单细胞转录组基础分析七：差异基因富集分析

单细胞测序技术是近年最大的生命科学突破之一，相关文章频繁发表于各大顶级期刊，然而单细胞数据的分析依然是大家普遍面临的障碍。本专题将针对10X Genomics单细胞转录组数据演示各种主流分析，包括基于Seurat的基础分析、以及基于clusterProfiler、Monocle、SingleR等R包的延伸分析。不足之处请大家批评指正，欢迎添加Kinesin微信交流探讨！

此前的分析我们按转录特征把细胞分成了很多类别，例如seurat聚类分析得到的按cluster分类，singleR分析得到的按细胞类型分类，monocle分析得到的按拟时状态（state）分类。不同的细胞类型之间，有哪些表达差异基因呢，这些差异基因有特别的意义吗？

基因差异表达分析

library(Seurat)
library(tidyverse)
library(patchwork)
library(monocle)
library(clusterProfiler)
library(org.Hs.eg.db)
rm(list=ls())
dir.create("enrich")
scRNA <- readRDS("scRNA.rds")
mycds <- readRDS("mycds.rds")
#比较cluster0和cluster1的差异表达基因
dge.cluster <- FindMarkers(scRNA,ident.1 = 0,ident.2 = 1)
sig_dge.cluster <- subset(dge.cluster, p_val_adj<0.01&abs(avg_logFC)>1)
#比较B_cell和T_cells的差异表达基因
dge.celltype <- FindMarkers(scRNA, ident.1 = 'B_cell', ident.2 = 'T_cells', group.by = 'celltype')
sig_dge.celltype <- subset(dge.celltype, p_val_adj<0.01&abs(avg_logFC)>1)
#比较拟时State1和State3的差异表达基因
p_data <- subset(pData(mycds),select='State')
scRNAsub <- subset(scRNA, cells=row.names(p_data))
scRNAsub <- AddMetaData(scRNAsub,p_data,col.name = 'State')
dge.State <- FindMarkers(scRNAsub, ident.1 = 1, ident.2 = 3, group.by = 'State')
sig_dge.State <- subset(dge.State, p_val_adj<0.01&abs(avg_logFC)>1)

差异基因GO富集分析

#差异基因GO富集分析
ego_ALL <- enrichGO(gene          = row.names(sig_dge.celltype),
                   #universe     = row.names(dge.celltype),
                   OrgDb         = 'org.Hs.eg.db',
                   keyType       = 'SYMBOL',
                   ont           = "ALL",
                   pAdjustMethod = "BH",
                   pvalueCutoff  = 0.01,
                   qvalueCutoff  = 0.05)
ego_all <- data.frame(ego_ALL)
write.csv(ego_all,'enrich/enrichGO.csv')           
ego_CC <- enrichGO(gene          = row.names(sig_dge.celltype),
                   #universe     = row.names(dge.celltype),
                   OrgDb         = 'org.Hs.eg.db',
                   keyType       = 'SYMBOL',
                   ont           = "CC",
                   pAdjustMethod = "BH",
                   pvalueCutoff  = 0.01,
                   qvalueCutoff  = 0.05)
ego_MF <- enrichGO(gene          = row.names(sig_dge.celltype),
                   #universe     = row.names(dge.celltype),
                   OrgDb         = 'org.Hs.eg.db',
                   keyType       = 'SYMBOL',
                   ont           = "MF",
                   pAdjustMethod = "BH",
                   pvalueCutoff  = 0.01,
                   qvalueCutoff  = 0.05)
ego_BP <- enrichGO(gene          = row.names(sig_dge.celltype),
                   #universe     = row.names(dge.celltype),
                   OrgDb         = 'org.Hs.eg.db',
                   keyType       = 'SYMBOL',
                   ont           = "BP",
                   pAdjustMethod = "BH",
                   pvalueCutoff  = 0.01,
                   qvalueCutoff  = 0.05)           
ego_CC@result$Description <- substring(ego_CC@result$Description,1,70)
ego_MF@result$Description <- substring(ego_MF@result$Description,1,70)
ego_BP@result$Description <- substring(ego_BP@result$Description,1,70)
p_BP <- barplot(ego_BP,showCategory = 10) + ggtitle("barplot for Biological process")
p_CC <- barplot(ego_CC,showCategory = 10) + ggtitle("barplot for Cellular component")
p_MF <- barplot(ego_MF,showCategory = 10) + ggtitle("barplot for Molecular function")
plotc <- p_BP/p_CC/p_MF
ggsave('enrich/enrichGO.png', plotc, width = 12,height = 10)

差异基kegg富集分析

genelist <- bitr(row.names(sig_dge.celltype), fromType="SYMBOL",
                           toType="ENTREZID", OrgDb='org.Hs.eg.db')
genelist <- pull(genelist,ENTREZID)               
ekegg <- enrichKEGG(gene = genelist, organism = 'hsa')
p1 <- barplot(ekegg, showCategory=20)
p2 <- dotplot(ekegg, showCategory=20)
plotc = p1/p2
ggsave("enrich/enrichKEGG.png", plot = plotc, width = 12, height = 10)

获取帮助

本教程的目的在于把常用的单细胞分析流程串起来，适合有一定R语言基础的朋友参考。分析原理和代码我没有详细解释，官网有详细的教程和权威的解释，翻译好的中文教程也有多个版本，有兴趣的可以搜索一下。如果您学习本教程有一定困难，可以点击 “阅读原文” 找到作者联系方式，获取帮助。

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