R EnhancedVolcano 绘制火山图
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)
legendVisible = FALSE 可以不展示图注
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。
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