介绍
文章提供画图代码和数据，本文记录
数据和代码
数据可从以下链接下载（画图所需要的所有数据）：
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百度云盘链接: https://pan.baidu.com/s/1peU1f8_TG2kUKXftkpYqug
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提取码: 7pjy
图1
#### Figure 1: Census of cell types of the mouse uterine tube ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
#### Distal and Proximal Datasets ####
Distal <- readRDS(file = "../dataset/Distal_Filtered_Cells.rds" , refhook = NULL)
Proximal <- readRDS( file = "../dataset/Proximal_Filtered_Cells.rds" , refhook = NULL)
#### Figure 1b: Cells of the Distal Uterine Tube ####
Distal_Named <- RenameIdents(Distal,
'0' = "Fibroblast 1",
'1' = "Fibroblast 2",
'2' = "Secretory Epithelial",
'3' = "Smooth Muscle",
'4' = "Ciliated Epithelial 1",
'5' = "Fibroblast 3",
'6' = "Stem-like Epithelial 1",
'7' = "Stem-like Epithelial 2",
'8' = "Ciliated Epithelial 2",
'9' = "Blood Endothelial",
'10' = "Pericyte",
'11' = "Intermediate Epithelial",
'12' = "T/NK Cell",
'13' = "Epithelial/Fibroblast",
'14' = "Macrophage",
'15' = "Erythrocyte",
'16' = "Luteal",
'17' = "Mesothelial",
'18' = "Lymph Endothelial/Epithelial") # Remove cluster due few data points and suspected doublet
Distal_Named@active.ident <- factor(x = Distal_Named@active.ident,
levels = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Pericyte',
'Blood Endothelial',
'Lymph Endothelial/Epithelial',
'Epithelial/Fibroblast',
'Stem-like Epithelial 1',
'Stem-like Epithelial 2',
'Intermediate Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial 1',
'Ciliated Epithelial 2',
'T/NK Cell',
'Macrophage',
'Erythrocyte',
'Mesothelial',
'Luteal'))
Distal_Named <- SetIdent(Distal_Named, value = Distal_Named@active.ident)
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
Muscle <- c('#E55451' , '#FFB7B2') # Reds
Endothelial <- c('#A0E6FF') # Reds
FiboEpi <- "#FFE0B3" # Reddish Brown
Epi <-c('#6E3E6E','#8A2BE2','#CCCCFF','#DA70D6','#DF73FF','#604791') # Blues/Purples
Immune <- c( '#5A5E6B' , '#B8C2CC' , '#FC86AA') # Yellowish Brown
Meso <- "#9EFFFF" # Pink
Lut <- "#9DCC00" # Green
colors <- c(Fibroblasts, Muscle, Endothelial, FiboEpi, Epi, Immune, Meso, Lut)
pie(rep(1,length(colors)), col=colors)
Distal_Named <- subset(Distal_Named,
idents = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Pericyte',
'Blood Endothelial',
'Epithelial/Fibroblast',
'Stem-like Epithelial 1',
'Stem-like Epithelial 2',
'Intermediate Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial 1',
'Ciliated Epithelial 2',
'T/NK Cell',
'Macrophage',
'Erythrocyte',
'Mesothelial',
'Luteal'))
p1 <- DimPlot(
Distal_Named,
reduction='umap',
cols=colors,
pt.size = 0.5,
label.size = 4,
label.color = "black",
repel = TRUE,
label=F) +
NoLegend() +
labs(x="UMAP_1",y="UMAP_2")
LabelClusters(p1, id="ident", color = "black", repel = T , size = 4, box.padding = .75)
ggsave(filename = "FIG1b_all_distal_umap.pdf", plot = p1, width = 8, height = 12, dpi = 600)
## Figure 1c: Distal Uterine Tube Features for Cell Type Identification ##
features <- c("Dcn","Col1a1", # Fibroblasts
"Acta2","Myh11","Myl9", # Smooth Muscle
"Pdgfrb","Mcam","Cspg4", # Pericyte
"Sele","Vwf","Tek", # Blood Endothelial
"Lyve1","Prox1","Icam1", # Lymph Endothelial
"Epcam","Krt8", # Epithelial
"Foxj1", # Ciliated Epithelial
"Ovgp1", # Secretory Epithelial
"Slc1a3","Pax8","Cd44","Itga6", # Stem-like Epithelieal
"Ptprc", # Immune
"Cd8a","Cd4","Cd3e", # T-Cell
"Klrc1","Runx3", # T/NK Cell
"Klrd1", # NK Cell
"Aif1","Cd68","Csf1r","Itgax", # Macrophage
"Hbb-bs", "Hba-a1", # Erythrocytes
"Fras1","Rspo1","Lrrn4","Msln", # Mesothelial
"Cyp11a1","Bcat1","Fkbp5","Spp1","Prlr") # Luteal Cells
all_dp <- DotPlot(object = Distal_Named, # Seurat object
assay = 'RNA', # Name of assay to use. Default is the active assay
features = features, # List of features (select one from above or create a new one)
# Colors to be used in the gradient
col.min = 0, # Minimum scaled average expression threshold (everything smaller will be set to this)
col.max = 2.5, # Maximum scaled average expression threshold (everything larger will be set to this)
dot.min = 0, # The fraction of cells at which to draw the smallest dot (default is 0)
dot.scale = 9, # Scale the size of the points
group.by = NULL, # How the cells are going to be grouped
split.by = NULL, # Whether to split the data (if you fo this make sure you have a different color for each variable)
scale = TRUE, # Whether the data is scaled
scale.by = "radius", # Scale the size of the points by 'size' or 'radius'
scale.min = NA, # Set lower limit for scaling
scale.max = NA # Set upper limit for scaling
)+
labs(x = NULL, y = NULL)+
scale_color_viridis_c(option="F",begin=.4,end=0.9, direction = -1)+
geom_point(aes(size=pct.exp), shape = 21, colour="black", stroke=0.6)+
#theme_linedraw()+
guides(x = guide_axis(angle = 90))+
theme(axis.text.x = element_text(size = 14 , face = "italic"))+
theme(axis.text.y = element_text(size = 14))+
scale_y_discrete(limits = rev(levels(Distal_Named)))+
theme(legend.title = element_text(size = 14))
ggsave(filename = "FIG1c_all_distal_dotplot.pdf", plot = all_dp, width = 18, height = 10, dpi = 600)
图2
#### Figure 2: Characterization of distal epithelial cell states ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
library(monocle3)
#### Load Distal Epithelial Dataset ####
Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
EpiSecStemMarkers <- FindMarkers(Epi_Named,
ident.1 = "Spdef+ Secretory",
ident.2 = "Slc1a3+ Stem/Progenitor")
write.csv(EpiSecStemMarkers, file = "20240319_Staining_Markers2.csv")
#### Figure 2a: Epithelial Cells of the Distal Uterine Tube ####
epi_umap <- DimPlot(object = Epi_Named, # Seurat object
reduction = 'umap', # Axes for the plot (UMAP, PCA, etc.)
repel = TRUE, # Whether to repel the cluster labels
label = FALSE, # Whether to have cluster labels
cols = c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6"), #9
pt.size = 0.6, # Size of each dot is (0.1 is the smallest)
label.size = 0.5) + # Font size for labels
# You can add any ggplot2 1customizations here
labs(title = 'Colored by Cluster')+ # Plot title
NoLegend() +
labs(x="UMAP_1",y="UMAP_2")
ggsave(filename = "Fig2a_epi_umap.pdf", plot = epi_umap, width = 15, height = 12, dpi = 600)
#### Figure 2c: Distal Uterine Tube Features for Epithelial Cell State Identification ####
distal_features <- c("Krt8","Epcam",
"Slc1a3","Cd44","Sox9",
"Ovgp1","Sox17","Pax8", "Egr1",
"Itga6", "Bmpr1b",
"Rhoj", "Klf6","Msln","Cebpd",
"Dpp6", "Sec14l3", "Fam161a",
"Prom1", "Ly6a", "Kctd8", "Adam8",
"Dcn", "Col1a1", "Col1a2", "Timp3", "Pdgfra","Lgals1",
"Upk1a", "Thrsp","Spdef","Lcn2",
"Selenop", "Gstm2",
"Foxj1","Fam183b",
"Rgs22","Dnali1", "Mt1" , "Dynlrb2","Cdh1")
epi_dp <- DotPlot(object = Epi_Named, # Seurat object
assay = 'RNA', # Name of assay to use. Default is the active assay
features = distal_features, # List of features (select one from above or create a new one)
# Colors to be used in the gradient
col.min = 0, # Minimum scaled average expression threshold (everything smaller will be set to this)
col.max = 2.5, # Maximum scaled average expression threshold (everything larger will be set to this)
dot.min = 0, # The fraction of cells at which to draw the smallest dot (default is 0)
dot.scale = 4, # Scale the size of the points
group.by = NULL, # How the cells are going to be grouped
split.by = NULL, # Whether to split the data (if you fo this make sure you have a different color for each variable)
scale = TRUE, # Whether the data is scaled
scale.by = "radius", # Scale the size of the points by 'size' or 'radius'
scale.min = NA, # Set lower limit for scaling
scale.max = NA )+ # Set upper limit for scaling
labs(x = NULL, # x-axis label
y = NULL)+
scale_color_viridis_c(option="F",begin=.4,end=0.9, direction = -1)+
geom_point(aes(size=pct.exp), shape = 21, colour="black", stroke=0.6)+
#theme_linedraw()+
guides(x = guide_axis(angle = 90))+
theme(axis.text.x = element_text(size = 8 , face = "italic"))+
theme(axis.text.y = element_text(size = 9))+
theme(legend.title = element_text(size = 9))+
theme(legend.text = element_text(size = 8))+
scale_y_discrete(limits = c("Ciliated 2",
"Ciliated 1",
"Selenop+/Gstm2high Secretory",
"Spdef+ Secretory",
"Fibroblast-like",
"Pax8low/Prom1+ Cilia-forming",
"Slc1a3med/Sox9+ Cilia-forming",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3+ Stem/Progenitor"))
ggsave(filename = "Fig2b_epi_dot_plot.pdf", plot = epi_dp, width = 8.3, height = 4.0625, dpi = 600)
#### Load Distal Epithelial Pseudotime Dataset ####
Distal_PHATE <- readRDS(file = "../dataset/Distal_Epi_PHATE.rds" , refhook = NULL)
Beeg_PHATE <- Distal_PHATE
Beeg_PHATE@reductions[["phate"]]@cell.embeddings <- Distal_PHATE@reductions[["phate"]]@cell.embeddings*100
cds <- readRDS(file = "../dataset/Distal_Epi_PHATE_Monocle3.rds" , refhook = NULL)
