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---
title: "R Notebook"
output: html_notebook
---
```{r}
library(ggplot2)
library(pheatmap)
library(RColorBrewer)
#display.brewer.all()
library(tidyverse)
library(ggpubr)
library(ggsignif)
library(openxlsx)
library(viridis)
library(data.table)
library(readxl)
library(janitor)
library(dplyr)
library(jcolors)
library(stringr)
```
```{r}
setwd("//atlas.uni.lux/users/isabel.rosety/GBA/Stainings/Plots/")
data1<-read.csv("//atlas.uni.lux/users/isabel.rosety/GBA/Stainings/Plots/488TUJ1_568LMNB1_Sox2647/IR_20211208_488TUJ1_568LMNB1_647Sox2_RS_e41e42e45d30_20211216_094400.csv",sep = ",")
data2<-read.csv("//atlas.uni.lux/users/isabel.rosety/GBA/Stainings/Plots/488TUJ1_568LMNB1_Sox2647/IR_20211208_488TUJ1_568LMNB1_647Sox2_RS_e43e35to36d30_20211208_110531.csv",sep = ";")
data3<-read.csv("//atlas.uni.lux/users/isabel.rosety/GBA/Stainings/Plots/488TUJ1_568LMNB1_Sox2647/IR_20211208_488TUJ1_568LMNB1_647Sox2_RS_e46e48d30_20220730_160215.csv",sep = ";")
data = bind_rows(data1,data2,data3)
tmp=do.call(rbind,strsplit(data$AreaName,"_")) #stringsplit will take the column Areaname and will split this depending on the underscore, then we put them otgeter one ofter the other
data$Condition = tmp[,1]
data %>%
mutate(Condition = str_replace_all(Condition,
pattern = "MUT", replacement = "GBA-PD")) %>%
mutate(Condition = str_replace_all(Condition,
pattern = "WT", replacement = "CTRL")) ->data
data %>%
mutate(CellLine = AreaName) ->data
data %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "WT_56", replacement = "CTRL1")) %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "WT_39", replacement = "CTRL2")) %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "WT_68", replacement = "CTRL3")) %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "MUT_309", replacement = "PD1")) %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "MUT_KTI6", replacement = "PD2")) %>%
mutate(CellLine = str_replace_all(CellLine,
pattern = "MUT_SGO1", replacement = "PD3")) ->data
toselect = c("Barcode","AreaName","Condition","Batch","OrganoidIdx","Day","Batch", "LMNB1hMFIinSox2")
data %>% #same
dplyr::select(toselect)->data
#feature_names<-colnames(data[,7:ncol(data)])
feature_names<-"LMNB1hMFIinSox2"
#Mean of replicates
data%>%
pivot_longer(feature_names,names_to = "Features", values_to = "Measure") -> data_all_long
data2 = drop_na(data_all_long)
data2 %>%
group_by(Condition,AreaName,Batch,Features, Barcode,Day) %>%
summarize(MeanFeatures = mean(Measure))-> data_Mean
#Normalizing to mean of controls
data_Mean %>%
dplyr::filter(Condition=="CTRL") %>%
#group_by(Condition, Day, Features,Batch) %>%
group_by(Condition, Features,Batch,Day) %>%
summarise(baseline = mean(MeanFeatures)) %>%
ungroup() %>%
dplyr::select(-Condition) %>%
#full_join(data_Mean, by=c("Day","Features","Batch")) %>%
full_join(data_Mean, by=c("Features","Batch","Day")) %>%
mutate(Foldchange = MeanFeatures /baseline) ->data_all_based
included_vars =c("AreaName", "Condition","Batch","Barcode","Day") # here we already have the mean of each section
data_all_based %>%
pivot_wider(all_of(included_vars),names_from = Features,values_from = Foldchange)->data_all_based_wide2
