文章目錄
火山圖（Volcano plots）是一種常用於可視化差異表達分析（Differential expression analysis）結果的呈現方式，在生物學、基因組學、藥物研發等領域都有廣泛的應用。通過火山圖，研究人員可以直觀地了解在不同條件下的基因或分子表達差異，並從中識別出在生物學過程、疾病發展等方面具有重要意義的變化。在這裡跟大家分享一個繪製火山圖的強大又直覺的R套件，EnhancedVolcano。
如何下載EnhancedVolcano套件?
# 下載EnhancedVolcano套件
if (!requireNamespace('BiocManager', quietly = TRUE))
install.packages('BiocManager')
BiocManager::install('EnhancedVolcano')
# 檢查版本
packageVersion("EnhancedVolcano")進行RNA-seq結果分析
1. 加載套件和載入RNA-seq結果
要使用任何 R 套件之前，需要先使用library()載入需要的套件。這次範例中所使用的RNA-seq檔案可以在這裡下載。
# 加載套件
library(EnhancedVolcano)
# 載入RNA-seq結果
RNAseq <- read.csv("C:/Users/USER/Desktop/RNAseq_Sample.csv", header = T)
head(RNAseq)
成功輸入RNA-seq結果後，就可以直接繪製火山圖囉，過程就是那麼簡單粗暴！
2. 繪製最基本的火山圖
# 繪製最基本的火山圖
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name),x = 'Log.fold.change', y = 'P.value')
3. 修改logFC和P值的截斷值、指定標題、調整點和標籤的大小
# 修改logFC和P值的截斷值、指定標題、調整點和標籤的大小
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5)
4. 調整點點的顏色以及alpha值
將同時通過logFC和P值閾值的變量更改成紅色，其他所有變量塗成黑色。此外，alpha值能控制點點的透明度：1 = 100% 不透明；0 = 100% 透明。
# 調整點點的顏色以及alpha值
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5, col=c('black', 'black', 'black', 'red3'), colAlpha = 1)
5. 調整標點形狀
可以通過shape()參數指定標點形狀。形狀代碼的更多信息，請參考ggplot2：shape。
# 調整標點形狀
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5, shape = c(1, 4, 23, 25), colAlpha = 1)
6. 調整圖例位置、大小和文字
# 調整圖例位置、大小和文字
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, cutoffLineType = 'twodash', cutoffLineWidth = 0.8,, pointSize = 3.0, labSize = 4.5, colAlpha = 1, legendLabels=c('Not sig.','Log (base 2) FC','p-value','p-value & Log (base 2) FC'),legendPosition = 'right',legendLabSize = 16,legendIconSize = 5.0)
7. 添加連接器來顯示更多標記
# 添加連接器來顯示更多標記
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', xlab = bquote(~Log[2]~ 'Log.fold.change'), title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5, legendIconSize = 3.0, drawConnectors = TRUE, widthConnectors = 0.5)
8. 只顯示特定標記
# 只顯示特定標記
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', selectLab = c('Hamp','Irx5','Myh7','Tecrl'), title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5)
9. 在標記外加框框
# 在標記外加框框
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', selectLab = c('Hamp','Irx5','Myh7','Tecrl'), title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5, labCol = 'black', labFace = 'bold', boxedLabels = TRUE, colAlpha = 4/5, legendLabSize = 14, legendIconSize = 4.0, drawConnectors = TRUE, widthConnectors = 1.0, colConnectors = 'black')
10. 將火山圖翻轉到其它面
# 將火山圖翻轉到其它面
EnhancedVolcano(RNAseq,lab = as.character(RNAseq$Name), x = 'Log.fold.change', y = 'P.value', selectLab = c('Hamp','Irx5','Myh7','Tecrl'), title = 'control versus treatment', pCutoff = 10e-4, FCcutoff = 1, pointSize = 3.0, labSize = 4.5, labCol = 'black', labFace = 'bold', boxedLabels = TRUE, colAlpha = 4/5, legendLabSize = 14, legendIconSize = 4.0, drawConnectors = TRUE, widthConnectors = 1.0, colConnectors = 'black') + coord_flip()
以上便是EnhancedVolcano R套件中最常用到的指令和參數，如果想要畫更與眾不同的火山圖，可以參考EnhancedVolcano 套件的詳細教學。
參考文獻
1. Blighe, K, S Rana, and M Lewis. EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. 2018.
2. EnhancedVolcano教學網站.