#### Figure 2b: PHATE embedding for differentiation trajectory of distal epithelial cells ####
phate_dif <- DimPlot(Beeg_PHATE ,
reduction = "phate",
cols = c("#B20224",
"#35EFEF",
"#00A1C6",
"#A374B5",
"#9000C6",
"#EA68E1",
"#59D1AF",
"#2188F7",
"#F28D86"),
pt.size = 0.7,
shuffle = TRUE,
seed = 0,
label = FALSE)+
labs(title = 'Colored by Cluster')+ # Plot title
NoLegend() +
labs(x="UMAP_1",y="UMAP_2")
ggsave(filename = "Fig3a_epi_phate.pdf", plot = phate_dif, width = 15, height = 12, dpi = 600)
#### Figure 2d: PHATE and Monocle3 differentiation trajectory path ####
pseudtotime <- plot_cells(cds,
color_cells_by = "pseudotime",
label_cell_groups=FALSE,
label_leaves=FALSE,
label_branch_points=FALSE,
graph_label_size=0,
cell_size = .01,
cell_stroke = 1)+
theme(axis.title.x = element_blank())+
theme(axis.title.y = element_blank())+
theme(axis.line.x = element_blank())+
theme(axis.line.y = element_blank())+
theme(axis.ticks.x = element_blank())+
theme(axis.ticks.y = element_blank())+
theme(axis.text.x = element_blank())+
theme(axis.text.y = element_blank())+
theme(legend.text = element_text(size = 12))
ggsave(filename = "Fig3b_epi_pseudtotime.pdf", plot = pseudtotime, width = 18, height = 12, dpi = 600)
#### Figure 2e: Slc1a3 PHATE Feature Plot ####
Slc1a3_PHATE <- FeaturePlot(Beeg_PHATE, features = c("Slc1a3"), reduction = "phate", pt.size = 0.5)+
scale_color_viridis_c(option="F",begin=.4,end=0.99, direction = -1)+
theme(plot.title = element_text(size = 32,face = "bold.italic"))+
theme(axis.title.x = element_blank())+
theme(axis.title.y = element_blank())+
theme(axis.line.x = element_blank())+
theme(axis.line.y = element_blank())+
theme(axis.ticks.x = element_blank())+
theme(axis.ticks.y = element_blank())+
theme(axis.text.x = element_blank())+
theme(axis.text.y = element_blank())+
theme(legend.text = element_text(size = 12))
ggsave(filename = "Fig3c_SLC1A3_PHATE.pdf", plot = Slc1a3_PHATE, width = 18, height = 9, dpi = 600)
#### Figure 2f: Pax8 PHATE Feature Plot ####
Pax8_PHATE <- FeaturePlot(Beeg_PHATE, features = c("Pax8"), reduction = "phate", pt.size = 0.5)+
scale_color_viridis_c(option="F",begin=.4,end=0.99, direction = -1)+
theme(plot.title = element_text(size = 32,face = "bold.italic"))+
theme(axis.title.x = element_blank())+
theme(axis.title.y = element_blank())+
theme(axis.line.x = element_blank())+
theme(axis.line.y = element_blank())+
theme(axis.ticks.x = element_blank())+
theme(axis.ticks.y = element_blank())+
theme(axis.text.x = element_blank())+
theme(axis.text.y = element_blank())+
theme(legend.text = element_text(size = 12))
ggsave(filename = "Fig3d_PAX8_PHATE.pdf", plot = Pax8_PHATE, width = 18, height = 9, dpi = 600)
图6
#### Figure 6: Identification of cancer-prone cell states ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
library(monocle3)
library(ComplexHeatmap)
library(ggExtra)
library(gridExtra)
library(egg)
library(scales)
#### Load and Prepare Distal Epithelial and Epithelial Pseudotime Datasets ####
Distal_PHATE <- readRDS(file = "../dataset/Distal_Epi_PHATE.rds" , refhook = NULL)
cds <- readRDS(file = "../dataset/Distal_Epi_PHATE_Monocle3.rds" , refhook = NULL)
Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
## Calculate Pseudotime Values ##
pseudo <- pseudotime(cds)
Distal_PHATE@meta.data$Pseudotime <- pseudo # Add to Seurat Metadata
## Subset Seurat Object ##
color_cells <- DimPlot(Distal_PHATE , reduction = "phate",
cols = c("#B20224", #1
"#35EFEF", #2
"#00A1C6", #3
"#A374B5", #4
"#9000C6", #5
"#EA68E1", #6
"lightgrey", #7
"#2188F7", #8
"#F28D86"),
pt.size = 0.7,
shuffle = TRUE,
seed = 0,
label = FALSE)
## Psuedotime and Lineage Assignment ##
cellID <- rownames(Distal_PHATE@reductions$phate@cell.embeddings)
phate_embeddings <- Distal_PHATE@reductions$phate@cell.embeddings
pseudotime_vals <- Distal_PHATE@meta.data$Pseudotime
combined_data <- data.frame(cellID, phate_embeddings, pseudotime_vals)
# Calculate the Average PHATE_1 Value for Pseudotime Points = 0 #
avg_phate_1 <- mean(phate_embeddings[pseudotime_vals == 0, 1])
# Pseudotime Values lower than avge PHATE_1 Embedding will be Negative to split lineages
combined_data$Split_Pseudo <- ifelse(phate_embeddings[, 1] < avg_phate_1, -pseudotime_vals, pseudotime_vals)
# Define Lineage #
combined_data$lineage <- ifelse(combined_data$PHATE_1 < avg_phate_1, "Secretory",
ifelse(combined_data$PHATE_1 > avg_phate_1, "Ciliogenic", "Progenitor"))
Distal_PHATE$Pseudotime_Adj <- combined_data$Split_Pseudo
Distal_PHATE$Lineage <- combined_data$lineage
# Subset #
Pseudotime_Lineage <- subset(Distal_PHATE,
idents = c("Secretory 1",
"Secretory 2",
"Msln+ Progenitor",
"Slc1a3+/Sox9+ Cilia-forming",
"Pax8+/Prom1+ Cilia-forming",
"Progenitor",
"Ciliated 1",
"Ciliated 2"))
## Set Bins ##
bins <- cut_number(Pseudotime_Lineage@meta.data$Pseudotime_Adj , 40) # Evenly distribute bins
Pseudotime_Lineage@meta.data$Bin <- bins # Metadata for Bins
## Set Idents to PSeudoime Bin ##
time_ident <- SetIdent(Pseudotime_Lineage, value = Pseudotime_Lineage@meta.data$Bin)
av.exp <- AverageExpression(time_ident, return.seurat = T)$RNA # Calculate Avg log normalized expression
# Calculates Average Expression for Each Bin #
# if you set return.seurat=T, NormalizeData is called which by default performs log-normalization #
# Reported as avg log normalized expression #
#### Figure 6c: PHATE embedding for differentiation trajectory of distal epithelial cells ####
# Create the stacked barplot
rainbow20 <- c('#FF0000',
'#FF6000',
'#FF8000',
'#FFA000',
'#FFC000',
'#FFE000',
'#FFFF00',
'#E0FF00',
'#C0FF00',
'#A0FF00',
'#00FF00',
'#00FFA0',
'#00F0FF',
'#00A0FF',
'#0020FF',
'#4000FF',
'#8000FF',
'#A000FF',
'#C000FF',
'#E000FF')
rainbow_pseudo <- DimPlot(Pseudotime_Lineage , reduction = "phate",
cols = c(rev(rainbow20),rainbow20),
pt.size = 1.2,
shuffle = TRUE,
seed = 0,
label = FALSE,
group.by = "Bin")+
NoLegend()
ggsave(filename = "rainbow_pseudo.pdf", plot = rainbow_pseudo, width = 20, height = 10, dpi = 600)
#### Figure 6d: PHATE embedding for differentiation trajectory of distal epithelial cells ####
## Pseudotime Scale Bar ##
list <- 1:40
colors = c(rev(rainbow20),rainbow20)
df <- data.frame(data = list, color = colors)
pseudo_bar <- ggplot(df, aes(x = 1:40, y = 1, fill = color)) +
geom_bar(stat = "identity",position = "fill", color = "black", size = 0, width = 1) +
scale_fill_identity() +
theme_void()+
theme(axis.line = element_blank(),
axis.ticks = element_blank(),
axis.text = element_blank(),
axis.title = element_blank())
ggsave(filename = "pseudo_bar.pdf", plot = pseudo_bar, width = 0.98, height = 0.19, dpi = 600)
## Plot gene list across pseudotime bin ##
features <- c('Upk1a', "Spdef", "Ovgp1", "Gstm2", "Selenop", "Msln", "Slc1a3",
"Itga6", "Pax8",'Ecrg4', 'Sox5', 'Pde4b', 'Lcn2','Klf6',
'Trp53' , 'Trp73','Krt5','Foxa2','Prom1','Clstn2','Spef2','Dnah12','Foxj1', 'Fam166c' , 'Cfap126',
'Fam183b')
# Create Bin List and expression of features #
bin_list <- unique(Pseudotime_Lineage@meta.data$Bin)
plot_info <- as.data.frame(av.exp[features, ]) # Call Avg Expression for features
z_score <- transform(plot_info, SD=apply(plot_info,1, mean, na.rm = TRUE))
z_score <- transform(z_score, MEAN=apply(plot_info,1, sd, na.rm = TRUE))
z_score1 <- (plot_info-z_score$MEAN)/z_score$SD
plot_info$y <- rownames(plot_info) # y values as features
z_score1$y <- rownames(plot_info)
plot_info <- gather(data = plot_info, x, expression, bin_list) #set plot
z_score1 <- gather(data = z_score1, x, z_score, bin_list) #set plot
# Create Cell Clusters DF #
Labeled_Pseudotime_Lineage <- RenameIdents(Pseudotime_Lineage,
'Secretory 1' = "Spdef+ Secretory",
'Progenitor' = "Slc1a3+ Stem/Progenitor",
'Msln+ Progenitor' = "Cebpdhigh/Foxj1- Progenitor",
'Ciliated 1' = "Ciliated 1",
'Ciliated 2' = "Ciliated 2",
'Pax8+/Prom1+ Cilia-forming' = "Pax8low/Prom1+ Cilia-forming",
'Fibroblast-like' = "Fibroblast-like", #removed
'Slc1a3+/Sox9+ Cilia-forming' = "Slc1a3med/Sox9+ Cilia-forming",
'Secretory 2' = "Selenop+/Gstm2high Secretory")
cluster_table <- table(Labeled_Pseudotime_Lineage@active.ident,
Labeled_Pseudotime_Lineage@meta.data$Bin)
clusters <- data.frame(cluster_table)
clusters <- clusters %>%
group_by(Var2) %>%
mutate(Perc = Freq / sum(Freq))
# Create Pseudotime DF #
pseudotime_table <- table(seq(1, length(bin_list), 1),
unique(Labeled_Pseudotime_Lineage@meta.data$Bin),
seq(1, length(bin_list), 1))
pseudotime_bins <- data.frame(pseudotime_table)
# calculate max and min z-scores
max_z <- max(z_score1$z_score, na.rm = TRUE)
min_z <- min(z_score1$z_score, na.rm = TRUE)
# set color for outliers
outlier_color <- ifelse(z_score1$z_score > max_z | z_score1$z_score < min_z, ifelse(z_score1$z_score > 0, "#AD1F24", "#51A6DC"), "#e2e2e2")
## Plot Gene Expression ##
# Set different na.value options for positive and negative values
na_color_pos <- "#AD1F24" # color for positive NA values
na_color_neg <- "#51A6DC" # color for negative NA values
