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Adverse effects in zebrafish by chronic exposure in relation to epigenetic processes_Final.R
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Adverse effects in zebrafish by chronic exposure in relation to epigenetic processes_Final.R
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##########Script for "Environmentally relevant exposure to PFAS during zebrafish development impacts behavior, fitness, and the transcriptome" ##########
####Call the librairies####
library(tibble)
library(DESeq2)
library("writexl")
library(ggplot2)
library(openxlsx)
library(forcats)
library(datasets)
library(RColorBrewer)
library(FSA)
library(dplyr)
library(ggpubr)
library(nortest)
library(lme4)
library(arm)
library(stringr)
library("survival")
library("survminer")
library(factoextra)
library("multcompView")
library(ggsignif)
library(tidyverse)
library(plyr)
library(reshape2)
library(grid)
library(gridExtra)
library(ape)
library(scales)
library(data.table) # different syntax to merge data
install.packages("readxl")
library("readxl")
library(car)
library(pheatmap)
library(dendextend)
library(VennDiagram)
#-------------------------
####Set working directory####
setwd("~/Papers/EpiTrans F0/R analysis")
#-------------------------
#####Sup.Fig.3-FET####
#Import data
surv<-read.xlsx("FET kaplan.xlsx")
#A. Survival curve
survival <- survfit(Surv(Timepoint, Statut) ~ Condition, data = surv, type ="kaplan-meier")
ggsurvplot(survival, conf.int = TRUE, risk.table = FALSE, ylim=c(0.45,1), pval = TRUE,pval.coord = c(20,0.7), data = surv,xlab = "Hours post fertilization",ylab = "Good health probability",legend=c(0.2, 0.2),palette = c("cornflowerblue","darkgreen","green","#FFCC33","#FFFF00"))
#B.Forest plot
CoxMod<-coxph(Surv(Timepoint,Statut)~Condition,data=surv)
ggforest(CoxMod)
#-------------------------
####Sup.Fig.4.A Spawning success#####
#Import data
spawn=read.xlsx("Spawning success_29072022.xlsx")
spawn$Condition <- fct_relevel(spawn$Condition, c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC", "PFBS_HC"))
Control<-subset(spawn,Condition=="Control")
PFOS_LC<-subset(spawn,Condition=="PFOS_LC")
PFOS_HC<-subset(spawn,Condition=="PFOS_HC")
PFBS_LC<-subset(spawn,Condition=="PFBS_LC")
PFBS_HC<-subset(spawn,Condition=="PFBS_HC")
shapiro.test(Control$Percentage_good_repro)
shapiro.test(PFOS_LC$Percentage_good_repro)
shapiro.test(PFOS_HC$Percentage_good_repro)
shapiro.test(PFBS_LC$Percentage_good_repro)
shapiro.test(PFBS_HC$Percentage_good_repro)
t.test(Control$Percentage_good_repro, PFOS_LC$Percentage_good_repro)
t.test(Control$Percentage_good_repro, PFOS_HC$Percentage_good_repro)
t.test(Control$Percentage_good_repro, PFBS_LC$Percentage_good_repro)
t.test(Control$Percentage_good_repro, PFBS_HC$Percentage_good_repro)
my_comparisons <- list( c("Control", "PFOS_LC"), c("Control", "PFOS_HC"), c("Control", "PFBS_LC"), c("Control", "PFBS_HC"))
Sup_Fig_4_A <- ggplot(spawn, aes(x = Condition, y = Percentage_good_repro, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("Spawning sucess (%)") + # stat_compare_means(comparisons = my_comparisons, method = "t.test") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Sup_Fig_4_A
#-------------------------
####Fig. 1.A Eggs production####
#Import data
eggs <- read.xlsx("Eggs production.xlsx")
#Eggs_total
eggs$Condition <- fct_relevel(eggs$Condition, c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC", "PFBS_HC"))
head(eggs)
hist(eggs$Eggs_total,prob=T,main="Eggs_total")
points(density(eggs$Eggs_total),type="l",col="red", lwd=2)
shapiro.test(eggs$Eggs_total)
#Subset to see difference between each condition according to sex
Control<-subset(eggs,Condition=="Control")
PFOS_LC<-subset(eggs,Condition=="PFOS_LC")
PFOS_HC<-subset(eggs,Condition=="PFOS_HC")
PFBS_LC<-subset(eggs,Condition=="PFBS_LC")
PFBS_HC<-subset(eggs,Condition=="PFBS_HC")
shapiro.test(Control$Eggs_total)
shapiro.test(PFOS_LC$Eggs_total)
shapiro.test(PFOS_HC$Eggs_total)
shapiro.test(PFBS_LC$Eggs_total)
shapiro.test(PFBS_HC$Eggs_total)
wilcox.test(Control$Eggs_total, PFOS_LC$Eggs_total)
wilcox.test(Control$Eggs_total, PFOS_HC$Eggs_total)
wilcox.test(Control$Eggs_total, PFBS_LC$Eggs_total)
wilcox.test(Control$Eggs_total, PFBS_HC$Eggs_total)
