火山图是用于差异表达分析结果可视化的一种有效方法。今天，我们来介绍一个用于增强火山图绘制的强大 R 包：EnhancedVolcano ，该包拥有强大的绘图功能，用户可以简单的通过设置颜色、形状、大小和阴影等参数定义不同的绘图属性，此外通过可以通过添加连线的方式有效避免数据点之间的重叠现象。使用 EnhancedVocalno 包绘制的火山图基本可以直接用于文献发表，可以说非常简单又实用的一款神器了。

1. 下载与安装

R 版本：3.6.1。从 Bioconductor 中下载包：

if (!requireNamespace('BiocManager', quietly = TRUE))    install.packages('BiocManager')    BiocManager::install('EnhancedVolcano')

2. 简单使用

2.1 输入数据格式

首先，我们先来介绍一下 EnhancedVolcan o 输入数据格式。EnhancedVolcano 包可以使用多种差异算法（例如 DESeq2 等）的结果作为输入，数据中需包含 log2FC 、Pvalue  或（和） qvalue 结果，示例数据如下：

2.2 基础绘图

library(EnhancedVolcano)res <- read.table(diffexpress,                  sep="\t",                  head=T,                  row.names=1,                  check.names=F,                  quote="")  EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4))

3、进阶功能

3.1 调整阈值，设置点及标签大小

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    transcriptPointSize = 3.0,    transcriptLabSize = 3.0)

3.2 调整颜色及点透明度

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    col = c('black', 'black', 'black', 'red3'),    colAlpha = 1)

3.3 调整绘图点形状

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    #shape = 8,   #点形状    shape = c(1, 4, 23, 25), #形状列表    colAlpha = 1)

3.4 改变截止线及添加阈值线

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'blank',    cutoffLineCol = 'black',    cutoffLineWidth = 0.8,    hline = c(10e-4, 10e-8, 10e-12, 10e-15),    hlineCol = c('grey0', 'grey25','grey50','grey75'),    hlineType = 'longdash',    hlineWidth = 0.8,    gridlines.major = FALSE,    gridlines.minor = FALSE)

3.5 调整标注位置、大小及文字

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    legendLabSize = 16,    legendIconSize = 5.0)

3.6 校正后的 p 值作图

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,     xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    legendLabSize = 16,    legendIconSize = 5.0)

3.7 添加连线用于展示更多标注

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    drawConnectors = TRUE,    legendLabSize = 16,    legendIconSize = 5.0)

3.8 只标注重要变量

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    selectLab = c('Spp1','S100a11','Mgp','LOC498555','Sh3bgrl',                  'Ring1','Apoe','Tcn2','Ager','Mc1r'),    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    drawConnectors = TRUE,    legendLabSize = 16,    legendIconSize = 5.0)

3.9 加框展示变量

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    selectLab = c('Spp1','S100a11','Mgp','LOC498555','Sh3bgrl',                  'Ring1','Apoe','Tcn2','Ager','Mc1r'),    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    drawConnectors = TRUE,    boxedlabels = TRUE,    legendLabSize = 16,    legendIconSize = 5.0)

3.10 针对特殊点设置颜色

colCustom 功能可针对特定位点设置颜色，例如上下调基因设置不同颜色，参考代码如下：

keyvals <- rep('black', nrow(res))

 # set the base name/label as 'Mid' names(keyvals) <- rep('Mid', nrow(res))

 # fold change > 1.5 & p-value < 0.0001 为高表达 keyvals[which(res$"log2(Fold_change)" > 1.5 & res$"p-value"<0.0001)] <- 'gold' names(keyvals)[which(res$"log2(Fold_change)" > 1.5 & res$"p-value"<0.0001)] <- 'high'

 # fold change < -1.5 & p-value < 0.0001为低表达 keyvals[which(res$"log2(Fold_change)" < -1.5 & res$"p-value"<0.0001)] <- 'royalblue' names(keyvals)[which(res$"log2(Fold_change)" < -1.5 & res$"p-value"<0.0001)] <- 'low'

EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = 'A versus B',    pCutoff = 10e-3,    FCcutoff = 1.5,    xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    selectLab = rownames(res)[which(names(keyvals) %in% c('high', 'low'))],    transcriptPointSize = 3.0,    transcriptLabSize = 3.0,    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    colCustom = keyvals,    border = 'full',    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    drawConnectors = FALSE,    boxedlabels = FALSE,    legendLabSize = 16,    legendIconSize = 5.0)

3.11 设置特定点的大小

p <- EnhancedVolcano(res,    lab = rownames(res),    x = 'log2(Fold_change)',    y = 'p-value',    xlim = c(-4, 4),    ylim = c(0,15),    title = '',    subtitle = '',    pCutoff = 10e-3,    FCcutoff = 1.5,    xlab = bquote(~Log[2]~ 'fold change'),    ylab = bquote(~-Log[10]~adjusted~italic(P)),    selectLab = rownames(res)[which(names(keyvals) %in% c('high', 'low'))],    transcriptLabSize = 3.0,    transcriptPointSize = c(ifelse((res$"log2(Fold_change)">2 |res$"log2(Fold_change)"< -2) & res$"p-value"<0.0001 , 3, 1)),    colAlpha = 1,    cutoffLineType = 'twodash',    cutoffLineWidth = 0.8,    colCustom = keyvals,    border = 'full',    legend=c('NS','Log (base 2) fold-change','P value',      'P value & Log (base 2) fold-change'),    legendPosition = 'right',    drawConnectors = FALSE,    boxedlabels = FALSE,    legendLabSize = 16,    legendIconSize = 5.0,    caption = "")p

3.12 自定义刻度

p +    ggplot2::coord_cartesian(xlim=c(-6, 6)) +    ggplot2::scale_x_continuous(      breaks=seq(-6,6, 1))

EnhancedVolcano 包绘制火山图就先介绍到这里，如果对你有所帮助，请点个赞吧。