custom_bin_names <- c(paste0("S", 20:1), paste0("C", 1:20))
figure <- ggplot(z_score1, aes(x, y, fill = z_score)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradientn(colors=c("#1984c5", "#e2e2e2", "#c23728"),
name = "Average Expression \nZ-Score", limits = c(-3,3),
na.value = ifelse(is.na(z_score1) & z_score1 > 0, na_color_pos,
ifelse(is.na(z_score1) & z_score1 < 0, na_color_neg, "grey50")),
oob = scales::squish)+
scale_x_discrete(limits= sort(bin_list) , labels= custom_bin_names)+
scale_y_discrete(limits= rev(features))+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 0, hjust = 0.5, size = 10, face = "bold"), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold.italic"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-0.5,1,1,1), "cm"))
## Plot Cluster Percentage ##
`Spdef+ Secretory` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Spdef+ Secretory")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(1,1,1,1), "cm"))
`Selenop+/Gstm2high Secretory` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Selenop+/Gstm2high Secretory")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Cebpdhigh/Foxj1- Progenitor` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Cebpdhigh/Foxj1- Progenitor")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Slc1a3+ Stem/Progenitor` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Slc1a3+ Stem/Progenitor")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Slc1a3med/Sox9+ Cilia-forming` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Slc1a3med/Sox9+ Cilia-forming")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Pax8low/Prom1+ Cilia-forming` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Pax8low/Prom1+ Cilia-forming")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Ciliated 1` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Ciliated 1")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Ciliated 2` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Ciliated 2")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
## Plot Pseudotime Color ##
list <- 1:40
colors = c(rev(rainbow20),rainbow20)
df <- data.frame(data = list, color = colors)
binning <- ggplot(df, aes(x = 1:40, y = 1, fill = color)) +
geom_bar(stat = "identity",position = "fill", color = "black", size = 1, width = 1) +
scale_fill_identity() +
theme_void()+
theme(axis.line = element_blank(),
axis.ticks = element_blank(),
axis.text = element_blank(),
axis.title = element_blank())+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Pseudotime Bin ")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust =1, vjust = .75, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
### Combine Plots ###
psuedotime_lineage <- ggarrange(`Spdef+ Secretory`,
`Selenop+/Gstm2high Secretory`,
`Cebpdhigh/Foxj1- Progenitor`,
`Slc1a3+ Stem/Progenitor`,
`Slc1a3med/Sox9+ Cilia-forming`,
`Pax8low/Prom1+ Cilia-forming`,
`Ciliated 1`,
`Ciliated 2`,
`binning`,
figure , ncol=1,
heights = c(2, 2, 2, 2, 2, 2, 2, 2, 2, (2*length(features)),
widths = c(3)),
padding = unit(0.01))
ggsave(filename = "FIG6d_psuedotime_lineage.pdf", plot = psuedotime_lineage, width = 18, height = 9, dpi = 600)
#### Figure 6e: Stacked violin plots for cancer-prone cell states ####
### Stacked Violin Plot Function ###
#https://divingintogeneticsandgenomics.rbind.io/post/stacked-violin-plot-for-visualizing-single-cell-data-in-seurat/
## remove the x-axis text and tick
## plot.margin to adjust the white space between each plot.
## ... pass any arguments to VlnPlot in Seurat
modify_vlnplot <- function(obj,
feature,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
p<- VlnPlot(obj, features = feature, pt.size = pt.size, ... ) +
xlab("") + ylab(feature) + ggtitle("") +
theme(legend.position = "none",
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
axis.title.y = element_text(size = rel(1), angle = 0, face = "bold.italic"),
axis.text.y = element_text(size = rel(1)),
plot.margin = plot.margin )
return(p)
}
## extract the max value of the y axis
extract_max<- function(p){
ymax<- max(ggplot_build(p)$layout$panel_scales_y[[1]]$range$range)
return(ceiling(ymax))
}
## main function
StackedVlnPlot<- function(obj, features,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
plot_list<- purrr::map(features, function(x) modify_vlnplot(obj = obj,feature = x, ...))
# Add back x-axis title to bottom plot. patchwork is going to support this?
plot_list[[length(plot_list)]]<- plot_list[[length(plot_list)]] +
theme(axis.text.x=element_text(angle = 60, hjust=1, vjust=0.95), axis.ticks.x = element_line())
# change the y-axis tick to only max value
ymaxs<- purrr::map_dbl(plot_list, extract_max)
plot_list<- purrr::map2(plot_list, ymaxs, function(x,y) x +
scale_y_continuous(breaks = c(y)) +
expand_limits(y = y))
p<- patchwork::wrap_plots(plotlist = plot_list, ncol = 1)
return(p)
}
features<- c("Slc1a3", "Pax8" , "Trp73" , "Prom1" )
features<- c("Pax8", "Ovgp1" , "Lcn2" , "Upk1a" , "Spdef" ,"Thrsp" )
colors <- c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
#"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6")
No_Fibro <- subset(x = Epi_Named, idents =
c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
#"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2"))
stack_vln <- StackedVlnPlot(obj = No_Fibro, features = features, slot = "data",
pt.size = 0,
cols = c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
#"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6"))+ #9
theme(plot.title = element_text(size = 32, face = "bold.italic"))+
scale_x_discrete(limits = c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
#"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2"))+
theme(axis.text.x = element_text(size = 16, angle = 60))+
theme(axis.text.y = element_text(size = 14))+
theme(axis.title.y.left = element_text(size = 16))
ggsave(filename = "FIG6e_stacked_vln_noFibro.pdf", plot = stack_vln, width = 18, height = 12, dpi = 600)
#### Figure 6f: Krt5 expression within epithelial cell states ####
ggsave(filename = "Krt5_dp_others.pdf", plot = Krt5_dp_others, width = 1.89*8, height = 3.06*8, dpi = 600)
Krt5_dp <- DotPlot(object = No_Fibro, # Seurat object
assay = 'RNA', # Name of assay to use. Default is the active assay
features = 'Krt5', # List of features (select one from above or create a new one)
# Colors to be used in the gradient
col.min = 0, # Minimum scaled average expression threshold (everything smaller will be set to this)
col.max = 2.5, # Maximum scaled average expression threshold (everything larger will be set to this)
dot.min = 0, # The fraction of cells at which to draw the smallest dot (default is 0)
dot.scale = 24, # Scale the size of the points
group.by = NULL, # How the cells are going to be grouped
split.by = NULL, # Whether to split the data (if you fo this make sure you have a different color for each variable)
scale = TRUE, # Whether the data is scaled
scale.by = "radius", # Scale the size of the points by 'size' or 'radius'
scale.min = NA, # Set lower limit for scaling
scale.max = NA )+ # Set upper limit for scaling
labs(x = NULL, # x-axis label
y = NULL)+
scale_color_viridis_c(option="F",begin=.4,end=0.9, direction = -1)+
geom_point(aes(size=pct.exp), shape = 21, colour="black", stroke=0.7)+
theme_linedraw(base_line_size = 5)+
guides(x = guide_axis(angle = 90))+
theme(axis.text.x = element_text(size = 32 , face = "italic"))+
theme(axis.text.y = element_text(size = 32))+
theme(legend.title = element_text(size = 12))+
scale_y_discrete(limits = c("Ciliated 2",
"Ciliated 1",
"Selenop+/Gstm2high Secretory",
"Spdef+ Secretory",
#"Fibroblast-like",
"Pax8low/Prom1+ Cilia-forming",
"Slc1a3med/Sox9+ Cilia-forming",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3+ Stem/Progenitor"))
ggsave(filename = "Krt5_dp_noFibro.pdf", plot = Krt5_dp, width = 1.89*8, height = 3.06*8, dpi = 600)
附图2
#### Figure Supp 2: Characterization of distal epithelial cell states ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
#### Unprocessed and Processed Distal Dataset ####
All.merged <- readRDS(file = "../dataset/Unfiltered_Mouse_Distal.rds", refhook = NULL) # Prior to Quality Control
Distal <- readRDS(file = "../dataset/Distal_Filtered_Cells.rds" , refhook = NULL) # After to Quality Control
Distal_Named <- RenameIdents(Distal,
'0' = "Fibroblast 1",
'1' = "Fibroblast 2",
'2' = "Secretory Epithelial",
'3' = "Smooth Muscle",
'4' = "Ciliated Epithelial 1",
'5' = "Fibroblast 3",
'6' = "Stem-like Epithelial 1",
'7' = "Stem-like Epithelial 2",
'8' = "Ciliated Epithelial 2",
'9' = "Blood Endothelial",
'10' = "Pericyte",
'11' = "Intermediate Epithelial",
'12' = "T/NK Cell",
'13' = "Epithelial/Fibroblast",
'14' = "Macrophage",
'15' = "Erythrocyte",
'16' = "Luteal",
'17' = "Mesothelial",
'18' = "Lymph Endothelial/Epithelial") # Remove cluster due few data points and suspected doublet
