R EnhancedVolcano 绘制火山图

章鱼猫先生

发布于 2021-10-15 13:46:33

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发布于 2021-10-15 13:46:33

文章被收录于专栏：BioIT爱好者

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，如果对你有所帮助，请点个赞吧。

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原始发表：2020-01-07，如有侵权请联系 cloudcommunity@tencent.com 删除

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