# script for figures 2C,D,E,F

## loading libraries
library(Seurat)
library(ggplot2)
library(ggplotify)
library(ggraph)
library(ggrepel)
library(ggpubr)
library(ComplexHeatmap)
library(gridExtra)
library(RColorBrewer)
library(dittoSeq)
library(openxlsx)
library(reshape2)
library(ggalluvial)
library(dplyr)

# load seurat object

obj <- readRDS("Fig2C_Dataset.rds")

# setting color palette:

colpalette <- c("#A6CEE3","#B2DF8A","#FDBF6F","#E78AC3","#FFFF99","#E31A1C","darkgrey")

## Figure 2C

pdf(file = "Fig2C.pdf", width = 4, height = 4)
dittoSeq::dittoDimPlot(object = obj, reduction.use = "umap.harmony.orig.ident",
                       var = "Population", opacity = 0.6, do.label = T,labels.size = 5,
                       labels.highlight = F, labels.repel = T, size = 0.6) + 
  scale_color_manual(values = colpalette) + 
  theme(legend.position = "none", plot.title = element_blank(),
        axis.text = element_text(size = 16, color = "black"))
dev.off()

## Figure 2D

pdf(file = "Fig2D.pdf", 
    width = 4, height = 4)
ggplot(data = g, aes(x = Sample, y = Frequency, alluvium = Population)) +
  geom_alluvium(aes(fill = Population),
                alpha = .5, decreasing = FALSE, width = 1/4) +
  geom_stratum(aes(stratum = Population, fill = Population), alpha = .50, decreasing = FALSE, width = 1/4) + 
  scale_fill_manual(values = alpha(colour = colpalette, alpha = 0.50)) + 
  theme_void() + 
  theme(legend.position = "none",
        plot.title = element_blank(),
        axis.title = element_blank(),
        panel.background = element_rect(fill = "white"),
        axis.text = element_text(size = 16, color = "black"))
dev.off()

## Figure 2E

obj_sorted <- subset(obj, cells = WhichCells(object = obj, expression = orig.ident %in% c("S10","S11","S12")))

obj_sorted$Sorting <- obj_sorted$orig.ident
obj_sorted$Sorting <- gsub(x = obj_sorted$Sorting, pattern = "S10", replacement = "CD38-")
obj_sorted$Sorting <- gsub(x = obj_sorted$Sorting, pattern = "S11", replacement = "GMP")
obj_sorted$Sorting <- gsub(x = obj_sorted$Sorting, pattern = "S12", replacement = "MEP")
obj_sorted$Sorting <- factor(obj_sorted$Sorting, levels = c("CD38-","GMP","MEP"))

cols_sorted <- list("CD38-" = "#008744",
                    "GMP" = "#0057e7",
                    "MEP" = "#d62d20"
)

obj_sorted <- SetIdent(obj_sorted, value = "Sorting")
obj_sorted$sortcol <- cols_sorted$`CD38-` 
obj_sorted$sortcol[which(obj_sorted$Sorting == "GMP")] <- cols_sorted$GMP
obj_sorted$sortcol[which(obj_sorted$Sorting == "MEP")] <- cols_sorted$MEP

p <- dittoDimPlot(object = obj_sorted, var = "Sorting",
                  color.panel = cols_sorted,
                  show.others = T,split.nrow = 1,
                  split.by = "Sorting",
                  labels.highlight = F,labels.repel = T,
                  contour.color = "darkgrey",
                  labels.size = 3.5,do.contour = T,contour.linetype = 2,
                  reduction.use = "umap.harmony.orig.ident", size = 0.5,
                  do.label = F) +
  theme(legend.position = "none",
        legend.key.size = unit(x = 0.1, units = "in"),
        strip.background.x = element_rect(fill = "white"),
        strip.text.x.bottom = element_text(size = 12),
        plot.title = element_blank(),
        axis.title = element_blank(),
        axis.text = element_text(size = 12),
        panel.border = element_rect())

pdf("Fig2E.pdf", width = 6, height = 2.5)
p
dev.off()

## Figure 2F

pdf(file = "Fig2F.pdf", width = 4, height = 4)
ggplot(data = g, aes(x = Sample, y = Frequency, fill = Population)) +
  geom_bar(stat = "identity", width = 0.5, color = "black") + 
  scale_fill_manual(values = alpha(colour = colpalette, alpha = 0.50)) + 
  theme_void() + 
  theme(legend.position = "none",
        plot.title = element_blank(),
        axis.title = element_blank(),
        panel.background = element_rect(fill = "white"),
        axis.text = element_text(size = 16, color = "black"))
dev.off()