Distal_Named@active.ident <- factor(x = Distal_Named@active.ident,
levels = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Pericyte',
'Blood Endothelial',
'Lymph Endothelial/Epithelial',
'Epithelial/Fibroblast',
'Stem-like Epithelial 1',
'Stem-like Epithelial 2',
'Intermediate Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial 1',
'Ciliated Epithelial 2',
'T/NK Cell',
'Macrophage',
'Erythrocyte',
'Mesothelial',
'Luteal'))
Distal_Named <- subset(Distal_Named,
idents = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Pericyte',
'Blood Endothelial',
'Epithelial/Fibroblast',
'Stem-like Epithelial 1',
'Stem-like Epithelial 2',
'Intermediate Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial 1',
'Ciliated Epithelial 2',
'T/NK Cell',
'Macrophage',
'Erythrocyte',
'Mesothelial',
'Luteal'))
Distal_Named <- SetIdent(Distal_Named, value = Distal_Named@active.ident)
Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
#### Figure Supp 2a: Unfilitered % MT Genes ####
unfiltered_MT <- VlnPlot(All.merged, features = c("percent.mt"), group.by = 'Sample', pt.size = 0,
cols = natparks.pals(name="Arches2",n=3))+
theme(legend.position = 'none')+
theme(axis.text.x = element_text(size = 16))+ # Change X-Axis Text Size
theme(axis.text.y = element_text(size = 16))+ # Change Y-Axis Text Size
theme(axis.title.y = element_text(size = 18))+ # Change Y-Axis Title Text Size
theme(plot.title = element_text(size = 32,face = "bold.italic"))+
theme(axis.title.x = element_blank())
ggsave(filename = "FIGs2a_unfiltered_MT.pdf", plot = unfiltered_MT, width = 12, height = 9, dpi = 600)
#exprs <- as.data.frame(FetchData(object = All.merged, vars = c('nCount_RNA' , "Sample")))
#df_new <- filter(exprs, Sample == 'mD1')
#mean(df_new$nCount_RNA)
#sd(df_new$nCount_RNA)
# Remove the original 'Value' column if needed
df_new <- df_new %>%
select(-Value)
x <- spread(exprs, Sample, percent.mt )
mean(exprs)
#### Figure Supp 2b: Unfilitered nFeature RNA ####
unfiltered_nFeature <- VlnPlot(All.merged, features = c("nFeature_RNA"), group.by = 'Sample', pt.size = 0,
cols = natparks.pals(name="Arches2",n=3))+
theme(legend.position = 'none')+
theme(axis.text.x = element_text(size = 16))+
theme(axis.text.y = element_text(size = 16))+
theme(axis.title.y = element_text(size = 18))+
theme(plot.title = element_text(size = 32,face = "bold.italic"))+
theme(axis.title.x = element_blank()) # Change object to visualize other samples
ggsave(filename = "FIGs2b_unfiltered_nFeature.pdf", plot = unfiltered_nFeature, width = 12, height = 9, dpi = 600)
#### Figure Supp 2c: Unfilitered nCount RNA ####
unfiltered_nCount <- VlnPlot(All.merged, features = c("nCount_RNA"), group.by = 'Sample', pt.size = 0,
cols = natparks.pals(name="Arches2",n=3))+
theme(legend.position = 'none')+
theme(axis.text.x = element_text(size = 16))+
theme(axis.text.y = element_text(size = 16))+
theme(axis.title.y = element_text(size = 18))+
theme(plot.title = element_text(size = 32,face = "bold.italic"))+
theme(axis.title.x = element_blank()) # Change object to visualize other samples
ggsave(filename = "FIGs2c_unfiltered_nCount.pdf", plot = unfiltered_nCount, width = 12, height = 9, dpi = 600)
#### Figure Supp 2d: Doublets All Cells ####
All_Doublet <- DimPlot(object = Distal,
reduction = 'umap',
group.by = "Doublet",
cols = c( "#ffb6c1", "#380b11"),
repel = TRUE,
label = F,
pt.size = 1.2,
order = c("Doublet","Singlet"),
label.size = 5) +
labs(x="UMAP_1",y="UMAP_2")
ggsave(filename = "FIGs2d1_All_Doublet_umap.pdf", plot = All_Doublet, width = 22, height = 17, dpi = 600)
## Stacked Bar Doublets ##
table <- table(Distal_Named@active.ident ,
Distal_Named@meta.data$Doublet) # Create a table of counts
df <- data.frame(table)
doublet <- ggplot(data = df, # Dataset to use for plot. Needs to be a data.frame
aes(x = Var1, # Variable to plot on the x-axis
y = Freq, # Variable to plot on the y-axis
fill = factor(Var2, # Variable to fill the bars
levels = c("Doublet","Singlet")))) + # Order of the stacked bars
theme_classic() + # ggplot2 theme
# Bar plot
geom_bar(position = 'fill', # Position of bars. Fill means the bars are stacked.
stat = "identity", # Height of bars represent values in the data
size = 1) + # Size of bars
# Color scheme
scale_fill_manual("Doublet", limits = c("Doublet","Singlet"),
values = c('#8B0000','#808080')) +
# Add plot labels
labs(x = NULL, # x-axis label
y = "Fraction of Cells") + # y-axis label
theme(text = element_text(size = 15), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 60, hjust = 1, size = 11), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1))+ # Text color and horizontal adjustment on y-axis
scale_x_discrete(limits = (c('Intermediate Epithelial',
'Epithelial/Fibroblast',
'Stem-like Epithelial 1',
'Ciliated Epithelial 1',
'Erythrocyte',
'Smooth Muscle',
'Stem-like Epithelial 2',
'Mesothelial',
'Blood Endothelial',
'Pericyte',
'Fibroblast 2',
'Secretory Epithelial',
'Fibroblast 1',
'Ciliated Epithelial 2',
'Fibroblast 3',
'T/NK Cell',
'Macrophage',
'Luteal')))+
coord_flip()
ggsave(filename = "FIGs2d2_doublet_quant.pdf", plot = doublet, width = 10, height = 16, dpi = 600)
#### Figure Supp 2e: Sample Distribution for All Cells ####
table <- table(Distal_Named@active.ident ,
Distal_Named@meta.data$Sample) # Create a table of counts
df <- data.frame(table)
table2 <- table(Epi_Named@meta.data$Sample)
all_sample_dist <- ggplot(data = df, # Dataset to use for plot. Needs to be a data.frame
aes(x = Var1, # Variable to plot on the x-axis
y = Freq, # Variable to plot on the y-axis
fill = factor(Var2, # Variable to fill the bars
levels = c("mD1","mD2","mD4")))) + # Order of the stacked bars
theme_classic() + # ggplot2 theme
# Bar plot
geom_bar(position = 'fill', # Position of bars. Fill means the bars are stacked.
stat = "identity", # Height of bars represent values in the data
size = 1) + # Size of bars
# Color scheme
scale_fill_manual("Location", limits = c("mD1","mD2","mD4"),
values = c(natparks.pals(name="Arches2",n=3))) +
# Add plot labels
labs(x = NULL, # x-axis label
y = "Fraction of Cells") + # y-axis label
theme(text = element_text(size = 15), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 60, hjust = 1, size = 11), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1)) # Text color and horizontal adjustment on y-axis
ggsave(filename = "FIGs2f_all_sample_dist.pdf", plot = epi_sample_dist, width = 16, height = 12, dpi = 600)
#### Figure Supp 2f: Doublets Epithelial Cells ####
Epi_Doublet <- DimPlot(object = Epi_Named,
reduction = 'umap',
group.by = "Doublet",
cols = c( "#ffb6c1", "#380b11"),
repel = TRUE,
label = F,
pt.size = 1.2,
order = c("Doublet","Singlet"),
label.size = 5) +
labs(x="UMAP_1",y="UMAP_2")
ggsave(filename = "FIGs2e1_Epi_Doublet_umap.pdf", plot = All_Doublet, width = 22, height = 17, dpi = 600)
## Stacked Bar Doublets ##
table <- table(Epi_Named@active.ident ,
Epi_Named@meta.data$Doublet) # Create a table of counts
df <- data.frame(table)
epi_doublet <- ggplot(data = df, # Dataset to use for plot. Needs to be a data.frame
aes(x = Var1, # Variable to plot on the x-axis
y = Freq, # Variable to plot on the y-axis
fill = factor(Var2, # Variable to fill the bars
levels = c("Doublet","Singlet")))) + # Order of the stacked bars
theme_classic() + # ggplot2 theme
# Bar plot
geom_bar(position = 'fill', # Position of bars. Fill means the bars are stacked.
stat = "identity", # Height of bars represent values in the data
size = 1) + # Size of bars
# Color scheme
scale_fill_manual("Doublet", limits = c("Doublet","Singlet"),
values = c('#8B0000','#808080')) +
# Add plot labels
labs(x = NULL, # x-axis label
y = "Fraction of Cells") + # y-axis label
theme(text = element_text(size = 15), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 60, hjust = 1, size = 11), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1))+ # Text color and horizontal adjustment on y-axis
scale_x_discrete(limits = (c("Slc1a3med/Sox9+ Cilia-forming",
"Fibroblast-like",
"Pax8low/Prom1+ Cilia-forming",
"Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Ciliated 1",
"Ciliated 2",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory")))+
coord_flip()
ggsave(filename = "FIGs2e2_epi_doublet_quant.pdf", plot = epi_doublet, width = 10, height = 16, dpi = 600)
#### Figure Supp 2g: Sample Distribution for Epithelial Cells ####
table <- table(Epi_Named@active.ident ,
Epi_Named@meta.data$Sample) # Create a table of counts
df <- data.frame(table)
table2 <- table(Epi_Named@meta.data$Samlpe)
epi_sample_dist <- ggplot(data = df, # Dataset to use for plot. Needs to be a data.frame
aes(x = Var1, # Variable to plot on the x-axis
y = Freq, # Variable to plot on the y-axis
fill = factor(Var2, # Variable to fill the bars
levels = c("mD1","mD2","mD4")))) + # Order of the stacked bars
theme_classic() + # ggplot2 theme
# Bar plot
geom_bar(position = 'fill', # Position of bars. Fill means the bars are stacked.
