Working_directory ="T:\\TimelapseData\\201029GA14"
setwd(Working_directory)

data = read.table("allquant.csv",header=TRUE, row.names=NULL, sep=";")


data$SumCh02 = ifelse(data$SumCh02 <=0, 0.00000001, data$SumCh02)


data$MeanCh02 = data$SumCh02/data$Area
data$MeanCh03 = data$SumCh03/data$Area


data$SumCh02_log = log10(data$SumCh02)
data$SumCh03_log = log10(data$SumCh03)

data$MeanCh02_log = log10(data$MeanCh02)
data$MeanCh03_log = log10(data$MeanCh03)


data$Mash1_log = log10(data$SumCh02)
data$mCherry_log = log10(data$SumCh03)

data$Mash1 = data$SumCh02
data$mCherry = data$SumCh03


data$PositionComment = as.character(data$PositionComment)
temp = strsplit(data$PositionComment,split = " ")
data$Comment = sapply(temp,"[[",1)

Mash1IRESDsRed_pos = c("B2","B4","B6",
                       "B10","F2","F6","F10")
pMash1IRESDsRed_pos = c("B3","B5","B7",
                       "B11","F3","F7","F11")


NoVirus_pos = c("C4")

data$Virus = NA

data[data$Comment %in% Mash1IRESDsRed_pos,]$Virus = "Mash1IRESDsRed"
data[data$Comment %in% pMash1IRESDsRed_pos,]$Virus = "pMash1IRESDsRed"
data[data$Comment %in% Novirus_pos,]$Virus ="No Virus"

data$StimCond = NA

Teststim_1 = c("B2","B3","C2","C3")
Teststim_2 = c("B6","B7","C6","C7")
No_light = c("B4","B5","C4","C5","F4","F5","G4","G5")
Teststim_3 = c("B10","B11","C10","C11")
Teststim_4 = c("F2","F3","G2","G3")
Teststim_5 = c("F6","G6","F7","G7")
Teststim_6 = c("F10","F11","G10","G11")

##### 6 hr on means "1 min on 15 min off" within that 6 hr duration

data[data$Comment %in% Teststim_1,]$StimCond = "3d on"
data[data$Comment %in% Teststim_2,]$StimCond = "6 hr on 12 hr off"
data[data$Comment %in% No_light,]$StimCond = "No light"

data[data$Comment %in% Teststim_3,]$StimCond = "12 hr on 12 hr off"
data[data$Comment %in% Teststim_4,]$StimCond = "24hr on"
data[data$Comment %in% Teststim_5,]$StimCond = "12 hr on"
data[data$Comment %in% Teststim_6,]$StimCond = "6hr on"




data$StimCond = factor(data$StimCond)


temp_Mash1_TP1 = median(data[data$Comment %in% No_light & data$Comment %in% Mash1IRESDsRed_pos 
     & data$TimePoint %in% c("1"),]$Mash1)

temp_pMash1_TP1 = median(data[data$Comment %in% No_light & data$Comment %in% pMash1IRESDsRed_pos 
                          & data$TimePoint %in% c("1"),]$Mash1)

temp_Mash1_TP2 = median(data[data$Comment %in% No_light & data$Comment %in% Mash1IRESDsRed_pos 
                          & data$TimePoint %in% c("2"),]$Mash1)

temp_pMash1_TP2 = median(data[data$Comment %in% No_light & data$Comment %in% pMash1IRESDsRed_pos 
                          & data$TimePoint %in% c("2"),]$Mash1)

data$Mash1_norm = NA

data[data$TimePoint %in% c("1") & data$Comment %in% Mash1IRESDsRed_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% Mash1IRESDsRed_pos,]$Mash1/temp_Mash1_TP1

data[data$TimePoint %in% c("1") & data$Comment %in% pMash1IRESDsRed_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% pMash1IRESDsRed_pos,]$Mash1/temp_pMash1_TP1

data[data$TimePoint %in% c("2") & data$Comment %in% Mash1IRESDsRed_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% Mash1IRESDsRed_pos,]$Mash1/temp_Mash1_TP2

data[data$TimePoint %in% c("2") & data$Comment %in% pMash1IRESDsRed_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% pMash1IRESDsRed_pos,]$Mash1/temp_pMash1_TP2

data[data$TimePoint %in% c("1") & data$Comment %in% NoVirus_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% NoVirus_pos,]$Mash1/temp_Mash1_TP1

data[data$TimePoint %in% c("2") & data$Comment %in% NoVirus_pos,]$Mash1_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% NoVirus_pos,]$Mash1/temp_Mash1_TP1


data$Mash1_norm_log = log10(data$Mash1_norm)





temp_DsRed_TP1 = median(data[data$Comment %in% No_light & data$Comment %in% Mash1IRESDsRed_pos 
                             & data$TimePoint %in% c("1"),]$mCherry)

