-
Notifications
You must be signed in to change notification settings - Fork 0
/
3.Epi_cells_stage.r
407 lines (352 loc) · 17.4 KB
/
3.Epi_cells_stage.r
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
library(Seurat)
library(dplyr)
library(patchwork)
library(ggplot2)
library(rtracklayer)
library(sctransform)
library(clustree)
library(harmony)
library(cowplot)
library(stringr)
#library(slingshot)
options(future.globals.maxSize = 10000 * 1024^2)
source("/share/pub/dengcy/Singlecell/CC_space/src/Functions.r")
source("/share/pub/dengcy/Singlecell/CC_space/src/library.r")
#"E:/OneDrive/colorectal_sc/CC_space/src/Functions.r"
#"E:/OneDrive/colorectal_sc/CC_space/3.Epi_cells_stage"
setwd("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage")
load("/share/pub/dengcy/Singlecell/CC_space/1.scRNA_seq_prepare/CRC_merged_all.RData")
Idents(CRC_merged_all)<-CRC_merged_all$Initial_annotation
############################
####1.get the epi cells
cancer_scrna<-subset(CRC_merged_all,Initial_annotation %in% c("Epithelial_cells"))
rm(CRC_merged_all)
cancer_scrna$stage<-"StageIII"
cancer_scrna$stage[cancer_scrna$samples=="CC1"]<-"StageI"
cancer_scrna$stage[cancer_scrna$orig.ident %in% c("KUL01-B","KUL01-T","KUL28-B","KUL28-T","KUL30-B","KUL30-T",
"SMC01-T","SMC05-T","SMC09-T","SMC10-T","SMC15-T","SMC18-T","scrEXT001","scrEXT002","scrEXT021",
"scrEXT022","scrEXT027","scrEXT028")]<-"StageII"
cancer_scrna$stage[cancer_scrna$orig.ident %in% c("KUL31-B","KUL31-T","SMC07-T","SMC24-T","scrEXT018","scrEXT019")]<-"StageI"
cancer_scrna_stage1<-subset(cancer_scrna,stage %in% c("StageI","StageII"))
cancer_scrna_stage2<-subset(cancer_scrna,stage== "StageIII")
cancer_scrna_stage1<- cluster_pca_umap(cancer_scrna_stage1, assay = "RNA",reduction="harmony",cluster_res = 0.1)
cancer_scrna_stage2<- cluster_pca_umap(cancer_scrna_stage2, assay = "RNA",reduction="harmony",cluster_res = 0.1)
save(cancer_scrna_stage1,file="cancer_scrna_stage1.RData")
save(cancer_scrna_stage2,file="cancer_scrna_stage2.RData")
color_CRC<-c("#7FBCD2","#A5F1E9","#FFEEAF","#7FB77E","#F7F6DC","#B1D7B4","#FFC090",
"#F3E0B5","#FFAE6D","#FECD70","#E3C770","#94B49F","#CEE5D0","#ECB390","#F2D7D9",
"#D3CEDF","#B2C8DF","#C4D7E0","#F8F9D7","#76BA99","#ADCF9F",
"#CED89E","#FFDCAE","#FFE3A9","#FFC3C3","#FF8C8C","#97C4B8","#F1F0C0","#B1BCE6")
############################
#####2. cluster
pdf(file="Malignant_cells_umap_0.3.pdf",height=8,width=8)
DimPlot(cancer_scrna,pt.size=0.8,label = TRUE, repel=TRUE,label.size = 6)+
umap_theme()+ labs(x="UMAP",y="")+
ggtitle("Malignant cells")+
scale_colour_manual(name = "Cluster", values = color_scanpy_viridis28[c(1,4,5,7,20,11)]) +
theme(aspect.ratio=1)
dev.off()
pdf(file="Malignant_cells_tsne_0.3.pdf",height=8,width=8)
DimPlot(cancer_scrna,pt.size=0.8,label = TRUE,reduction="tsne", repel=TRUE,label.size = 6)+
umap_theme()+ labs(x="TSNE",y="")+
ggtitle("Malignant cells")+
scale_colour_manual(name = "Cluster", values = color_scanpy_viridis28[c(1,4,5,7,20,11)]) +
theme(aspect.ratio=1)
dev.off()
pdf(file="Malignant_cells_samples_0.3.pdf",height=8,width=8)
DimPlot(cancer_scrna,group.by = "samples",pt.size=0.8,label = TRUE, repel=TRUE)+ umap_theme()+
ggtitle("Malignant cells in samples")+labs(x="UMAP",y="")+
scale_colour_manual(name = "Samples", values =c("#E05D5D","#00A19D") ) +
theme(aspect.ratio=1)
dev.off()
####################
# inferCNV
#######################