stat = "identity", # Height of bars represent values in the data
size = 1) + # Size of bars
# Color scheme
scale_fill_manual("Location", limits = c("mD1","mD2","mD4"),
values = c(natparks.pals(name="Arches2",n=3))) +
# Add plot labels
labs(x = NULL, # x-axis label
y = "Fraction of Cells") + # y-axis label
theme(text = element_text(size = 15), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 60, hjust = 1, size = 11), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1)) # Text color and horizontal adjustment on y-axis
ggsave(filename = "FIGs2g_epi_sample_dist.pdf", plot = epi_sample_dist, width = 16, height = 12, dpi = 600)
#### Figure Supp 2h: Distal Tile Mosaic ####
library(treemap)
dist_cell_types <- table(Idents(Distal_Named), Distal_Named$orig.ident)
dist_cell_type_df <- as.data.frame(dist_cell_types)
## Colors ##
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
FiboEpi <- "#FFE0B3" # Reddish Brown
Muscle <- c('#E55451' , '#FFB7B2') # Reds
Endothelial <- c('#A0E6FF') # Reds
Epi <-c('#6E3E6E','#8A2BE2','#604791','#CCCCFF','#DA70D6','#DF73FF') # Blues/Purples
Immune <- c( '#5A5E6B' , '#B8C2CC' , '#FC86AA') # Yellowish Brown
Meso <- "#9EFFFF" # Pink
Lut <- "#9DCC00" # Green
colors <- c(Fibroblasts, FiboEpi, Muscle, Endothelial, Epi, Immune, Meso, Lut)
## Tile Mosaic ##
distal_treemap <- treemap(dist_cell_type_df, index = 'Var1', vSize= 'Freq', vColor = colors, palette = colors)
ggsave(filename = "20240612_all_distal_tile.pdf", plot = distal_treemap, width = 12, height = 8, dpi = 600)
#### Figure Supp 2i: Epi Markers All Distal Cells ####
### Stacked Violin Plot Function ###
#https://divingintogeneticsandgenomics.rbind.io/post/stacked-violin-plot-for-visualizing-single-cell-data-in-seurat/
## remove the x-axis text and tick
## plot.margin to adjust the white space between each plot.
## ... pass any arguments to VlnPlot in Seurat
modify_vlnplot <- function(obj,
feature,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
p<- VlnPlot(obj, features = feature, pt.size = pt.size, ... ) +
xlab("") + ylab(feature) + ggtitle("") +
theme(legend.position = "none",
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
axis.title.y = element_text(size = rel(1), angle = 0, face = "bold.italic"),
axis.text.y = element_text(size = rel(1)),
plot.margin = plot.margin )
return(p)
}
## extract the max value of the y axis
extract_max<- function(p){
ymax<- max(ggplot_build(p)$layout$panel_scales_y[[1]]$range$range)
return(ceiling(ymax))
}
## main function
StackedVlnPlot<- function(obj, features,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
plot_list<- purrr::map(features, function(x) modify_vlnplot(obj = obj,feature = x, ...))
# Add back x-axis title to bottom plot. patchwork is going to support this?
plot_list[[length(plot_list)]]<- plot_list[[length(plot_list)]] +
theme(axis.text.x=element_text(angle = 60, hjust=1, vjust=0.95), axis.ticks.x = element_line())
# change the y-axis tick to only max value
ymaxs<- purrr::map_dbl(plot_list, extract_max)
# plot_list<- purrr::map2(plot_list, ymaxs, function(x,y) x +
plot_list<- purrr::map2(plot_list, c(5,5,8,5), function(x,y) x +
scale_y_continuous(breaks = c(y)) +
expand_limits(y = y))
p<- patchwork::wrap_plots(plotlist = plot_list, ncol = 1)
return(p)
}
features<- c("Epcam", "Krt8" , "Ovgp1" , "Foxj1" )
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
Muscle <- c('#E55451' , '#FFB7B2') # Reds
Endothelial <- c('#A0E6FF') # Reds
FiboEpi <- "#FFE0B3" # Reddish Brown
Epi <-c('#6E3E6E','#8A2BE2','#604791','#CCCCFF','#DA70D6','#DF73FF') # Blues/Purples
Immune <- c( '#5A5E6B' , '#B8C2CC' , '#FC86AA') # Yellowish Brown
Meso <- "#9EFFFF" # Pink
Lut <- "#9DCC00" # Green
colors <- c(Fibroblasts, FiboEpi, Muscle, Endothelial, Epi, Immune, Meso, Lut)
stack_vln <- StackedVlnPlot(obj = Distal_Named, features = features, slot = "data",
pt.size = 0,
cols = colors)+ #9
theme(plot.title = element_text(size = 32, face = "bold.italic"))+
scale_x_discrete(limits = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Epithelial/Fibroblast',
'Smooth Muscle',
'Pericyte',
'Blood Endothelial',
'Stem-like Epithelial 1',
'Stem-like Epithelial 2',
'Intermediate Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial 1',
'Ciliated Epithelial 2',
'T/NK Cell',
'Macrophage',
'Erythrocyte',
'Mesothelial',
'Luteal'))+
theme(axis.text.x = element_text(size = 16, angle = 60))+
theme(axis.text.y = element_text(size = 14))+
theme(axis.title.y.left = element_text(size = 16))
ggsave(filename = "20240612_All_Distal_stacked_vln.pdf", plot = stack_vln, width = 18, height = 12, dpi = 600)
#### Figure Supp 2j: Epi Markers Distal Epi Cells ####
Epi_Sub <- subset(Epi_Named,
idents = c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2"))
colors <- c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6")
stack_vln <- StackedVlnPlot(obj = Epi_Named, features = features, slot = "data",
pt.size = 0,
cols = c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6"))+ #9
theme(plot.title = element_text(size = 32, face = "bold.italic"))+
scale_x_discrete(limits = c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2"))+
theme(axis.text.x = element_text(size = 16, angle = 60))+
theme(axis.text.y = element_text(size = 14))+
theme(axis.title.y.left = element_text(size = 16))
ggsave(filename = "20240612_Distal_Epi_stacked_vln.pdf", plot = stack_vln, width = 18, height = 12, dpi = 600)
附图3
#### Figure Supp 3: Doublet detection of fibroblast and epithelial markers ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
#### Load Distal Epithelial Dataset ####
Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
table(Epi_Named@active.ident)
#### Plot Compilation ####
feature_scatter <- FeatureScatter( Fibroblast, "Krt8","Col1a1",
cols = c("#35EFEF", #1
"#00A1C6", #2
"#2188F7", #3
"#EA68E1", #4
"#59D1AF", #5
"#B20224", #6
"#F28D86", #7
"#A374B5", #8
"#9000C6"))
x <- DotPlot(Epi_Named , features = c("Krt8" , "Col1a1"))
write.csv(x$data , "doublet_data.csv")
Fibroblast <- subset(Epi_Named,
idents = c("Fibroblast-like"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( Fibroblast, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Fibroblast_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Stem ##
c("Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
Stem <- subset(Epi_Named,
idents = c("Slc1a3+ Stem/Progenitor"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( Stem, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Slc1a3_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Prog ##
c("Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
Prog <- subset(Epi_Named,
idents = c("Cebpdhigh/Foxj1- Progenitor"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( Prog, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Cebpd_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Cilia-forming ##
c("Pax8low/Prom1+ Cilia-forming",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
trans <- subset(Epi_Named,
idents = c("Slc1a3med/Sox9+ Cilia-forming"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( trans, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_SlcCilia_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Pax8 Cilia-forming ##
c("Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
cancer <- subset(Epi_Named,
idents = c("Pax8low/Prom1+ Cilia-forming"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( cancer, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Prom1_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Spdef Secretory ##
c("Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
sec1 <- subset(Epi_Named,
idents = c("Spdef+ Secretory"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( sec1, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Spdef_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Selenop Secretory ##
c("Ciliated 1",
"Ciliated 2")
sec2 <- subset(Epi_Named,
idents = c("Selenop+/Gstm2high Secretory"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( sec2, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Selenop_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Ciliated 1 ##
c("Ciliated 2")
cil1 <- subset(Epi_Named,
idents = c("Ciliated 1"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( cil1, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Ciliated_1_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
## Ciliated 2 ##
cil2 <- subset(Epi_Named,
idents = c("Ciliated 2"))
custom_labels <- function(x) {
ifelse(x %% 1 == 0, as.character(x), "")
}
feature_scatter <- FeatureScatter( cil2, "Krt8","Col1a1",
cols = "black")+ # Scatter plot
NoLegend()+
labs(title = NULL)+