temp_pDsRed_TP1 = median(data[data$Comment %in% No_light & data$Comment %in% pMash1IRESDsRed_pos 
                              & data$TimePoint %in% c("1"),]$mCherry)

temp_DsRed_TP2 = median(data[data$Comment %in% No_light & data$Comment %in% Mash1IRESDsRed_pos 
                             & data$TimePoint %in% c("2"),]$mCherry)

temp_pDsRed_TP2 = median(data[data$Comment %in% No_light & data$Comment %in% pMash1IRESDsRed_pos 
                              & data$TimePoint %in% c("2"),]$mCherry)

data$mCherry_norm = NA

data[data$TimePoint %in% c("1") & data$Comment %in% Mash1IRESDsRed_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% Mash1IRESDsRed_pos,]$mCherry/temp_DsRed_TP1

data[data$TimePoint %in% c("1") & data$Comment %in% pMash1IRESDsRed_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% pMash1IRESDsRed_pos,]$mCherry/temp_pDsRed_TP1

data[data$TimePoint %in% c("2") & data$Comment %in% Mash1IRESDsRed_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% Mash1IRESDsRed_pos,]$mCherry/temp_DsRed_TP2

data[data$TimePoint %in% c("2") & data$Comment %in% pMash1IRESDsRed_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% pMash1IRESDsRed_pos,]$mCherry/temp_pDsRed_TP2

data[data$TimePoint %in% c("1") & data$Comment %in% NoVirus_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("1") & data$Comment %in% NoVirus_pos,]$mCherry/temp_DsRed_TP1

data[data$TimePoint %in% c("2") & data$Comment %in% NoVirus_pos,]$mCherry_norm = 
  data[data$TimePoint %in% c("2") & data$Comment %in% NoVirus_pos,]$mCherry/temp_DsRed_TP1


data$mCherry_norm_log = log10(data$mCherry_norm)

data = data[!is.na(data$StimCond),]

# data$M1 = NA
# data[which(data$SumCh02_log<=1.25),]$M1 = 0
# data[which(data$SumCh02_log>1.25),]$M1 = 1
# 
# data$mCh = NA
# data[which(data$SumCh03_log<=1.19),]$mCh = 0
# data[which(data$SumCh03_log>1.19),]$mCh = 1
# 
# data$B3 = NA
# data[which(data$SumCh04_log<=1.63),]$B3 = 0
# data[which(data$SumCh04_log>1.63),]$B3 = 1



library("ggplot2")

Mash1_negthreshold_TP1_Rep1 = data.frame(matrix(ncol=2,nrow=1, dimnames=list(NULL, c("Pos", "Threshold_Norm"))))
Postobeanalyzed = NoVirus_pos

for (i in 1:length(Postobeanalyzed)){
  
  temp = quantile(data[data$Comment %in% Postobeanalyzed[i] & data$TimePoint %in% c("1"),]$Mash1_norm_log,0.99)
  Mash1_negthreshold_TP1_Rep1 = rbind(Mash1_negthreshold_TP1_Rep1, c(Postobeanalyzed[i],temp))
}

mCherry_negthreshold_TP1_Rep1 = data.frame(matrix(ncol=2,nrow=1, dimnames=list(NULL, c("Pos", "Threshold_Norm"))))
Postobeanalyzed = NoVirus_pos

for (i in 1:length(Postobeanalyzed)){
  
  temp = quantile(data[data$Comment %in% Postobeanalyzed[i] & data$TimePoint %in% c("2"),]$mCherry_norm_log,0.99)
  mCherry_negthreshold_TP1_Rep1 = rbind(mCherry_negthreshold_TP1_Rep1, c(Postobeanalyzed[i],temp))
}


cbbPalette<- c("#000000", "#0072B2", "#CC79A7","#F0E442","#009E73", "#E69F00", "#56B4E9", "#D55E00")

cbbPalette_mod <- c("#000000", "#E69F00", "#56B4E9", "#D55E00","#F0E442","#009E73", "#0072B2", "#CC79A7")
#Pos_tobeplotted = Novirus_pos
#Pos_tobeplotted = c("B2","B6","B10","B4","F2","F6")



Pos_tobeplotted = Mash1IRESDsRed_pos
TimePoint_tobeplotted = c(1)
Idx = data$Comment %in% Pos_tobeplotted & data$TimePoint %in% TimePoint_tobeplotted
#data$temp = log(data$SumBaSiCWithRatioflatCh04)

ggplot(data[Idx,], aes(x = Mash1_norm_log)) +
  geom_density(size=1) +
  #scale_x_log10()+
  facet_wrap(~ StimCond+Virus+Comment, scales = "fixed") +
  theme_bw() +
  coord_cartesian(xlim=c(-1,2))+
  geom_vline(xintercept = 0.4, color="#CC79A7",size=1)+
  theme(axis.text.x = element_text(angle = 90, hjust = 1),
        strip.text = element_text(size = 10, face ="bold"), axis.text = element_text(size = 20, face = "bold"),
        axis.title=element_text(size=14,face="bold")) +
  labs(x = "Mash1 (log transformed)")+
  scale_color_manual(values = cbbPalette_mod)