setwd("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage")
load("/share/pub/dengcy/Singlecell/CC_space/1.scRNA_seq_prepare/CRC_merged_all.RData")
library(AnnoProbe)
library(SeuratDisk)
library(Seurat)
Idents(CRC_merged_all)<-CRC_merged_all$Initial_annotation
a1<-unlist(lapply(c("Macrophage","Fibroblasts"),function(x){
a<-colnames(CRC_merged_all)[Idents(CRC_merged_all)==x]
a<-sample(a,2000)
return(a)
}))
CRC_immune_subset<-CRC_merged_all[,a1]
rm(CRC_merged_all)
load("cancer_scrna_stage1.RData")
CC_infercnv<-merge(cancer_scrna_stage1,CRC_immune_subset)
CC_infercnv$cnv_annotation<-c(cancer_scrna_stage1$seurat_clusters,CRC_immune_subset$Initial_annotation)
a<-GetAssayData(CC_infercnv,assay = "RNA",slot="counts")
a <- CreateSeuratObject(counts = a,
min.cells = 3,
meta.data [email protected],
min.features = 200)
DefaultAssay(a) <- "RNA"
SaveH5Seurat(a, "cancer_scrna_stage1.h5seurat")
Convert("cancer_scrna_stage1.h5seurat", dest="h5ad")
###
load("cancer_scrna_stage2.RData")
CC_infercnv<-merge(cancer_scrna_stage2,CRC_immune_subset)
CC_infercnv$cnv_annotation<-c(cancer_scrna_stage2$seurat_clusters,CRC_immune_subset$Initial_annotation)
rm(cancer_scrna_stage2,CRC_immune_subset)
a<-GetAssayData(CC_infercnv,assay = "RNA",slot="counts")
a <- CreateSeuratObject(counts = a,
min.cells = 3,
meta.data [email protected],
min.features = 200)
DefaultAssay(a) <- "RNA"
SaveH5Seurat(a, "cancer_scrna_stage2.h5seurat")
Convert("cancer_scrna_stage2.h5seurat", dest="h5ad")
########################
###remove mormal cells
#####################
load("cancer_scrna_stage1.RData")
##16115个细胞
stage1_t<-read.csv("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/cancer_infercnv_stage1.csv")
tn<-intersect(colnames(cancer_scrna_stage1),stage1_t$X[stage1_t$cnv_status=="tumor"])
cancer_scrna_stage1<-cancer_scrna_stage1[,tn]
save(cancer_scrna_stage1,file="cancer_scrna_stage1.RData")
##11826个细胞
load("cancer_scrna_stage2.RData")
#18267
stage2_t<-read.csv("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/cancer_infercnv_stage2.csv")
tn<-intersect(colnames(cancer_scrna_stage2),stage2_t$X[stage2_t$cnv_status=="tumor"])
cancer_scrna_stage2<-cancer_scrna_stage2[,tn]
#14026
save(cancer_scrna_stage2,file="cancer_scrna_stage2.RData")
cancer_scrna_stage1<- cluster_pca_umap(cancer_scrna_stage1, assay = "RNA",reduction="harmony",cluster_res = 0.2)
cancer_scrna_stage2<- cluster_pca_umap(cancer_scrna_stage2, assay = "RNA",reduction="harmony",cluster_res = 0.2)
pdf(file="stage1_magliant_clusters_umap_0.2.pdf",height=5,width=5)
DimPlot(cancer_scrna_stage1,group.by="seurat_clusters",pt.size=0.3,label = TRUE, repel=TRUE,label.size = 6)+
umap_theme()+ labs(x="UMAP",y="")+
ggtitle("StageI/II Epithelial cells")+
scale_colour_manual(name = "seurat_clusters", values = color_CRC) +
theme(aspect.ratio=1)
dev.off()
pdf(file="stage2_magliant_clusters_umap_0.2.pdf",height=5,width=5)
DimPlot(cancer_scrna_stage2,group.by="seurat_clusters",pt.size=0.3,label = TRUE, repel=TRUE,label.size = 6)+
umap_theme()+ labs(x="UMAP",y="")+
ggtitle("StageIII Epithelial cells")+
scale_colour_manual(name = "seurat_clusters", values = color_CRC) +
theme(aspect.ratio=1)
dev.off()
save(cancer_scrna_stage2,file="cancer_scrna_stage2.RData")
save(cancer_scrna_stage1,file="cancer_scrna_stage1.RData")
############################
#4.monocle analysis for each stage
########################
setwd("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage")