theme(panel.grid.major = element_line(color = "grey", size = 0.5),
panel.grid.minor = element_blank())+
theme(axis.text.x = element_text(color = 'black', size = 12),
axis.title.x = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'black'),
axis.text.y = element_text(color = 'black', size = 12),
axis.title.y = element_text(color = 'black', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'black'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) + # Set breaks every 0.5 units on x-axis
scale_y_continuous(limits = c(0,5) , breaks = seq(0, 10, by = 0.5),
labels = custom_labels) # Set breaks every 0.5 units on y-axis
plot_data <- as.data.frame(feature_scatter$data)
# Create density plot for x-axis
density_x <- ggplot(plot_data, aes(x = Krt8 , fill = 'black')) +
geom_density() +
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
axis.ticks.x = element_blank(),
axis.text.y = element_text(color = 'white', size = 12),
axis.title.y = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.y = element_line(color = 'white'),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
scale_y_continuous(labels = function(y) sprintf("%.0f", y))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,4) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on x-axis
# Create density plot for y-axis
density_y <- ggplot(plot_data, aes(x = Col1a1 , fill = 'black')) +
geom_density() +
coord_flip() + # Flip axes for y-density plot
theme(axis.line = element_line(color='white'),
panel.background = element_blank()) +
theme(axis.text.x = element_text(color = 'white', size = 12),
axis.title.x = element_text(color = 'white', size = 14, face = "bold.italic"),
axis.ticks.x = element_line(color = 'white'),
axis.text.y = element_blank(),
axis.title.y = element_blank(),
axis.ticks.y = element_blank(),
plot.margin = unit(c(0, 0, 0, 0), "mm"))+
NoLegend()+
scale_fill_manual(values = c("grey"))+
scale_x_continuous(limits = c(0,5))+
scale_y_continuous(limits = c(0,1) , breaks = seq(0, 10, by = 0.5)) # Set breaks every 0.5 units on y-axis
# Arrange plots
top_row <- cowplot::plot_grid(
density_x, NULL,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(0.5,0.5))
bottom_row <- cowplot::plot_grid(
feature_scatter,density_y,
nrow = 1, rel_widths = c(3, 1), rel_heights = c(3,3))
combined_plot <- cowplot::plot_grid(
top_row , bottom_row,
nrow = 2 , rel_widths = c(3, 1), rel_heights = c(0.25,3))
ggsave(filename = "20240221_Ciliated_2_Doublet.pdf", plot = combined_plot, width = 12, height = 12, dpi = 600)
附图4
#### Figure Supp 4: Census of cell types of the mouse uterine tube ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
#### Proximal Datasets ####
Proximal <- readRDS( file = "../dataset/Proximal_Filtered_Cells.rds" , refhook = NULL)
Proximal_Named <- RenameIdents(Proximal,
'0' = "Fibroblast 1",
'1' = "Stem-like Epithelial",
'2' = "Fibroblast 2",
'3' = "Fibroblast 3",
'4' = "Immune",
'5' = "Secretory Epithelial",
'6' = "Endothelial",
'7' = "Ciliated Epithelial",
'8' = "Mesothelial",
'9' = "Smooth Muscle")
Proximal_Named@active.ident <- factor(x = Proximal_Named@active.ident,
levels = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Endothelial',
'Stem-like Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial',
'Immune',
'Mesothelial'))
Proximal_Named <- SetIdent(Proximal_Named, value = Proximal_Named@active.ident)
Epi_Filter <- readRDS(file = "../dataset/Proximal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Dbi+/Spdefhigh Secretory",
'1' = "Bmpr1b+ Progenitor",
'2' = "Wfdc2+ Secretory",
'3' = "Ciliated",
'4' = "Sox17high Secretory",
'5' = "Kcne3+ Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c("Ciliated",
"Dbi+/Spdefhigh Secretory",
"Kcne3+ Secretory",
"Sox17high Secretory",
"Wfdc2+ Secretory",
"Bmpr1b+ Progenitor"))
#### Figure Supp 4a: Proximal All Cell Types ####
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
Muscle <- c('#E55451') # Reds
Endothelial <- c('#A0E6FF') # Blues
Epi <-c('#6E3E6E','#CCCCFF','#DF73FF') # Purples
Immune <- c( '#5A5E6B' ) # Grey
Meso <- "#1F51FF" # Neon BLue
colors <- c(Fibroblasts, Muscle, Endothelial, Epi, Immune, Meso)
p1 <- DimPlot(
Proximal_Named,
reduction='umap',
cols=colors,
pt.size = 1.4,
label.size = 4,
label.color = "black",
repel = TRUE,
label=F) +
NoLegend() +
labs(x="UMAP_1",y="UMAP_2")
ggsave(filename = "FIGs3a_all_proximal_umap.pdf", plot = p1, width = 15, height = 12, dpi = 600)
#### Figure Supp 4b: Proximal Tile Mosaic ####
library(treemap)
Proximal_Named <- RenameIdents(Proximal,
'0' = "Fibroblast 1",
'1' = "Stem-like Epithelial",
'2' = "Fibroblast 2",
'3' = "Fibroblast 3",
'4' = "Immune",
'5' = "Secretory Epithelial",
'6' = "Endothelial",
'7' = "Ciliated Epithelial",
'8' = "Mesothelial",
'9' = "Smooth Muscle")
Proximal_Named@active.ident <- factor(x = Proximal_Named@active.ident,
levels = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Endothelial',
'Stem-like Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial',
'Immune',
'Mesothelial'))
Proximal_Named <- SetIdent(Proximal_Named, value = Proximal_Named@active.ident)
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
Muscle <- c('#E55451') # Reds
Endothelial <- c('#A0E6FF') # Blues
Epi <-c('#6E3E6E','#CCCCFF','#DF73FF') # Purples
Immune <- c( '#5A5E6B' ) # Grey
Meso <- "#1F51FF" # Neon BLue
colors <- c(Fibroblasts, Muscle, Endothelial, Epi, Immune, Meso)
prox_cell_types <- table(Idents(Proximal_Named), Proximal_Named$orig.ident)
prox_cell_type_df <- as.data.frame(prox_cell_types)
## Tile Mosaic ##
prox_treemap <- treemap(prox_cell_type_df, index = 'Var1', vSize= 'Freq', vColor = colors, palette = colors)
ggsave(filename = "20240612_all_prox_tile.pdf", plot = prox_cell_type_df, width = 8, height = 12, dpi = 600)
#### Figure Supp 4c: Epi Markers All Distal Cells ####
### Stacked Violin Plot Function ###
#https://divingintogeneticsandgenomics.rbind.io/post/stacked-violin-plot-for-visualizing-single-cell-data-in-seurat/
## remove the x-axis text and tick
## plot.margin to adjust the white space between each plot.
## ... pass any arguments to VlnPlot in Seurat
modify_vlnplot <- function(obj,
feature,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
p<- VlnPlot(obj, features = feature, pt.size = pt.size, ... ) +
xlab("") + ylab(feature) + ggtitle("") +
theme(legend.position = "none",
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
axis.title.y = element_text(size = rel(1), angle = 0, face = "bold.italic"),
axis.text.y = element_text(size = rel(1)),
plot.margin = plot.margin )
return(p)
}
## extract the max value of the y axis
extract_max<- function(p){
ymax<- max(ggplot_build(p)$layout$panel_scales_y[[1]]$range$range)
return(ceiling(ymax))
}
## main function
StackedVlnPlot<- function(obj, features,
pt.size = 0,
plot.margin = unit(c(-0.75, 0, -0.75, 0), "cm"),
...) {
plot_list<- purrr::map(features, function(x) modify_vlnplot(obj = obj,feature = x, ...))
# Add back x-axis title to bottom plot. patchwork is going to support this?
plot_list[[length(plot_list)]]<- plot_list[[length(plot_list)]] +
theme(axis.text.x=element_text(angle = 60, hjust=1, vjust=0.95), axis.ticks.x = element_line())
# change the y-axis tick to only max value
ymaxs<- purrr::map_dbl(plot_list, extract_max)
# plot_list<- purrr::map2(plot_list, ymaxs, function(x,y) x +
plot_list<- purrr::map2(plot_list, c(5,5,8,5), function(x,y) x +
scale_y_continuous(breaks = c(y)) +
expand_limits(y = y))
p<- patchwork::wrap_plots(plotlist = plot_list, ncol = 1)
return(p)
}
features<- c("Epcam", "Krt8" , "Ovgp1" , "Foxj1" )
Fibroblasts <- c('#FF9D00' , '#FFB653' , '#FFCB9A') # Oranges
Muscle <- c('#E55451') # Reds
Endothelial <- c('#A0E6FF') # Blues
Epi <-c('#6E3E6E','#CCCCFF','#DF73FF') # Purples
Immune <- c( '#5A5E6B' ) # Grey
Meso <- "#1F51FF" # Neon BLue
colors <- c(Fibroblasts, Muscle, Endothelial, Epi, Immune, Meso)
stack_vln <- StackedVlnPlot(obj = Proximal_Named, features = features, slot = "data",
pt.size = 0,
cols = colors)+ #9
theme(plot.title = element_text(size = 32, face = "bold.italic"))+
scale_x_discrete(limits = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Endothelial',
'Stem-like Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial',
'Immune',
'Mesothelial'))+
theme(axis.text.x = element_text(size = 16, angle = 60))+
theme(axis.text.y = element_text(size = 14))+
theme(axis.title.y.left = element_text(size = 16))
ggsave(filename = "20240612_All_Prox_stacked_vln.pdf", plot = stack_vln, width = 18, height = 12, dpi = 600)
#### Figure Supp 3d: Proximal Epi Cell Types ####
epi_umap <- DimPlot(object = Epi_Named, # Seurat object
reduction = 'umap', # Axes for the plot (UMAP, PCA, etc.)