Pos_tobeplotted = Mash1IRESDsRed_pos
TimePoint_tobeplotted = c(2)
Idx = data$Comment %in% Pos_tobeplotted & data$TimePoint %in% TimePoint_tobeplotted
#data$temp = log(data$SumBaSiCWithRatioflatCh04)

ggplot(data[Idx,], aes(x = mCherry_norm_log)) +
  geom_density(size=1) +
  #scale_x_log10()+
  facet_wrap(~ StimCond+Virus+Comment, scales = "fixed") +
  theme_bw() +
  coord_cartesian(xlim=c(-0.5,0.75))+
  geom_vline(xintercept = 0.14, color="#CC79A7",size=1)+
  theme(axis.text.x = element_text(angle = 90, hjust = 1),
        strip.text = element_text(size = 10, face ="bold"), axis.text = element_text(size = 20, face = "bold"),
        axis.title=element_text(size=14,face="bold")) +
  labs(x = "Dsred (log transformed)")+
  scale_color_manual(values = cbbPalette_mod)



Pos_tobeplotted = Mash1IRESDsRed_pos
TimePoint_tobeplotted = c(2)
Idx = data$Comment %in% Pos_tobeplotted & data$TimePoint %in% TimePoint_tobeplotted
#data$temp = log(data$SumBaSiCWithRatioflatCh04)

ggplot(data[Idx,], aes(x = mCherry_norm_log, y= Mash1_norm_log)) +
  geom_point() +
  #scale_x_log10()+
  facet_wrap(~ StimCond+Virus+Comment, scales = "fixed") +
  theme_bw() +
  #coord_cartesian(xlim=c(-0.5,0.75))+
  geom_vline(xintercept = 0.14, color="#CC79A7",size=1)+
  geom_hline(yintercept = 0.4, color="#CC79A7",size=1)+
  theme(axis.text.x = element_text(angle = 90, hjust = 1),
        strip.text = element_text(size = 10, face ="bold"), axis.text = element_text(size = 20, face = "bold"),
        axis.title=element_text(size=14,face="bold")) +
  labs(x = "Dsred (log transformed)",y="Mash1(log transformed)")+
  scale_color_manual(values = cbbPalette_mod)


data$M1 = NA
data[which(data$TimePoint==1 & data$Comment %in% Mash1IRESDsRed_pos & data$Mash1_norm_log<=0.43),]$M1 = 0
data[which(data$TimePoint==1 & data$Comment %in% Mash1IRESDsRed_pos & data$Mash1_norm_log>0.43),]$M1 = 1


data[which(data$TimePoint==2 & data$Comment %in% Mash1IRESDsRed_pos & data$Mash1_norm_log<=0.4),]$M1 = 0
data[which(data$TimePoint==2 & data$Comment %in% Mash1IRESDsRed_pos & data$Mash1_norm_log>0.4),]$M1 = 1


data$DR = NA
data[which(data$TimePoint==1 & data$Comment %in% Mash1IRESDsRed_pos & data$mCherry_norm_log<=0.225),]$DR = 0
data[which(data$TimePoint==1 & data$Comment %in% Mash1IRESDsRed_pos & data$mCherry_norm_log>0.225),]$DR = 1


data[which(data$TimePoint==2 & data$Comment %in% Mash1IRESDsRed_pos & data$mCherry_norm_log<=0.14),]$DR = 0
data[which(data$TimePoint==2 & data$Comment %in% Mash1IRESDsRed_pos & data$mCherry_norm_log>0.14),]$DR = 1

write.csv(data,"allquant_withthresholds.csv", row.names=FALSE)


library(dplyr)
count_data = data %>%
  group_by(TimePoint,Virus,StimCond,Comment,M1,DR) %>%
  summarise (n = n()) %>%
  mutate(freq = n / sum(n))





write.csv(count_data,"count_data_Mash1posorneg.csv", row.names=FALSE)


data_TP2$M1 = NA
data_TP2[which(data_TP2$SumCh02_log<=1.15),]$M1 = 0
data_TP2[which(data_TP2$SumCh02_log>1.15),]$M1 = 1
library(dplyr)
count_data_TP2 = data_TP2 %>%
  group_by(Virus,StimCond,M1) %>%
  summarise (n = n()) %>%
  mutate(freq = n / sum(n))

write.csv(count_data_TP2,"count_data_TP2_Mash1posorneg.csv", row.names=FALSE)