base 2) fold-change"),

        legendPosition = "right",

        legendLabSize = 14,

        legendIconSize = 5.0)

Example 7: plot adjusted p-values:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "padj",

        xlab = bquote(~Log[2]~ "fold change"),

        ylab = bquote(~-Log[10]~adjusted~italic(P)),

        pCutoff = 0.0001,

        FCcutoff = 1.0,

        xlim=c(-6,6),

        transcriptLabSize = 3.0,

        colAlpha = 1,

        legend=c("NS","Log2 FC","Adjusted p-value",

            "Adjusted p-value & Log2 FC"),

        legendPosition = "bottom",

        legendLabSize = 10,

        legendIconSize = 3.0)

Example 8: fit more labels by adding connectors:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "padj",

        xlab = bquote(~Log[2]~ "fold change"),

        ylab = bquote(~-Log[10]~adjusted~italic(P)),

        pCutoff = 0.0001,

        FCcutoff = 2.0,

        xlim = c(-6,6),

        transcriptLabSize = 3.0,

        colAlpha = 1,

        legend=c("NS","Log2 FC","Adjusted p-value",

            "Adjusted p-value & Log2 FC"),

        legendPosition = "bottom",

        legendLabSize = 10,

        legendIconSize = 3.0,

        DrawConnectors = TRUE,

        widthConnectors = 0.5,

        colConnectors = "black")

Example 9: only label key transcripts:


    EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "padj",

        selectLab = c("ENSG00000106565","ENSG00000187758"),

        xlab = bquote(~Log[2]~ "fold change"),

        ylab = bquote(~-Log[10]~adjusted~italic(P)),

        pCutoff = 0.0001,

        FCcutoff = 2.0,

        xlim = c(-6,6),

        transcriptLabSize = 5.0,

        colAlpha = 1,

        legend=c("NS","Log2 FC","Adjusted p-value",

            "Adjusted p-value & Log2 FC"),

        legendPosition = "bottom",

        legendLabSize = 10,

        legendIconSize = 3.0)

Example 10: plot multiple volcanos on the same page:


    p1 <- EnhancedVolcano(res1,

        lab = rownames(res1),

        x = "log2FoldChange",

        y = "pvalue",

        pCutoff = 10e-24,

        FCcutoff = 2.0,

        transcriptLabSize = 2.0,

        colAlpha = 1,

        legendPosition = "bottom",

        legendLabSize = 10,

        legendIconSize = 3.0)

    p2 <- EnhancedVolcano(res2,

        lab = rownames(res2),

        x = "log2FoldChange",

        y = "padj",

        selectLab = c("ENSG00000106565","ENSG00000187758"),

        xlab = bquote(~Log[2]~ "fold change"),

        ylab = bquote(~-Log[10]~adjusted~italic(P)),

        pCutoff = 0.0001,

        FCcutoff = 2.0,

        xlim = c(-6,6),

        transcriptLabSize = 5.0,

        colAlpha = 1,

        legend=c("NS","Log2 FC","Adjusted p-value",

            "Adjusted p-value & Log2 FC"),

        legendPosition = "bottom",

        legendLabSize = 10,

        legendIconSize = 3.0)


    library(gridExtra)

    library(grid)

    grid.arrange(p1, p2, ncol=2, top="EnhancedVolcano")

    grid.rect(gp=gpar(fill=NA))

Acknowledgments

The development of EnhancedVolcano has benefited from contributions and suggestions from:

Sharmila Rana, Myles Lewis

Session info


sessionInfo()

References

@EnhancedVolcano