#group.by = "Patient", # Labels to color the cells by ("seurat_clusters", "Age", "Time.Point)
repel = TRUE, # Whether to repel the cluster labels
label = FALSE, # Whether to have cluster labels
cols = c( "#35EFEF",
"#E95FE0",
"#B20224",
"#F28D86",
"#FB1111",
"#FEB0DB"),
pt.size = 1.6, # Size of each dot is (0.1 is the smallest)
label.size = 2) + # Font size for labels
# You can add any ggplot2 1customizations here
labs(title = 'Colored by Cluster')+ # Plot title
NoLegend()
ggsave(filename = "FIGs3b_epi_proximal_umap.pdf", plot = epi_umap, width = 15, height = 12, dpi = 600)
#### Figure Supp 4e: Epi Markers All Distal Cells ####
P_Epi_Filter <- readRDS(file = "../Proximal/20220914_Proximal_Epi_Cells.rds" , refhook = NULL)
P_Epi_Named <- RenameIdents(P_Epi_Filter,
'0' = "Dbi+/Spdefhigh Secretory",
'1' = "Bmpr1b+ Progenitor",
'2' = "Wfdc2+ Secretory",
'3' = "Ciliated",
'4' = "Sox17high Secretory",
'5' = "Kcne3+ Secretory")
P_Epi_Named@active.ident <- factor(x = P_Epi_Named@active.ident, levels = c("Bmpr1b+ Progenitor",
"Wfdc2+ Secretory",
"Sox17high Secretory",
"Kcne3+ Secretory",
"Dbi+/Spdefhigh Secretory",
"Ciliated"))
stack_vln <- StackedVlnPlot(obj = P_Epi_Named, features = features, slot = "data",
pt.size = 0,
cols = c( "#35EFEF",
"#F28D86",
"#FB1111",
"#FEB0DB",
"#B20224",
"#E95FE0"
))+
theme(plot.title = element_text(size = 32, face = "bold.italic"))+
scale_x_discrete(limits = c("Bmpr1b+ Progenitor",
"Wfdc2+ Secretory",
"Sox17high Secretory",
"Kcne3+ Secretory",
"Dbi+/Spdefhigh Secretory",
"Ciliated"))+
theme(axis.text.x = element_text(size = 16, angle = 60))+
theme(axis.text.y = element_text(size = 14))+
theme(axis.title.y.left = element_text(size = 16))
ggsave(filename = "20240612_Prox_Epi_stacked_vln.pdf", plot = stack_vln, width = 18, height = 12, dpi = 600)
#### Figure Supp 4f: Proximal Epi Features####
named_features <- c("Krt8","Epcam", "Msln",
"Slc1a3","Sox9","Itga6", "Bmpr1b",
"Ovgp1","Sox17","Pax8", "Egr1",
"Wfdc2","Dbi","Gsto1","Fxyd4","Vim","Kcne3",
"Spdef","Lgals1","Upk1a", "Thrsp",
"Selenop", "Gstm2",
"Anpep", "Klf6",
"Id2",
"Ifit1",
"Prom1", "Ly6a", "Kctd8", "Adam8",
"Foxj1","Fam183b",
"Rgs22","Dnali1", "Mt1" , "Dynlrb2")
prox_dp <- DotPlot(object = Epi_Named, # Seurat object
assay = 'RNA', # Name of assay to use. Default is the active assay
features = named_features, # List of features (select one from above or create a new one)
# Colors to be used in the gradient
col.min = 0, # Minimum scaled average expression threshold (everything smaller will be set to this)
col.max = 2.5, # Maximum scaled average expression threshold (everything larger will be set to this)
dot.min = 0, # The fraction of cells at which to draw the smallest dot (default is 0)
dot.scale = 6, # Scale the size of the points
group.by = NULL, # How the cells are going to be grouped
split.by = NULL, # Whether to split the data (if you fo this make sure you have a different color for each variable)
scale = TRUE, # Whether the data is scaled
scale.by = "radius", # Scale the size of the points by 'size' or 'radius'
scale.min = NA, # Set lower limit for scaling
scale.max = NA # Set upper limit for scaling
)+ labs(x = NULL, # x-axis label
y = NULL)+
scale_color_viridis_c(option="F",begin=.4,end=0.9, direction = -1)+
geom_point(aes(size=pct.exp), shape = 21, colour="black", stroke=0.6)+
theme_linedraw()+
guides(x = guide_axis(angle = 90))+
theme(axis.text.x = element_text(size = 12 , face = "italic"))+
theme(axis.text.y = element_text(size = 12))+
theme(legend.title = element_text(size = 12))+
scale_y_discrete(limits = c("Ciliated",
"Dbi+/Spdefhigh Secretory",
"Kcne3+ Secretory",
"Sox17high Secretory",
"Wfdc2+ Secretory",
"Bmpr1b+ Progenitor"
))
ggsave(filename = "FIGs3c_epi_proximal_dotplot.pdf", plot = prox_dp, width = 18, height = 10, dpi = 600)
附图5
#### Figure Supp 5: Distal and proximal epithelial cell correlation ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
#### Proximal Datasets ####
Proximal <- readRDS( file = "../dataset/Proximal_Filtered_Cells.rds" , refhook = NULL)
Proximal_Named <- RenameIdents(Proximal,
'0' = "Fibroblast 1",
'1' = "Stem-like Epithelial",
'2' = "Fibroblast 2",
'3' = "Fibroblast 3",
'4' = "Immune",
'5' = "Secretory Epithelial",
'6' = "Endothelial",
'7' = "Ciliated Epithelial",
'8' = "Mesothelial",
'9' = "Smooth Muscle")
Proximal_Named@active.ident <- factor(x = Proximal_Named@active.ident,
levels = c('Fibroblast 1',
'Fibroblast 2',
'Fibroblast 3',
'Smooth Muscle',
'Endothelial',
'Stem-like Epithelial',
'Secretory Epithelial',
'Ciliated Epithelial',
'Immune',
'Mesothelial'))
Proximal_Named <- SetIdent(Proximal_Named, value = Proximal_Named@active.ident)
Epi_Filter <- readRDS(file = "../dataset/Proximal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Dbi+/Spdefhigh Secretory",
'1' = "Bmpr1b+ Progenitor",
'2' = "Wfdc2+ Secretory",
'3' = "Ciliated",
'4' = "Sox17high Secretory",
'5' = "Kcne3+ Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c("Ciliated",
"Dbi+/Spdefhigh Secretory",
"Kcne3+ Secretory",
"Sox17high Secretory",
"Wfdc2+ Secretory",
"Bmpr1b+ Progenitor"))
#### Proximal vs Distal Cluster Correlation ####
Distal_Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Distal_Epi_Named <- RenameIdents(Distal_Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Distal_Epi_Named@active.ident <- factor(x = Distal_Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
prox_avg_exp <- AverageExpression(Epi_Named)$RNA
distal_avg_exp <- AverageExpression(Distal_Epi_Named)$RNA
cor.exp <- as.data.frame(cor(x = prox_avg_exp , y = distal_avg_exp))
cor.exp$x <- rownames(cor.exp)
cor.df <- tidyr::gather(data = cor.exp, y, correlation, c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2"))
distal_cells <- c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")
prox_cells <- c("Bmpr1b+ Progenitor",
"Ciliated",
"Dbi+/Spdefhigh Secretory",
"Wfdc2+ Secretory",
"Sox17high Secretory",
"Kcne3+ Secretory")
corr_matrix <- ggplot(cor.df, aes(x, y, fill = correlation)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_viridis_c(values = c(0,1),option="rocket", begin=.4,end=0.99, direction = -1,)+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 60, hjust = 1, size = 12, face = "bold"), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 12, face = "bold.italic"))+
theme(plot.title = element_blank())+
scale_y_discrete(limits = c("Ciliated 2",
"Ciliated 1",
"Selenop+/Gstm2high Secretory",
"Spdef+ Secretory",
"Fibroblast-like",
"Pax8low/Prom1+ Cilia-forming",
"Slc1a3med/Sox9+ Cilia-forming",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3+ Stem/Progenitor"))+
scale_x_discrete(limits = c("Bmpr1b+ Progenitor",
"Wfdc2+ Secretory",
"Sox17high Secretory",
"Kcne3+ Secretory",
"Dbi+/Spdefhigh Secretory",
"Ciliated"))+
geom_text(aes(x, y, label = round(correlation, digits = 2)), color = "black", size = 4)
ggsave(filename = "FIGs3d_epi_cluster_corr.pdf", plot = corr_matrix, width = 18, height = 10, dpi = 600)
附图6
#### Figure Supp 6 and 10: Stem and Cancer Markers ####
#### Packages Load ####
library(dplyr)
library(patchwork)
library(Seurat)
library(harmony)
library(ggplot2)
library(cowplot)
library(SoupX)
library(DoubletFinder)
library(data.table)
library(parallel)
library(tidyverse)
library(SoupX)
library(ggrepel)
library(ggplot2)
library(gplots)
library(RColorBrewer)
library(viridisLite)
library(Polychrome)
library(circlize)
library(NatParksPalettes)
library(monocle3)
library(ComplexHeatmap)
library(ggExtra)
library(gridExtra)
library(egg)
library(scales)
#### Distal Epithelial and Pseudotime Dataset ####
Epi_Filter <- readRDS(file = "../dataset/Distal_Epi_Cells.rds" , refhook = NULL)
Epi_Named <- RenameIdents(Epi_Filter,
'0' = "Spdef+ Secretory",
'1' = "Slc1a3+ Stem/Progenitor",
'2' = "Cebpdhigh/Foxj1- Progenitor",
'3' = "Ciliated 1",
'4' = "Ciliated 2",
'5' = "Pax8low/Prom1+ Cilia-forming",
'6' = "Fibroblast-like",
'7' = "Slc1a3med/Sox9+ Cilia-forming",
'8' = "Selenop+/Gstm2high Secretory")
Epi_Named@active.ident <- factor(x = Epi_Named@active.ident, levels = c( c("Slc1a3+ Stem/Progenitor",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3med/Sox9+ Cilia-forming",
"Pax8low/Prom1+ Cilia-forming",
"Fibroblast-like",
"Spdef+ Secretory",
"Selenop+/Gstm2high Secretory",
"Ciliated 1",
"Ciliated 2")))
cds <- readRDS(file = "../dataset/Distal_Epi_PHATE_Monocle3.rds" , refhook = NULL)
#### Figure Supp 4: Stem Dot Plot ####
stem_features <- c("Krt5","Krt17","Cd44","Prom1","Kit","Aldh1a1","Aldh1a2","Aldh1a3",
"Efnb1","Ephb1","Trp63","Sox2","Sox9","Klf4","Rnf43","Foxm1",
"Pax8","Nanog","Itga6","Psca","Tcf3","Tcf4","Nrp1","Slc1a3","Tnfrsf19",
"Smo","Lrig1","Ezh2","Egr1","Tacstd2","Dusp1","Slc38a2","Malat1",
"Btg2","Cdkn1c","Pdk4","Nedd9","Fos","Jun","Junb","Zfp36",
"Neat1","Gadd45g","Gadd45b")
stem_dp <- DotPlot(object = Epi_Named, # Seurat object
assay = 'RNA', # Name of assay to use. Default is the active assay
features = stem_features, # List of features (select one from above or create a new one)
# Colors to be used in the gradient
col.min = 0, # Minimum scaled average expression threshold (everything smaller will be set to this)
col.max = 2.5, # Maximum scaled average expression threshold (everything larger will be set to this)
dot.min = 0, # The fraction of cells at which to draw the smallest dot (default is 0)
dot.scale = 6, # Scale the size of the points
group.by = NULL, # How the cells are going to be grouped
split.by = NULL, # Whether to split the data (if you fo this make sure you have a different color for each variable)
scale = TRUE, # Whether the data is scaled
scale.by = "radius", # Scale the size of the points by 'size' or 'radius'
scale.min = NA, # Set lower limit for scaling
scale.max = NA )+ # Set upper limit for scaling
labs(x = NULL, # x-axis label
y = NULL)+
scale_color_viridis_c(option="F",begin=.4,end=0.9, direction = -1)+