Fig_1_A <- ggplot(eggs, aes(x = Condition, y = Eggs_total, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("Number of eggs") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "wilcox.test", label = "{p.format} {p.signif}", hide.ns = TRUE, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Fig_1_A
#-------------------------
####Sup.Fig.4.B Eggs quality####
#Eggs_good
ggdensity(eggs$Eggs_good,
main = "Density plot of good eggs",
xlab = "Number of good eggs")
shapiro.test(eggs$Eggs_good)
wilcox.test(Control$Eggs_good, PFOS_LC$Eggs_good)
wilcox.test(Control$Eggs_good, PFOS_HC$Eggs_good)
wilcox.test(Control$Eggs_good, PFBS_LC$Eggs_good)
wilcox.test(Control$Eggs_good, PFBS_HC$Eggs_good)
Nb_Good <- ggplot(eggs, aes(x = Condition, y = Eggs_good, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("Number of eggs") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "wilcox.test", label = "p = {p.format} {p.signif}", hide.ns = TRUE, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Nb_Good
#Percentage_Eggs_good
ggdensity(eggs$Percentage_good,
main = "Density plot of good eggs",
xlab = "Number of good eggs")
shapiro.test(eggs$Percentage_good)
wilcox.test(Control$Percentage_good, PFOS_LC$Percentage_good)
wilcox.test(Control$Percentage_good, PFOS_HC$Percentage_good)
wilcox.test(Control$Percentage_good, PFBS_LC$Percentage_good)
wilcox.test(Control$Percentage_good, PFBS_HC$Percentage_good)
Sup_Fig_4_B <- ggplot(eggs, aes(x = Condition, y = Percentage_good, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("Percentage of good eggs") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "wilcox.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Sup_Fig_4_B
#-------------------------
####Sup.Fig.4.C BCF global####
rm(list = ls())
BodyCondition<-read.table("F0_onlyPairs_BodyConditionOrgans.txt",h=T)
BodyCondition$Condition <- fct_relevel(BodyCondition$Condition, c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC", "PFBS_HC"))
#Subset to see difference between each condition according to sex
Control<-subset(BodyCondition,Condition=="Control")
PFOS_LC<-subset(BodyCondition,Condition=="PFOS_LC")
PFOS_HC<-subset(BodyCondition,Condition=="PFOS_HC")
PFBS_LC<-subset(BodyCondition,Condition=="PFBS_LC")
PFBS_HC<-subset(BodyCondition,Condition=="PFBS_HC")
#Body Condition
hist(Control$Condition_Factor_K)
sd(Control$Condition_Factor_K)
shapiro.test(Control$Condition_Factor_K)
ggdensity(Control$Condition_Factor_K,
main = "Density plot of Total_distance moved (cm)",
xlab = "Total distance moved cm")
hist(PFOS_HC$Condition_Factor_K,prob=T,main="Condition_Factor_K")
points(density(PFOS_HC$Condition_Factor_K),type="l",col="red", lwd=2)
shapiro.test(Control$Condition_Factor_K)
shapiro.test(PFOS_LC$Condition_Factor_K)
shapiro.test(PFOS_HC$Condition_Factor_K)
shapiro.test(PFBS_LC$Condition_Factor_K)
shapiro.test(PFBS_HC$Condition_Factor_K)
t.test(Control$Condition_Factor_K, PFOS_LC$Condition_Factor_K)
t.test(Control$Condition_Factor_K, PFOS_HC$Condition_Factor_K)
t.test(Control$Condition_Factor_K, PFBS_LC$Condition_Factor_K)
t.test(Control$Condition_Factor_K, PFBS_HC$Condition_Factor_K)
my_comparisons <- list( c("Control", "PFOS_HC"), c("Control", "PFBS_HC"))
Sup_Fig_4_C <- ggplot(BodyCondition, aes(x = Condition, y = Condition_Factor_K, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("BCF") + geom_hline(yintercept = 1, linetype = 2) +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Sup_Fig_4_C
#-------------------------
####Fig. 1.B BCF males####
###Per conditions in males
males<-subset(BodyCondition,Sex=="Male")
summary(males)
males$Condition <- fct_relevel(males$Condition, c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC", "PFBS_HC"))
#subset males for pairwise test
malesC<-subset(males,Condition=="Control") #To select only the females
malesPFOSLC<-subset(males,Condition=="PFOS_LC") #To select only the females
malesPFOSHC<-subset(males,Condition=="PFOS_HC") #To select only the females
malesPFBSLC<-subset(males,Condition=="PFBS_LC") #To select only the females
malesPFBSHC<-subset(males,Condition=="PFBS_HC") #To select only the females
hist(males$Condition_Factor_K)
sd(males$Condition_Factor_K)
summary(males)
shapiro.test(rnorm(1000, mean = 1.0042 , sd = 0.1366105))
hist(PFOS_HC$Condition_Factor_K,prob=T,main="Distance moved")
points(density(PFOS_HC$Condition_Factor_K),type="l",col="red", lwd=2)
#pairwise test for significances