#write.csv(data_all_based_wide2, file = 'All_data_normalized.csv')
#If I want to get the wide table without normalizing to mean of controls:
data_Mean %>%
full_join(data_Mean, by=c("Features","MeanFeatures","Batch","AreaName", "Barcode", "Condition","Day")) ->data_Mean
included_vars =c("AreaName", "Condition","Batch","Barcode","Day")
data_Mean %>%
pivot_wider(all_of(included_vars),names_from = Features,values_from = MeanFeatures)->data_Mean_wide
#write.csv(data_Mean_wide, file = 'All_data_not_normalized.csv')
for (i in 1:length(feature_names)) {
data_all_based_wide2 %>%
pivot_longer(cols=feature_names, names_to = "feature", values_to = "value") %>%
filter(feature %in% feature_names[i]) %>%
ggplot( aes(x=factor(Condition, level = c("CTRL", "GBA-PD")), y=value)) +
#geom_violin( aes(fill=Condition),show.legend = T, trim=T),
geom_violin( aes(fill=Condition),show.legend = T,scale = "width", trim=F)+
geom_dotplot(binaxis = "y",stackdir = "center",dotsize=0.8)+
#scale_fill_manual(values= c("#bdd7e7","#2171b5"),name = "Condition", guide = FALSE)+
scale_fill_manual(values= alpha(c("#1565C0","#CC0000"),0.75),name = "Condition",guide = "none")+
#geom_point(aes(color=Batch),shape=18, size=3,show.legend = T)+
#scale_color_manual(values = rev(brewer.pal(n=6, name="OrRd")))+
scale_color_jcolors("pal7")+
#scale_color_viridis(option = "D", discrete=TRUE)+
#geom_point(shape = 1,size = 3,colour = "black")+
theme(legend.key=element_blank()) +
geom_signif(comparisons = list(c("CTRL", "GBA-PD")), test='wilcox.test',
vjust=0.6, size=0.5, margin_top=0.5, textsize=9, map_signif_level=c("***"=0.001, "**"=0.01, "*"=0.05, " "=2) ) +
#facet_grid(~fct_relevel(Day, "d30","d60"), scales="free") +
labs(x = "",
y = paste(feature_names[i]),
#y = paste(names[i]),
fill = "Condition",
#title = paste(feature_names[i])) +
title = "" )+
theme_bw() +
theme(
axis.line = element_line(colour = 'black', size = 0.5) ,
axis.title.x = element_blank(),
axis.text.x = element_text(size=12, color="black"),
axis.title.y = element_text(size = 12),
axis.text.y = element_text(size=10, color="black"),
axis.ticks.y = element_line(),
axis.ticks.length=unit(.25, "cm"),
#change legend text font size)
#legend.key.size = unit(0.7, "cm"),
#legend.key.width = unit(0.6,"cm"),
legend.key=element_blank(),
panel.grid.major = element_blank(),
panel.grid.minor = element_blank(),
panel.border = element_blank(),
plot.title = element_text(size = 20, hjust=0.5, vjust= 1, face = "bold"),
plot.subtitle = element_blank(),#element_text(size = 2, hjust=0.5)
strip.text = element_text(size=12, vjust=0.5),
strip.background = element_rect(fill="lightgray"),
# panel.border = element_rect(fill = NA, color = "black"),
panel.spacing.y = unit(0.8, "lines"),
strip.switch.pad.wrap=unit(20, "lines"),
legend.position="right",
legend.text = element_text(size=17),
legend.title = element_text(size=19)
) -> p
#t<- cowplot::ggdraw(cowplot::add_sub(p, "Wilcox-test, ***p=0.001, **p=0.01, *p=0.05",hjust=-0.2, size=13))
print(p)
##ggsave(paste0(Sys.Date(),"_", names[i], ".pdf"), plot=t)
#ggsave(paste0(Sys.Date(),(sprintf("Plot_%s.pdf",feature_names[i]))),height=3.5,width=3)
}
```
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