其实之前我们细胞的分群是很粗糙的，只是一个大概的方向，随着深入的研究，需要对特定细胞的更多亚群进行分析，这里我们选择免疫细胞进行分析，主要是为了跟随文章的脚步，也好完成后续一些示例，比如细胞互作，转录因子、拟时分析等。
首先提取免疫细胞群，然后跑一遍Seurat流程，重新聚类分群。
library(Seurat)immune <- subset(scedata, celltype=="Immune")immune <- ScaleData(immune, vars.to.regress = c("nCount_RNA", "percent.mt"), verbose = FALSE)immune <- FindVariableFeatures(immune, nfeatures = 4000)immune <- RunPCA(immune, npcs = 50, verbose = FALSE)immune <- FindNeighbors(immune, reduction = "pca", dims = 1:50)immune <- FindClusters(immune,resolution = seq(from = 0.1,to = 1.0,by = 0.2))immune <- RunUMAP(immune, reduction = "pca", dims = 1:50)library(clustree)clustree(immune)Idents(immune) <- "RNA_snn_res.0.5"immune$seurat_clusters <- immune@active.identDimPlot(immune, label = T,pt.size = 1)
查看下免疫细胞marker的表达。
immune_cellmarker <- c("CD3D",'CD3E','CD2',"CD4","CD8A",#T cell'CD79A','MZB1','MS4A1','CD79B',#B cell'FOXP3',"IL32",'TNFRSF18','TNFRSF4',#Treg'IL17A','IL17F','CD40LG',#Th17'S100A8','CXCL8','SOD2','NAMPT',#Neutrophil'SEPP1','C1QA','APOE','CD14','RNASE1',#Macrophage'TPSAB1','TPSB2','CPA3','HPGDS',#Mast'HLA-DRA','HLA-DPB1','CST3','HLA-DPA1',#mDC'PTGDS','SOX4','GZMB','IRF7',#pDC'IGHA1','IGHG1',"IGHG2",#Plasma'KLRF1','KLRD1','XCL2','XCL1'#NK)library(ggplot2)DotPlot(immune, features = immune_cellmarker)+theme_bw()+theme(panel.grid = element_blank(), axis.text.x=element_text(hjust = 1,vjust=0.5,angle=90))+labs(x=NULL,y=NULL)+guides(size=guide_legend(order=3))+scale_color_gradientn(values = seq(0,1,0.2),colours = c('#330066','#336699','#66CC66','#FFCC33'))
然后对细胞进行定群。
immune <- subset(immune, idents = c("1","8","9"), invert = TRUE)new.cluster.ids <- c("0"="Macrophage","2"="T cell","3"="Macrophage","4"="mDC","5"="Neutrophil","6"="Macrophage","7"="Macrophage","10"="Mast")immune <- RenameIdents(immune, new.cluster.ids)immune$celltype <- immune@active.identDimPlot(immune, label = T,pt.size = 1,group.by = "celltype")
以上并不是新内容，亚群分析之后还可以和之前一样，做比例等。不过今天这里我们演示下细胞互作，用Cellcall这个比较简单的包。
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将GM和BM分开做互作，可以看看不同状态下细胞互作之间的区别。
library(devtools)devtools::install_github("ShellyCoder/cellcall")library(cellcall)GM_immune <- subset(immune, group=="GM")test <- CreateObject_fromSeurat(Seurat.object= GM_immune, #seurat对象slot="counts",cell_type="celltype", #细胞类型data_source="UMI",scale.factor = 10^6,Org = "Homo sapiens") #物种信息mt <- TransCommuProfile(object = test,pValueCor = 0.05,CorValue = 0.1,topTargetCor=1,p.adjust = 0.05,use.type="median",probs = 0.9,method="mean",IS_core = TRUE,Org = 'Homo sapiens')#有多少细胞类型就设置多少个颜色cell_color <- data.frame(color=c("#FF34B3","#BC8F8F","#20B2AA","#00F5FF","#FFA500"), stringsAsFactors = FALSE)rownames(cell_color) <- c("Macrophage","T cell","mDC","Neutrophil","Mast")#绘制互作图ViewInterCircos(object = mt, font = 2, cellColor = cell_color,lrColor = c("#F16B6F", "#84B1ED"),arr.type = "big.arrow",arr.length = 0.04,trackhight1 = 0.05, slot="expr_l_r_log2_scale",linkcolor.from.sender = TRUE,linkcolor = NULL, gap.degree = 0.5, #细胞类型多的话设置小点，不然图太大画不出来trackhight2 = 0.032, track.margin2 = c(0.01,0.12), DIY = FALSE)#可视化互作受配体关系viewPheatmap(object = mt, slot="expr_l_r_log2_scale", show_rownames = T,show_colnames = T,treeheight_row=0, treeheight_col=10,cluster_rows = T,cluster_cols = F,fontsize = 12,angle_col = "45",main="score")
BM的互作结果为，变化还是挺大的。
除了这些，cellcall还可以做其他的事情，具体参考：
https://github.com/ShellyCoder/cellcall
做细胞互作的工具很多，比如iTALK，Cellchat，CellphoneDB等，感兴趣的可以自己取探索下。好了，今天的分享就到这里了，其实这篇分享不是很严谨，主要是演示单细胞数据进一步分析思路，希望对大家有启发。
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