t.test(malesC$Condition_Factor_K, malesPFOSLC$Condition_Factor_K)
t.test(malesC$Condition_Factor_K, malesPFOSHC$Condition_Factor_K)
t.test(malesC$Condition_Factor_K, malesPFBSLC$Condition_Factor_K)
t.test(malesC$Condition_Factor_K, malesPFBSHC$Condition_Factor_K)
compare_means(Condition_Factor_K ~ Condition, data = males, method = "t.test")
#Condition_Factor_K Control PFOS HC p = 0.0219
#Condition_Factor_K Control PFBS LC p = 0.0170
my_comparisons <- list( c("Control", "PFOS_HC"), c("Control", "PFBS_LC"))
Fig_2_B <- ggplot(males, aes(x = Condition, y = Condition_Factor_K, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("Boxplot of the total number of eggs produced by reproduction (n=30)") +
ylab("BCF in males") + geom_hline(yintercept = 1, linetype = 2) +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format} {p.signif}", hide.ns = TRUE, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Fig_2_B
#-------------------------
####Fig. 1.C BCF males vs. females####
#By sex
# Now make a interaction between two factors
# on x axis
BodyCondition$Factor1Factor2 <- interaction(BodyCondition$Condition,BodyCondition$Sex)
my_comparisons <- list( c("Control.Female", "Control.Male"), c("PFOS_LC.Female", "PFOS_LC.Male"), c("PFOS_HC.Female", "PFOS_HC.Male"), c("PFBS_LC.Female", "PFBS_LC.Male"), c("PFBS_HC.Female","PFBS_HC.Male"))
# now Plot Boxplot with fill color according
# to factor1 and factor2
BodyCondition$Factor1Factor2 <- fct_relevel(BodyCondition$Factor1Factor2, c("Control.Female", "Control.Male","PFOS_LC.Female","PFOS_LC.Male","PFOS_HC.Female","PFOS_HC.Male","PFBS_LC.Female","PFBS_LC.Male","PFBS_HC.Female","PFBS_HC.Male"))
Fig_2_C<-ggplot(aes(y = Condition_Factor_K , x = Factor1Factor2), data = BodyCondition) +
geom_boxplot(aes(fill=Factor1Factor2))+
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() + stat_compare_means(comparisons = my_comparisons, label = "p.format", label.y = 1.60,size=5,method = "t.test",hide.ns = FALSE)+
scale_fill_manual(values = c("cornflowerblue","cornflowerblue","#FFFF00","#FFFF00","#FFCC33","#FFCC33","green","green","darkgreen","darkgreen"))+
ylab("BCF") + geom_hline(yintercept = 1, linetype = 2) + xlab("Condition & Sex")+
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))
Fig_2_C
#-------------------------
####Sup.Fig.4.D & E gonad somatic index females and males####
#Gonad somatic Index
###Per conditions in females
females<-subset(BodyCondition,Sex=="Female")
boxplot(females$Gonade_somatic)$out
Q <- quantile(females$Gonade_somatic, probs=c(.25, .75), na.rm = FALSE)
iqr <- IQR(females$Gonade_somatic)
Gonade_somatic_without_outliers_females<- subset(females, females$Gonade_somatic > (Q[1] - 1.5*iqr) & females$Gonade_somatic < (Q[2]+1.5*iqr))
boxplot(Gonade_somatic_without_outliers_females$Gonade_somatic)
hist(Gonade_somatic_without_outliers_females$Gonade_somatic)
sd(Gonade_somatic_without_outliers_females$Gonade_somatic)
summary(Gonade_somatic_without_outliers_females)
shapiro.test(rnorm(1000, mean = 14.986 , sd = 3.397044))
hist(Gonade_somatic_without_outliers_females$Gonade_somatic,prob=T,main="Gonade somatic",ylim = c(0,0.15))
points(density(Gonade_somatic_without_outliers_females$Gonade_somatic),type="l",col="red", lwd=2)
femalesC<-subset(BodyCondition,Condition=="Control")
femalesPFOSLC<-subset(BodyCondition,Condition=="PFOS_LC")
femalesPFOSHC<-subset(BodyCondition,Condition=="PFOS_HC")
femalesPFBSLC<-subset(BodyCondition,Condition=="PFBS_LC")
femalesPFBSHC<-subset(BodyCondition,Condition=="PFBS_HC")
#pairwise test for significances
t.test(femalesC$Gonade_somatic, femalesPFOSLC$Gonade_somatic)
t.test(femalesC$Gonade_somatic, femalesPFOSHC$Gonade_somatic)
t.test(femalesC$Gonade_somatic, femalesPFBSLC$Gonade_somatic)
t.test(femalesC$Gonade_somatic, femalesPFBSHC$Gonade_somatic)
compare_means(Gonade_somatic ~ Condition, data = Gonade_somatic_without_outliers_females, method = "t.test")
Gonade_somatic_females <- ggplot(Gonade_somatic_without_outliers_females, aes(x = Condition, y = Gonade_somatic, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("F0: Gonade_somatic in females") +
ylab("Gonade somatic in females") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Gonade_somatic_females
###Per conditions in males
boxplot(males$Gonade_somatic)$out
Q <- quantile(males$Gonade_somatic, probs=c(.25, .75), na.rm = FALSE)
iqr <- IQR(males$Gonade_somatic)
