R语言绘图包系列：
1. 介绍
火山图（Volcano plot）用于展示基因表达差异的分布，横轴为LogFC（Log2 Fold Change，Fold Chang是处理组平均值/对照组平均值），越偏离中心表示差异倍数越大；纵轴为-Log10(P.Value)，值越大差异越显著。一般横轴越偏离中心的点其纵轴值也会比较大，因此呈现火山喷发的形状。（火山图其实就是一种特殊的点图）
2. 安装
if (!requireNamespace('BiocManager', quietly = TRUE))
install.packages('BiocManager')
BiocManager::install('EnhancedVolcano')
# to install development version:
# devtools::install_github('kevinblighe/EnhancedVolcano')
library(EnhancedVolcano)
3. 绘图
数据准备
library(airway)
library(magrittr)
data('airway')
airway$dex %<>% relevel('untrt')
根据gene symbols添加Ensembl IDs
ens <- rownames(airway)
library(org.Hs.eg.db)
symbols <- mapIds(org.Hs.eg.db, keys = ens,
column = c('SYMBOL'), keytype = 'ENSEMBL')
symbols <- symbols[!is.na(symbols)]
symbols <- symbols[match(rownames(airway), names(symbols))]
rownames(airway) <- symbols
keep <- !is.na(rownames(airway))
airway <- airway[keep,]
使用Deseq2进行差异分析
library('DESeq2')
dds <- DESeqDataSet(airway, design = ~ cell + dex)
dds <- DESeq(dds, betaPrior=FALSE)
res <- results(dds,
contrast = c('dex','trt','untrt'))
res <- lfcShrink(dds,
contrast = c('dex','trt','untrt'), res=res, type = 'normal')
3.1 绘制最基本的火山图
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue')
4. 绘图细节调整
4.1 调整log2FC和P值的cutoffs，设置图片标题，调整点和标签的大小
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
title = 'N061011 versus N61311',
pCutoff = 10e-32,
FCcutoff = 0.5,
pointSize = 3.0,
labSize = 6.0)
4.2 调整点的颜色和透明度
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
title = 'N061011 versus N61311',
pCutoff = 10e-16,
FCcutoff = 1.5,
pointSize = 3.0,
labSize = 6.0,
col=c('black', 'black', 'black', 'red3'),
colAlpha = 1)
4.3 调整点的形状
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
title = 'N061011 versus N61311',
pCutoff = 10e-16,
FCcutoff = 1.5,
pointSize = 4.0,
labSize = 6.0,
shape = 8,
colAlpha = 1)
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
title = 'N061011 versus N61311',
pCutoff = 10e-16,
FCcutoff = 1.5,
pointSize = 3.0,
labSize = 6.0,
shape = c(1, 4, 23, 25),
colAlpha = 1)
4.4 调整p值的cutoff
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
xlim = c(-6, 6),
title = 'N061011 versus N61311',
subtitle = paste0('p-value cutoff (red line) drawn ',
'at equivalent of adjusted p=0.0001'),
pCutoff = 0.0001,
FCcutoff = 1.5,
pointSize = 3.0,
labSize = 6.0,
colAlpha = 1,
cutoffLineType = 'solid',
cutoffLineCol = 'red2',
cutoffLineWidth = 2.5,
hline = c(10e-20,
10e-20 * 10e-30,
10e-20 * 10e-60,
10e-20 * 10e-90),
hlineCol = c('black', 'black', 'black', 'black'),
hlineType = c('longdash', 'longdash', 'dotdash', 'dotdash'),
hlineWidth = c(0.4, 0.4, 0.8, 0.8),
gridlines.major = FALSE,
gridlines.minor = FALSE)
4.5 调整图例位置大小和文字
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
pCutoff = 10e-12,
FCcutoff = 1.5,
cutoffLineType = 'twodash',
cutoffLineWidth = 0.8,
pointSize = 4.0,
labSize = 6.0,
colAlpha = 1,
legendLabels=c('Not sig.','Log (base 2) FC','p-value',
'p-value & Log (base 2) FC'),
legendPosition = 'right',
legendLabSize = 16,
legendIconSize = 5.0)
不显示图例
legendPosition = 'none'
4.6 通过添加connectors来加入更多标签
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
xlab = bquote(~Log[2]~ 'fold change'),
pCutoff = 10e-32,
FCcutoff = 2.0,
pointSize = 4.0,
labSize = 6.0,
colAlpha = 1,
legendPosition = 'right',
legendLabSize = 12,
legendIconSize = 4.0,
drawConnectors = TRUE,
widthConnectors = 0.75)
4.7 仅对重要变量添加注释
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
selectLab = c('TMEM176B','ADH1A'),
xlab = bquote(~Log[2]~ 'fold change'),
pCutoff = 10e-14,
FCcutoff = 2.0,
pointSize = 4.0,
labSize = 6.0,
shape = c(4, 35, 17, 18),
colAlpha = 1,
legendPosition = 'right',
legendLabSize = 14,
legendIconSize = 5.0)
4.8 添加盒型注释
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
selectLab = c('VCAM1','KCTD12','ADAM12',
'CXCL12','CACNB2','SPARCL1','DUSP1','SAMHD1','MAOA'),
xlab = bquote(~Log[2]~ 'fold change'),
pCutoff = 10e-14,
FCcutoff = 2.0,
pointSize = 4.0,
labSize = 6.0,
labCol = 'black',
labFace = 'bold',
boxedLabels = TRUE,
colAlpha = 4/5,
legendPosition = 'right',
legendLabSize = 14,
legendIconSize = 4.0,
drawConnectors = TRUE,
widthConnectors = 1.0,
colConnectors = 'black')
4.9 添加斜体标签，转置火山图
lab_italics <- paste0("italic('", rownames(res), "')")
selectLab_italics = paste0(
"italic('",
c('VCAM1','KCTD12','ADAM12', 'CXCL12','CACNB2','SPARCL1','DUSP1','SAMHD1','MAOA'),
"')")
EnhancedVolcano(res,
lab = lab_italics,
x = 'log2FoldChange',
y = 'pvalue',
selectLab = selectLab_italics,
xlab = bquote(~Log[2]~ 'fold change'),
pCutoff = 10e-14,
FCcutoff = 1.0,
pointSize = 3.0,
labSize = 6.0,
labCol = 'black',
labFace = 'bold',
boxedLabels = TRUE,
col = c('black', 'pink', 'purple', 'red3'),
colAlpha = 4/5,
legendPosition = 'bottom',
legendLabSize = 14,
legendIconSize = 4.0,
drawConnectors = TRUE,
widthConnectors = 1.0,
colConnectors = 'black') + coord_flip()
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