#source("/share/pub/dengcy/Singlecell/CC_space/src/STrectal/Functions.r")
load("cancer_scrna_stage1.RData")
#BiocManager::install("monocle")
output="/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/"
library(monocle)
library(Seurat)
library(dplyr)
library(patchwork)
library(ggplot2)
library(rtracklayer)
library(sctransform)
library(clustree)
library(harmony)
library(cowplot)
library(stringr)
seuTocds<-function(seu_obj=cancer_scrna_stage1,
output="/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/",
index=""){
expr_mat1<-as(as.matrix(seu_obj@assays$RNA@counts),'sparseMatrix')
p_data<[email protected]
p_data$celltype<-seu_obj$Initial_annotation
f_data<-data.frame(gene_short_name=rownames(seu_obj),row.names=rownames(seu_obj))
pd<-new('AnnotatedDataFrame',data=p_data)
pf<-new('AnnotatedDataFrame',data=f_data)
cds<-newCellDataSet(expr_mat1,phenoData=pd,featureData=pf,lowerDetectionLimit=0.5,expressionFamily=negbinomial.size())
cds <- estimateSizeFactors(cds)
cds <- estimateDispersions(cds)
cds <- detectGenes(cds, min_expr = 0.1)
#聚类分群
disp_table <- dispersionTable(cds)
unsup_clustering_genes <- subset(disp_table, mean_expression >= 0.1)
cds <- setOrderingFilter(cds, unsup_clustering_genes$gene_id)
cds<- reduceDimension(cds, max_components = 2, num_dim = 8, reduction_method = 'tSNE', verbose = T,check_duplicates = FALSE)
cds<- clusterCells(cds, num_clusters = 8)
expressed_genes <- row.names(subset(fData(cds), num_cells_expressed >= 10))
clustering_DEG_genes <- differentialGeneTest(cds[expressed_genes,],fullModelFormulaStr = '~Cluster',cores = 8)
ordering_genes <- row.names(clustering_DEG_genes)[order(clustering_DEG_genes$qval)][1:800] #排序后取前800个基因
cds <- setOrderingFilter(cds,ordering_genes = ordering_genes)
cds <- reduceDimension(cds, method = 'DDRTree')
cds <- orderCells(cds)
return(cds)
}
output="/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/"
index=1
pdf(paste0(output,index,"_cell_trajectoryPseudotime.pdf"))
plot_cell_trajectory(cds1, cell_size=1,color_by = "Pseudotime")+
scale_color_gradient(low = "#f4e2d8",high = "#ba5370")
dev.off()
pdf(paste0(output,index,"_cell_trajectoryCluster.pdf"))
plot_cell_trajectory(cds1, cell_size=2,color_by = "Cluster")+
scale_colour_manual(name = "Cluster", values = color_CRC[1:7] )
dev.off()
pdf(paste0(output,index,"_cell_trajectoryState.pdf"))
plot_cell_trajectory(cds1, cell_size=2,color_by = "State")+
scale_colour_manual(name = "State", values = c("#f38181","#fce38a","#eaffd0","#95e1d3","#64958f") )
dev.off()
cds1<-seuTocds(seu_obj=cancer_scrna_stage1,index="1")
save(cds1,file=paste0(output,"cds_cancer_scrna_stage1.RData"))
load(paste0(output,"cds_cancer_scrna_stage1.RData"))
#########################
###############得到emt相关的基因做交集
###############
#2）.hallmark genes
x <- readLines("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/h.all.v7.4.symbols.gmt")
res <- strsplit(x, "\t")
names(res) <- vapply(res, function(y) y[1], character(1))
genes.by.pathway.h <- lapply(res, "[", -c(1:2))
save(genes.by.pathway.h,file="genes.by.pathway.h.RData")
genes.pathway.h <- unique(unlist(genes.by.pathway.h))
load("/share/pub/dengcy/Singlecell/CC_space/4.stage_visualize/genes.by.pathway.h.RData")
#4）EMT
EMT_genes<-read.delim("/share/pub/dengcy/Singlecell/CC_space/4.stage_visualize/EMTgene.txt")
#intersecton
dehallgene<-intersect(unique(unlist(genes.by.pathway.h)),EMT_genes$x)