Gonade_somatic_without_outliers<- subset(males, males$Gonade_somatic > (Q[1] - 1.5*iqr) & males$Gonade_somatic < (Q[2]+1.5*iqr))
boxplot(Gonade_somatic_without_outliers$Gonade_somatic)
hist(Gonade_somatic_without_outliers$Gonade_somatic)
sd(Gonade_somatic_without_outliers$Gonade_somatic)
summary(Gonade_somatic_without_outliers)
shapiro.test(rnorm(1000, mean = 1.1098 , sd = 0.3870416))
hist(Gonade_somatic_without_outliers$Gonade_somatic,prob=T,main="Gonade_somatic",ylim=c(0,1.5))
points(density(Gonade_somatic_without_outliers$Gonade_somatic),type="l",col="red", lwd=2)
#pairwise test for significances
t.test(malesC$Gonade_somatic, malesPFOSLC$Gonade_somatic)
t.test(malesC$Gonade_somatic, malesPFOSHC$Gonade_somatic)
t.test(malesC$Gonade_somatic, malesPFBSLC$Gonade_somatic)
t.test(malesC$Gonade_somatic, malesPFBSHC$Gonade_somatic)
compare_means(Gonade_somatic ~ Condition, data = Gonade_somatic_without_outliers, method = "t.test")
Gonade_somatic_males <- ggplot(Gonade_somatic_without_outliers, aes(x = Condition, y = Gonade_somatic, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("F0: Gonade_somatic in males") +
ylab("Gonade somatic in males") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Gonade_somatic_males
#-------------------------
####Fig. 1. D Liver males####
#Liver somatic Index
###Per conditions in males
hist(males$Liver_somatic)
sd(males$Liver_somatic,na.rm = "TRUE")
Liver_somatic_males_wt_NA<-na.omit(males$Liver_somatic)
summary(males)
shapiro.test(rnorm(1000, mean = 0.5313 , sd = 0.2103225))
hist(males$Liver_somatic,prob=T,main="Liver_somatic")
points(density(Liver_somatic_males_wt_NA),type="l",col="red", lwd=2)
#pairwise test for significances
t.test(malesC$Liver_somatic, malesPFOSLC$Liver_somatic)
t.test(malesC$Liver_somatic, malesPFOSHC$Liver_somatic)
t.test(malesC$Liver_somatic, malesPFBSLC$Liver_somatic)
t.test(malesC$Liver_somatic, malesPFBSHC$Liver_somatic)
compare_means(Liver_somatic ~ Condition, data = males, method = "t.test")
my_comparisons <- list(c("Control", "PFBS_HC"))
Fig_1_D <- ggplot(males, aes(x = Condition, y = Liver_somatic, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("F0: Total Liver_somatic in males") +
ylab("Liver somatic in males") + # stat_compare_means(comparisons = my_comparisons, method = "t.test")+
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format} {p.signif}", hide.ns = TRUE, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Fig_1_D
#-------------------------
####Sup.Fig.4.F####
#Brain somatic Index
###Per conditions in females
hist(females$Brain_somatic)
sd(females$Brain_somatic,na.rm="TRUE")
summary(females)
shapiro.test(rnorm(1000, mean = 0.8265, sd = 0.3163376))
hist(females$Brain_somatic,prob=T,main="Brain_somatic")
Brain_somatic_females_wt_NA<-na.omit(females$Brain_somatic)
points(density(Brain_somatic_females_wt_NA),type="l",col="red", lwd=2)
#pairwise test for significances
t.test(femalesC$Brain_somatic, femalesPFOSLC$Brain_somatic)
t.test(femalesC$Brain_somatic, femalesPFOSHC$Brain_somatic)
t.test(femalesC$Brain_somatic, femalesPFBSLC$Brain_somatic)
t.test(femalesC$Brain_somatic, femalesPFBSHC$Brain_somatic)
compare_means(Brain_somatic ~ Condition, data = females, method = "t.test")
my_comparisons <- list( c("Control", "PFOS_HC"), c("Control", "PFBS_HC"))
Fig_4_F <- ggplot(females, aes(x = Condition, y = Brain_somatic, fill=Condition)) +
geom_boxplot(width=0.3) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","#FFFF00","#FFCC33","green","darkgreen")) +
#ggtitle("F0: Brain_somatic in females") +
ylab("Brain somatic in females") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
geom_pwc(method = "t.test", label = "{p.format}", hide.ns = F, tip.length = 0, p.adjust.method = "none", ref.group = "Control", label.size = 6)
Fig_4_F
#-------------------------
####Fig. 1. E####
#By sex
# Now make a interaction between two factors
# on x axis
my_comparisons <- list( c("Control.Female", "Control.Male"), c("PFOS_LC.Female", "PFOS_LC.Male"), c("PFOS_HC.Female", "PFOS_HC.Male"), c("PFBS_LC.Female", "PFBS_LC.Male"), c("PFBS_HC.Female","PFBS_HC.Male"))
# now Plot Boxplot with fill color according
# to factor1 and factor2
BodyCondition$Factor1Factor2 <- fct_relevel(BodyCondition$Factor1Factor2, c("Control.Female", "Control.Male","PFOS_LC.Female","PFOS_LC.Male","PFOS_HC.Female","PFOS_HC.Male","PFBS_LC.Female","PFBS_LC.Male","PFBS_HC.Female","PFBS_HC.Male"))