geom_point(aes(size=pct.exp), shape = 21, colour="black", stroke=0.6)+
#theme_linedraw()+
guides(x = guide_axis(angle = 90))+
theme(axis.text.x = element_text(size = 8 , face = "italic"))+
theme(axis.text.y = element_text(size = 9))+
theme(legend.title = element_text(size = 9))+
theme(legend.text = element_text(size = 8))+
scale_y_discrete(limits = c("Ciliated 2",
"Ciliated 1",
"Selenop+/Gstm2high Secretory",
"Spdef+ Secretory",
"Fibroblast-like",
"Pax8low/Prom1+ Cilia-forming",
"Slc1a3med/Sox9+ Cilia-forming",
"Cebpdhigh/Foxj1- Progenitor",
"Slc1a3+ Stem/Progenitor"))
ggsave(filename = "FIGs4_stem_dp.pdf", plot = stem_dp, width = 12, height = 6, dpi = 600)
x <- stem_dp$data
write.csv( x , 'stem_dp_data.csv')
#### Figure Supp 6: HGSC Driver Gene by Pseudotime ####
## Calculate Pseudotime Values ##
pseudo <- pseudotime(cds)
Distal_PHATE@meta.data$Pseudotime <- pseudo # Add to Seurat Metadata
## Subset Seurat Object ##
color_cells <- DimPlot(Distal_PHATE , reduction = "phate",
cols = c("#B20224", #1
"#35EFEF", #2
"#00A1C6", #3
"#A374B5", #4
"#9000C6", #5
"#EA68E1", #6
"lightgrey", #7
"#2188F7", #8
"#F28D86"),
pt.size = 0.7,
shuffle = TRUE,
seed = 0,
label = FALSE)
## Psuedotime and Lineage Assignment ##
cellID <- rownames(Distal_PHATE@reductions$phate@cell.embeddings)
phate_embeddings <- Distal_PHATE@reductions$phate@cell.embeddings
pseudotime_vals <- Distal_PHATE@meta.data$Pseudotime
combined_data <- data.frame(cellID, phate_embeddings, pseudotime_vals)
# Calculate the Average PHATE_1 Value for Pseudotime Points = 0 #
avg_phate_1 <- mean(phate_embeddings[pseudotime_vals == 0, 1])
# Pseudotime Values lower than avge PHATE_1 Embedding will be Negative to split lineages
combined_data$Split_Pseudo <- ifelse(phate_embeddings[, 1] < avg_phate_1, -pseudotime_vals, pseudotime_vals)
# Define Lineage #
combined_data$lineage <- ifelse(combined_data$PHATE_1 < avg_phate_1, "Secretory",
ifelse(combined_data$PHATE_1 > avg_phate_1, "Ciliogenic", "Progenitor"))
Distal_PHATE$Pseudotime_Adj <- combined_data$Split_Pseudo
Distal_PHATE$Lineage <- combined_data$lineage
# Subset #
Pseudotime_Lineage <- subset(Distal_PHATE,
idents = c("Secretory 1",
"Secretory 2",
"Msln+ Progenitor",
"Slc1a3+/Sox9+ Cilia-forming",
"Pax8+/Prom1+ Cilia-forming",
"Progenitor",
"Ciliated 1",
"Ciliated 2"))
## Set Bins ##
bins <- cut_number(Pseudotime_Lineage@meta.data$Pseudotime_Adj , 40) # Evenly distribute bins
Pseudotime_Lineage@meta.data$Bin <- bins # Metadata for Bins
## Set Idents to PSeudoime Bin ##
time_ident <- SetIdent(Pseudotime_Lineage, value = Pseudotime_Lineage@meta.data$Bin)
av.exp <- AverageExpression(time_ident, return.seurat = T)$RNA # Calculate Avg log normalized expression
# Calculates Average Expression for Each Bin #
# if you set return.seurat=T, NormalizeData is called which by default performs log-normalization #
# Reported as avg log normalized expression #
## Pseudotime Scale Bar ##
list <- 1:40
colors = c(rev(rainbow20),rainbow20)
df <- data.frame(data = list, color = colors)
pseudo_bar <- ggplot(df, aes(x = 1:40, y = 1, fill = color)) +
geom_bar(stat = "identity",position = "fill", color = "black", size = 0, width = 1) +
scale_fill_identity() +
theme_void()+
theme(axis.line = element_blank(),
axis.ticks = element_blank(),
axis.text = element_blank(),
axis.title = element_blank())
ggsave(filename = "pseudo_bar.pdf", plot = pseudo_bar, width = 0.98, height = 0.19, dpi = 600)
## Plot HGSC driver gene list across pseudotime bin ##
features <- c("Trp53", "Brca1", "Brca2", "Csmd3", "Nf1", "Fat3", "Gabra6", "Rb1", "Apc", "Lrp1b",
"Prim2", "Cdkn2a", "Crebbp", "Wwox", "Ankrd11",
"Map2k4", "Fancm", "Fancd2", "Rad51c", "Pten")
# Create Bin List and expression of features #
bin_list <- unique(Pseudotime_Lineage@meta.data$Bin)
plot_info <- as.data.frame(av.exp[features,]) # Call Avg Expression for features
z_score <- transform(plot_info, SD=apply(plot_info,1, mean, na.rm = TRUE))
z_score <- transform(z_score, MEAN=apply(plot_info,1, sd, na.rm = TRUE))
z_score1 <- (plot_info-z_score$MEAN)/z_score$SD
plot_info$y <- rownames(plot_info) # y values as features
z_score1$y <- rownames(plot_info)
plot_info <- gather(data = plot_info, x, expression, bin_list) #set plot
z_score1 <- gather(data = z_score1, x, z_score, bin_list) #set plot
# Create Cell Clusters DF #
Labeled_Pseudotime_Lineage <- RenameIdents(Pseudotime_Lineage,
'Secretory 1' = "Spdef+ Secretory",
'Progenitor' = "Slc1a3+ Stem/Progenitor",
'Msln+ Progenitor' = "Cebpdhigh/Foxj1- Progenitor",
'Ciliated 1' = "Ciliated 1",
'Ciliated 2' = "Ciliated 2",
'Pax8+/Prom1+ Cilia-forming' = "Pax8low/Prom1+ Cilia-forming",
'Fibroblast-like' = "Fibroblast-like", #removed
'Slc1a3+/Sox9+ Cilia-forming' = "Slc1a3med/Sox9+ Cilia-forming",
'Secretory 2' = "Selenop+/Gstm2high Secretory")
cluster_table <- table(Labeled_Pseudotime_Lineage@active.ident,
Labeled_Pseudotime_Lineage@meta.data$Bin)
clusters <- data.frame(cluster_table)
clusters <- clusters %>%
group_by(Var2) %>%
mutate(Perc = Freq / sum(Freq))
# Create Pseudotime DF #
pseudotime_table <- table(seq(1, length(bin_list), 1),
unique(Labeled_Pseudotime_Lineage@meta.data$Bin),
seq(1, length(bin_list), 1))
pseudotime_bins <- data.frame(pseudotime_table)
# calculate max and min z-scores
max_z <- max(z_score1$z_score, na.rm = TRUE)
min_z <- min(z_score1$z_score, na.rm = TRUE)
# set color for outliers
outlier_color <- ifelse(z_score1$z_score > max_z | z_score1$z_score < min_z, ifelse(z_score1$z_score > 0, "#AD1F24", "#51A6DC"), "#e2e2e2")
## Plot Gene Expression ##
# Set different na.value options for positive and negative values
na_color_pos <- "#AD1F24" # color for positive NA values
na_color_neg <- "#51A6DC" # color for negative NA values
custom_bin_names <- c(paste0("S", 20:1), paste0("C", 1:20))
figure <- ggplot(z_score1, aes(x, y, fill = z_score)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradientn(colors=c("#1984c5", "#e2e2e2", "#c23728"),
name = "Average Expression \nZ-Score", limits = c(-3,3),
na.value = ifelse(is.na(z_score1) & z_score1 > 0, na_color_pos,
ifelse(is.na(z_score1) & z_score1 < 0, na_color_neg, "grey50")),
oob = scales::squish)+
scale_x_discrete(limits= sort(bin_list) , labels= custom_bin_names)+
scale_y_discrete(limits= rev(features))+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"), # Text size throughout the plot
axis.text.x = element_text(color = 'black', angle = 0, hjust = 0.5, size = 10, face = "bold"), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold.italic"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-0.5,1,1,1), "cm"))
## Plot Cluster Percentage ##
`Spdef+ Secretory` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Spdef+ Secretory")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(1,1,1,1), "cm"))
`Selenop+/Gstm2high Secretory` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Selenop+/Gstm2high Secretory")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Cebpdhigh/Foxj1- Progenitor` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Cebpdhigh/Foxj1- Progenitor")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Slc1a3+ Stem/Progenitor` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Slc1a3+ Stem/Progenitor")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Slc1a3med/Sox9+ Cilia-forming` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Slc1a3med/Sox9+ Cilia-forming")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Pax8low/Prom1+ Cilia-forming` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Pax8low/Prom1+ Cilia-forming")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Ciliated 1` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Ciliated 1")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
`Ciliated 2` <- ggplot(clusters, aes(Var2, Var1, fill = Perc)) +
geom_tile(color = "black",
lwd = 1,
linetype = 1) +
scale_fill_gradient2(low="white", high="#000000", mid = "white", midpoint = 0, name = "Percentage")+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Ciliated 2")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust = 1, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
## Plot Pseudotime Color ##
list <- 1:40
colors = c(rev(rainbow20),rainbow20)
df <- data.frame(data = list, color = colors)
binning <- ggplot(df, aes(x = 1:40, y = 1, fill = color)) +
geom_bar(stat = "identity",position = "fill", color = "black", size = 1, width = 1) +
scale_fill_identity() +
theme_void()+
theme(axis.line = element_blank(),
axis.ticks = element_blank(),
axis.text = element_blank(),
axis.title = element_blank())+
scale_x_discrete(limits= sort(bin_list) , labels= seq(1, length(bin_list), 1))+
scale_y_discrete(limits= "Pseudotime Bin ")+
theme(panel.background = element_blank())+
labs(title = "Expression of Genes by Pseudotime Bin" ,
x = element_blank(),
y = element_blank())+
theme(text = element_text(size = 12, face = "bold"),# Text size throughout the plot
axis.ticks.x = element_blank(),
axis.ticks.y = element_blank(),
axis.text.x = element_blank(), # Text color, angle, and horizontal adjustment on x-axis
axis.text.y = element_text(color = 'black', hjust =1, vjust = .75, size = 14, face = "bold"))+
theme(plot.title = element_blank(),
plot.margin=unit(c(-1.25,1,1,1), "cm"))
### Combine Plots ###
psuedotime_lineage <- ggarrange(`Spdef+ Secretory`,
`Selenop+/Gstm2high Secretory`,
`Cebpdhigh/Foxj1- Progenitor`,
`Slc1a3+ Stem/Progenitor`,
`Slc1a3med/Sox9+ Cilia-forming`,
`Pax8low/Prom1+ Cilia-forming`,
`Ciliated 1`,
`Ciliated 2`,
`binning`,
figure , ncol=1,
heights = c(2, 2, 2, 2, 2, 2, 2, 2, 2, (2*length(features)),
widths = c(3)),
padding = unit(0.01))
ggsave(filename = "FIGs6_psuedotime_driver_gene.pdf", plot = psuedotime_lineage, width = 18, height = 9, dpi = 600)
write.csv(z_score1 , 'cancer_pseudotime.csv')
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