###提取轨迹上的差异基因
#expressed_genes <- row.names(subset(fData(cds1), num_cells_expressed >= 10))
dehallgene<-intersect(dehallgene,expressed_genes)
cds1_sub <- cds1[,pData(cds1)$State %in% c('1','3','4','5')] #提取State1、2、6上的细胞
pseudotime_de1 <- differentialGeneTest(cds1_sub[dehallgene,], fullModelFormulaStr = "~sm.ns(Pseudotime)") #求拟时序上基因基因的显著性
pseudotime_de1.1 <- subset(pseudotime_de1, qval < 1e-4) #取q值小于0.0001的基因
save(pseudotime_de1,pseudotime_de1.1,file=paste0(output,"pseudotime_de1.RData"))
library(RColorBrewer)
#做热图
pdf("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/cds1_pseudotime_heatmap.pdf")
plot_pseudotime_heatmap(cds1_sub[pseudotime_de1.1$gene_short_name,],
return_heatmap=T,
num_clusters=4,
hmcols = colorRampPalette(rev(brewer.pal(11, "Spectral")))(70),
show_rownames = F)
dev.off()
load("cancer_scrna_stage2.RData")
cds2<-seuTocds(seu_obj=cancer_scrna_stage2,index="2")
output="/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/"
index=2
pdf(paste0(output,index,"_cell_trajectoryPseudotime.pdf"))
plot_cell_trajectory(cds2, cell_size=2,color_by = "Pseudotime")+
scale_color_gradient(low = "#f4e2d8",high = "#ba5370")
dev.off()
pdf(paste0(output,index,"_cell_trajectoryCluster.pdf"))
plot_cell_trajectory(cds2, cell_size=2,color_by = "Cluster")+
scale_colour_manual(name = "Cluster", values = color_CRC[1:7] )
dev.off()
pdf(paste0(output,index,"_cell_trajectoryState.pdf"))
plot_cell_trajectory(cds2, cell_size=2,color_by = "State")+
scale_colour_manual(name = "State", values = c("#f38181","#fce38a","#95e1d3","#64958f") )
dev.off()
save(cds2,file=paste0(output,"cds_cancer_scrna_stage2.RData"))
load(paste0(output,"cds_cancer_scrna_stage2.RData"))
#expressed_genes <- row.names(subset(fData(cds2), num_cells_expressed >= 10))
#cds2_sub <- cds2[,pData(cds2)$State %in% c('1','3','4','5')] #提取State1、2、6上的细胞
pseudotime_de2 <- differentialGeneTest(cds2[dehallgene,], fullModelFormulaStr = "~sm.ns(Pseudotime)") #求拟时序上基因基因的显著性
pseudotime_de2.1 <- subset(pseudotime_de2, qval < 1e-4) #取q值小于0.0001的基因
save(pseudotime_de2,pseudotime_de2.1,file=paste0(output,"pseudotime_de2.RData"))
#做热图
pdf("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/monocle/cds2_pseudotime_heatmap.pdf")
p = plot_pseudotime_heatmap(cds2[pseudotime_de2.1$gene_short_name,],
return_heatmap=T,
num_clusters=4,
hmcols = colorRampPalette(rev(brewer.pal(11, "Spectral")))(70),
show_rownames = F)
dev.off()
load(paste0(output,"pseudotime_de2.RData"))
write.csv(pseudotime_de2,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pseudotime_de2.csv")
load(paste0(output,"pseudotime_de1.RData"))
write.csv(pseudotime_de1,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pseudotime_de1.csv")
###################
#选择两组基因的交集
emt_pseudotim_gene<-intersect(pseudotime_de2.1$gene_short_name,pseudotime_de1.1$gene_short_name)
#365
save(emt_pseudotim_gene,file="emt_pseudotim_gene.RData")
###############
#Pathway heatmap for stage
################
library(Seurat)
library(SeuratObject)
library("dplyr")
library("foreach")
library("ggplot2")
library(reshape2) # melt
library(gridExtra) # arrangeGrob
library(ggpubr) # as_ggplot
library(cowplot) # draw_plot_label
library(ggtext)
library(testSctpa)
library(VISION)
library(AUCell)
setwd("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage")
load("cancer_scrna_stage1.RData")
counts = load_counts()
se_oj = CreateSeuratObject(counts)
se_oj = cal_PAS(seurat_object = se_oj,