Fig_1_E<-ggplot(aes(y = Brain_somatic , x = Factor1Factor2), data = BodyCondition) +
geom_boxplot(aes(fill=Factor1Factor2))+
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.5)+
theme_light() +
scale_fill_manual(values = c("cornflowerblue","cornflowerblue","#FFFF00","#FFFF00","#FFCC33","#FFCC33","green","green","darkgreen","darkgreen"))+
ylab("Brain somatic") + geom_hline(yintercept = 1, linetype = 2) + xlab("Condition & Sex")+
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))+
stat_compare_means(comparisons = my_comparisons, label = "p.format", label.y = 1.60,size=5,method = "t.test",hide.ns = FALSE)
Fig_1_E
#-------------------------
####Sup Fig. 5. A Larvae behavioral test checking####
rm(list = ls())
#Set the working directory
B1<- read.table("StatsLight120ALL.txt",h=T)
summary(B1)
# Remove all rows with too high values due to instrument error
B<-B1[!(B1$Well=="F8" & B1$Plate=="Trial_1"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="H2" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="F9" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="A9" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E2" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="A5" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="B6" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="H5" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="B11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E9" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D1" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="G12" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C6" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="H8" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="G6" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="B12" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C2" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="H2" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_7"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_7"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="G11" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="G10" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="A2" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="B5" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F5" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="G7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="H1" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="F2" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="G2" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="H6" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="B1" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C2" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="B6" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A2" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A1" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="E5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="G5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B5" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="C5" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B10" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D5" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="H6" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="A10" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G12" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="B10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E5" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E8" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="H11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F6" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E8" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="H11" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="A11" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="H12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="B7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="D1" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G7" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G10" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="F9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_20"),]
B1<-B
