scRNA基础分析-1:安装包、导入数据、过滤质控 -
scRNA基础分析-2：降维与聚类 -
scRNA基础分析-3：鉴定细胞类型 -
scRNA基础分析-4：细胞亚类再聚类、注释 -
scRNA基础分析-5：伪时间分析 -
scRNA基础分析-6：富集分析 -
在之前的分析中，已经将细胞分为多种不同的类型，例如cluster、cell type、stage等。以cluster为例，不同的cluster有哪些基因表达差异显著，有何特别的意义？富集分析可以帮助我们挖掘一些信息。
之前学习转录组时，已了解有关富集分析的基本概念，详见笔记。
library(Seurat)
library(tidyverse)
library(patchwork)
library(monocle)
library(clusterProfiler)
library(org.Hs.eg.db)
rm(list=ls())
scRNA <- readRDS("scRNA.rds")
mycds <- readRDS("mycds.rds")
1、差异分析
- 差异分析是富集分析的基础，基于前面的结果，我们可以进行多种差异分析。
#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，后面的演示以此数据为例
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)
2、go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)
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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)
#截取每个description的前70个字符，方便后面作图排版
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
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3、kegg分析
genelist <- bitr(row.names(sig_dge.celltype), fromType="SYMBOL",
toType="ENTREZID", OrgDb='org.Hs.eg.db')
# kegg分析的基因名必须要是ENTREZID
genelist <- pull(genelist,ENTREZID)
ekegg <- enrichKEGG(gene = genelist, organism = 'hsa')
p1 <- barplot(ekegg, showCategory=20)
p2 <- dotplot(ekegg, showCategory=20)
plotc = p1/p2
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