tool = 'AUCell', ## GSVA, ssGSEA, plage, zscore or Vision
normalize = 'log',
species = 'mouse',
pathway='kegg')
DefaultAssay(object = cancer_scrna_stage1) <- "RNA"
gmt<-"/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/h.all.v7.5.1.symbols.gmt"
cancer_scrna_stage1 = ScaleData(cancer_scrna_stage1)
cancer_scrna_stage1 = cal_PAS(seurat_object = cancer_scrna_stage1,
tool = 'AUCell', ## GSVA, ssGSEA, plage, zscore or Vision
normalize = "log",
gmt_file=gmt)
library(dplyr)
Idents(cancer_scrna_stage1)<-cancer_scrna_stage1$State
markers = FindAllMarkers(cancer_scrna_stage1,logfc.threshold = 0)
pathways = markers %>% group_by(cluster) %>% top_n(n=5,wt=avg_log2FC) #For Seurat>4.0, set "wt=avg_logFC"
write.csv(pathways,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pathways_stage1_State.csv")
pdf("state_hallmark_pathway.pdf")
DoHeatmap(cancer_scrna_stage1,features=pathways$gene,slot="data",
group.colors = c("#f38181","#fce38a","#eaffd0","#95e1d3","#64958f"))
dev.off()
Idents(cancer_scrna_stage1)<-cancer_scrna_stage1$Cluster
markers = FindAllMarkers(cancer_scrna_stage1,logfc.threshold = 0)
pathways = markers %>% group_by(cluster) %>% top_n(n=5,wt=avg_log2FC) #For Seurat>4.0, set "wt=avg_logFC"
write.csv(pathways,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pathways_stage1_Cluster.csv")
pdf("cluster_hallmark_pathway.pdf")
DoHeatmap(cancer_scrna_stage1,features=pathways$gene,slot="data",
group.colors = color_CRC[1:7])
dev.off()
###############
load("cancer_scrna_stage2.RData")
cancer_scrna_stage2 = ScaleData(cancer_scrna_stage2)
DefaultAssay(object = cancer_scrna_stage2) <- "RNA"
cancer_scrna_stage2 = cal_PAS(seurat_object = cancer_scrna_stage2,
tool = 'AUCell', ## GSVA, ssGSEA, plage, zscore or Vision
normalize = "log",
gmt_file=gmt)
library(dplyr)
Idents(cancer_scrna_stage2)<-cancer_scrna_stage2$State
markers = FindAllMarkers(cancer_scrna_stage2,logfc.threshold = 0)
pathways = markers %>% group_by(cluster) %>% top_n(n=5,wt=avg_log2FC) #For Seurat>4.0, set "wt=avg_logFC"
write.csv(pathways,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pathways_stage3_State.csv")
pdf("state_hallmark_pathway2.pdf")
DoHeatmap(cancer_scrna_stage2,features=pathways$gene,slot="data",
group.colors = c("#f38181","#fce38a","#eaffd0","#95e1d3","#64958f"))
dev.off()
Idents(cancer_scrna_stage2)<-cancer_scrna_stage2$Cluster
markers = FindAllMarkers(cancer_scrna_stage2,logfc.threshold = 0)
pathways = markers %>% group_by(cluster) %>% top_n(n=5,wt=avg_log2FC) #For Seurat>4.0, set "wt=avg_logFC"
write.csv(pathways,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/pathways_stage3_cluster.csv")
pdf("cluster_hallmark_pathway2.pdf")
DoHeatmap(cancer_scrna_stage2,features=pathways$gene,slot="data",
group.colors = color_CRC[1:7])
dev.off()
##########
#infer cnv output
infercnv_stage1<-read.csv("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/cancer_infercnv_stage1.csv")
infercnv_stage1<-infercnv_stage1[,c("X","cnv_annotation","cnv_leiden","cnv_score","cnv_status")]
infercnv_stage2<-read.csv("/share/pub/dengcy/Singlecell/CC_space/3.Epi_cells_stage/cancer_infercnv_stage2.csv")
infercnv_stage2<-infercnv_stage2[,c("X","cnv_annotation","cnv_leiden","cnv_score","cnv_status")]
infercnv_stage<-rbind(infercnv_stage1,infercnv_stage2)
write.csv(infercnv_stage,file="/share/pub/dengcy/Singlecell/CC_space/SupplementFiles10.26/infercnv_stage_result.csv")