summary(B1) # check if all values are numeric and if range of values is ok
LON1<-B1[B1$Condition=="LON_1",]
LOFF<-B1[B1$Condition=="LOFF",]
LON2<-B1[B1$Condition=="LON_2",]
B1$Condition <- fct_relevel(B1$Condition, c("LON_1", "LOFF", "LON_2"))
B1$Expo <- fct_relevel(B1$Expo, c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC", "PFBS_HC"))
levels(B1$Condition)
levels(B1$Expo)
Sup_Fig_5_A <- ggplot(B1, aes(x = Expo, y = Dmcptot, fill=Condition)) + scale_y_continuous(limits=c(0,180))+
geom_boxplot() +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.1)+
theme_light() +
scale_fill_manual(values = c("yellow", "darkgrey","yellow")) +
xlab("Condition") + ylab("Total distance moved (cm)") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))
Sup_Fig_5_A + guides(fill=guide_legend(title="Light exposure"))
#ggsave("bp.png", width = 16, height = 9, bp)
LOFF$Expo <- factor(LOFF$Expo,levels = c("Control", "PFOS_LC", "PFOS_HC", "PFBS_LC","PFBS_HC"))
# Tapping test
B2<- read.table("StatsTap120ALLN.txt",h=T)
summary (B2)
# remove all values, where embryos were not hatched, coagulated, or deformed
B<-B2[!(B2$Well=="F8" & B2$Plate=="Trial_1"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_1"),]
B<-B[!(B$Well=="F9" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="A9" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E2" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="A5" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="B6" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_2"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="H5" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="B11" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_3"),]
B<-B[!(B$Well=="E9" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="E9" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D1" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="B12" & B$Plate=="Trial_4"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="G12" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C6" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="H8" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="C2" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="G6" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_5"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="G6" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="B12" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="C2" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="H2" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="A2" & B$Plate=="Trial_6"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_7"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_7"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="G11" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_8"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="G10" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="A2" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="B5" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F5" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_9"),]
B<-B[!(B$Well=="F1" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="G7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C10" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="H1" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="F2" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="G2" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_10"),]
B<-B[!(B$Well=="A6" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="H6" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="G12" & B$Plate=="Trial_11"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="B1" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C2" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="H10" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_12"),]
B<-B[!(B$Well=="B6" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A2" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="G2" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A1" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="E5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="G5" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="B8" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="C8" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="H7" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_13"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B5" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="C5" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B10" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="A5" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_14"),]
B<-B[!(B$Well=="B11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D5" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="H6" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="A10" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="D11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G11" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="G12" & B$Plate=="Trial_15"),]
B<-B[!(B$Well=="E6" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="B10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E1" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E5" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="C7" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E8" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="D10" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_16"),]
B<-B[!(B$Well=="G9" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F10" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="H11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D7" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F11" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F6" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D8" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="E8" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="D9" & B$Plate=="Trial_17"),]
B<-B[!(B$Well=="F8" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="C1" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="D12" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="H11" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="H12" & B$Plate=="Trial_18"),]
B<-B[!(B$Well=="E7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="C11" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="A11" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="F12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="H12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="F7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="B7" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="D1" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="E12" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="G1" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="A1" & B$Plate=="Trial_19"),]
B<-B[!(B$Well=="C12" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G8" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G7" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="H9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="G10" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="E10" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="D6" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="B9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="F9" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="A8" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="B2" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="A12" & B$Plate=="Trial_20"),]
B<-B[!(B$Well=="B11" & B$Plate=="Trial_21"),]
B2<-B[!(B$Well=="H9" & B$Plate=="Trial_21"),]
summary(B2)
# subset off and on
AT1<-subset(B2, Condition== "AT1")
AT5<-subset(B2, Condition== "AT5")
AT10<-subset(B2, Condition== "AT10")
BT<-subset(B2, Condition== "BT")
B2$Condition <- fct_relevel(B2$Condition, c("BT","AT1","AT5","AT10"))
B2$Expo <- fct_relevel(B2$Expo, c("Control","PFOS_LC","PFOS_HC","PFBS_LC", "PFBS_HC"))
levels(B2$Condition)
Sup_Fig_5_B <- ggplot(B2, aes(x=Expo, y=Dmcptot, fill=Condition)) + scale_y_continuous(limits=c(0,1.5))+ # oder colour for outer lines
geom_boxplot() + scale_fill_manual(name="Periods",breaks = c("BT", "AT1", "AT5","AT10"),
values=c("white", "magenta", "red", "darkred")) +
geom_dotplot(binaxis='y', stackdir='center', dotsize=0.1)+
# theme(legend.position = "none")+
theme_light() +
xlab("Condition") + ylab("Distance moved (cm)") +
theme(axis.text.x = element_text(size=18,angle = 60, vjust = 1, hjust=1),
axis.text.y = element_text(size=18),
axis.title.x = element_text(color = "black", size = 20, angle = 0, face = "bold"),
axis.title.y = element_text(color = "black", size = 20, angle = 90, face = "bold"))