Plots
June 2026, R version 4.5.3
Install and Load packages, functions and color palettes
library(readxl)
library(tidyverse)
library(readr)
library(ggplot2)
library(grid)
library(scales)
remove_outliers <- function(x, na.rm = TRUE, ...) {
qnt <- quantile(x, probs = c(.25, .75), na.rm = na.rm, ...)
val <- 1.25 * IQR(x, na.rm = na.rm)
y <- x
y[x < (qnt[1] - val)] <- NA
y[x > (qnt[2] + val)] <- NA
y
}
my_cols <- c( #options for colors
"#66C2A5",
"#3288BD",
"#6A51A3",
"#ABDDA4",
"#5E4FA2",
"#9EBCDA",
"#8DD3C7"
)
my_cols_12 <- c( #options for colors
"#ABDDA4",
"#66C2A5",
"#8DD3C7",
"#3288BD",
"#5DA5DA",
"#9EBCDA",
"#6A51A3",
"#5E4FA2",
"#8073AC",
"#B2ABD2",
"#C7E9B4",
"#A6DBA0"
)Set working directory and import data
setwd("...")
data <- read.csv("Assays_calculated.csv") %>%
dplyr::select(-c(Signal,Sample_type,Signal_corr)) #remove un-necessary columns
data_clean <- data %>% #remove outliers based on function
group_by(Assay, Sample_ID, RNA, Assay_comments)%>%
mutate(Concentration = case_when(
Sample_ID == "Oocyte" ~ remove_outliers(Concentration),
TRUE ~ Concentration))%>%
ungroup() %>%
filter(!is.na(Concentration)) Create subsets of data for plots. In this example the columns Assay indicate the different assays performed.
Assay_1 <- filter(data_clean, Assay == "E11-1")
Assay_2 <- filter(data_clean, Assay == "E11-2")
Assay_3 <- filter(data_clean, Assay == "E11-3")
Assay_4 <- filter(data_clean, Assay == "E11-4")Assay 1 - Expression time
Assay_1 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = RNA, y = Concentration, fill = Assay_comments)) +
geom_boxplot(alpha = 0.5, outlier.shape = NA) +
geom_point(position=position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8), aes(color = Assay_comments)) +
theme_classic() +
theme(
aspect.ratio = 1,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+
labs(
x = "RNA",
y = "Esculin (µM)",
title = "",
fill = "Expression time", color = "Expression time"
)+
scale_color_manual(values = c(my_cols))+ scale_fill_manual(values = c(my_cols))
Assay 2 - Time course
Option 1
Assay_2$Assay_comments <- factor( Assay_2$Assay_comments, levels = c( "5 min", "10 min", "15 min", "20 min", "30 min", "45 min", "1 hour" ))
Assay_2 <- Assay_2 %>%mutate(Time = recode(as.character(Assay_comments), "5 min" = "5","10 min" = "10", "15 min" = "15","20 min" = "20","30 min" = "30","45 min" = "45","1 hour" = "60" ) |> as.numeric())
Assay_2 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = Time, y = Concentration, color = RNA, group = RNA)) +
geom_boxplot(
aes(fill = RNA, group = interaction(Time, RNA)),width = 3.5,alpha = 0.25,
position = position_identity(), outlier.shape = NA) +
stat_summary(fun = mean, geom = "line", size = 1) +
geom_point(size = 2, position = position_jitter(width = 0.1)) +
theme_classic() +
labs(
x = "Time (min)",
y = "Esculin (µM)",
color = "RNA"
)+
scale_color_manual(values = my_cols) + scale_fill_manual(values = my_cols)+
theme(aspect.ratio = 1.2,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+scale_x_continuous( breaks = c(5, 10, 15, 20, 30, 45, 60),labels = c("5", "10", "15", "20", "30", "45", "60")
)## Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
## ℹ Please use `linewidth` instead.
## This warning is displayed once per session.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
Option 2
Assay_2 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = RNA, y = Concentration, fill = Assay_comments)) +
geom_boxplot(alpha = 0.5, outlier.shape = NA) +
geom_point(position=position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8), aes(color = Assay_comments)) +
theme_classic() +
theme(
aspect.ratio = 1,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+
labs(
x = "RNA",
y = "Esculin (µM)",
title = "",
fill = "Time course", color = "Time course"
)+ scale_color_manual(values = c(my_cols_12))+ scale_fill_manual(values = c(my_cols_12))
Assay 3 - pH assay
Option 1
Assay_3 <- Assay_3 %>%mutate(pH = recode(as.character(Assay_comments), "pH 4" = 4,"pH 4.5" = 4.5, "pH 5" = 5, "pH 5.5" = 5.5,
"pH 6" = 6, "pH 6.5" = 6.5, "pH 7.4" = 7.4 ) |> as.factor())
Assay_3 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = pH, y = Concentration, fill = RNA)) +
geom_boxplot(alpha = 0.5, outlier.shape = NA) +
geom_point(position=position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8), aes(color = RNA)) +
theme_classic() +
theme(
aspect.ratio = 1,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+
labs(
x = "pH",
y = "Esculin (µM)",
title = "",
fill = "RNA", color = "RNA"
)+ scale_color_manual(values = c(my_cols))+ scale_fill_manual(values = c(my_cols))
Same plot, but with different colors - in a scale format
library(ggnewscale)
cols_pH <- c(
"#2F7F6E",
"#3E8F7C",
"#4D9F8A",
"#5CAF98",
"#6BBFA6",
"#7ACFB4",
"#89DFC2",
"#1F5F8A",
"#2C6E9B",
"#397DAC",
"#468CBD",
"#53A0CE",
"#60B4DF",
"#6DC8F0"
)
Assay_3 <- Assay_3 %>%
mutate(RNA_pH = paste0(RNA, "_", Assay_comments))
Assay_3 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = pH, y = Concentration)) +
geom_boxplot(aes(fill = RNA_pH), alpha = 0.5, outlier.shape = NA) +
geom_point(aes(color = RNA_pH), position = position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8)) +
scale_fill_manual(values = cols_pH, guide = "none") +
scale_color_manual(values = cols_pH, guide = "none") +
new_scale_color() +
new_scale_fill() +
geom_point(aes(color = RNA, fill = RNA), alpha = 0, size = 4, show.legend = TRUE) +
scale_color_manual( name = "RNA", values = c("Mock" = "#5CAF98", "SUC1" ="#468CBD")) +
scale_fill_manual( name = "RNA", values = c("Mock" = "#5CAF98", "SUC1" = "#468CBD"),
guide = guide_legend(override.aes = list(alpha = 1, size = 4) )) +
theme_classic() +
labs(
x = "pH",
y = "Esculin (µM)"
)+
theme(
aspect.ratio = 1,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)
Option 2
Assay_3 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = RNA, y = Concentration, fill = Assay_comments)) +
geom_boxplot(alpha = 0.5, outlier.shape = NA) +
geom_point(position=position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8), aes(color = Assay_comments)) +
theme_classic() +
theme(
aspect.ratio = 1,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+
labs(
x = "RNA",
y = "Esculin (µM)",
title = "",
fill = "pH", color = "pH"
)+ scale_color_manual(values = c(my_cols_12))+ scale_fill_manual(values = c(my_cols_12))
Assay 4 - Competition assay + dose response
Esculin alone = 100 µM esculin in dose response
Assay4_1 <- filter(Assay_4, Assay_comments %in% c("Esculin alone", "Esculin + 2.5 mM sucrose", "Esculin + 2.5 mM glucose"))
Assay4_2<- filter(Assay_4, !Assay_comments %in% c( "Esculin + 2.5 mM sucrose", "Esculin + 2.5 mM glucose"))
Assay4_1$Assay_comments <- factor(Assay4_1$Assay_comments,levels = c( "Esculin alone", "Esculin + 2.5 mM sucrose", "Esculin + 2.5 mM glucose")
)
Assay4_1 %>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = RNA, y = Concentration, fill = Assay_comments)) +
geom_boxplot(alpha = 0.5, outlier.shape = NA, position = position_dodge(width = 0.8)) +
geom_point( aes(color = Assay_comments), position = position_jitterdodge(jitter.width = 0.2, dodge.width = 0.8), size = 2 )+
theme_classic() +
theme(
aspect.ratio = 2,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
) +
labs( x = "",y = "Esculin (µM)", fill = "Competition", color = "Competition")+ scale_color_manual(values = c(my_cols))+ scale_fill_manual(values = c(my_cols))
Assay4_2 <- Assay4_2 %>%
mutate(Conc = case_when(Assay_comments == "Esculin alone" ~ 100,TRUE ~ as.numeric(gsub(" µM", "", Assay_comments))))## Warning: There was 1 warning in `mutate()`.
## ℹ In argument: `Conc = case_when(...)`.
## Caused by warning:
## ! NAs introduced by coercionAssay4_2%>%
filter(Sample_ID == "Oocyte") %>%
ggplot(aes(x = Conc, y = Concentration, color = RNA, group = RNA)) +
geom_boxplot(
aes(fill = RNA, group = interaction(Conc, RNA)),width = 30,alpha = 0.25,
position = position_identity(), outlier.shape = NA) +
stat_summary(fun = mean, geom = "line", size = 1) +
geom_point(size = 2, position = position_jitter(width = 0.1)) +
theme_classic() +
labs(
x = "Concentration (µM)",
y = "Esculin (µM)",
color = "RNA"
)+
scale_color_manual(values = my_cols) + scale_fill_manual(values = my_cols)+
theme(aspect.ratio = 1.2,
axis.title = element_text(size = 14),
axis.text = element_text(size = 12),
legend.title = element_text(size = 12),
legend.text = element_text(size = 11)
)+scale_x_continuous(breaks = c(10, 50, 100, 250, 500))
# R studio Version
rstudioapi::versionInfo()$version## [1] '2026.7.1.147'# Session report
sessioninfo::session_info()## ─ Session info ───────────────────────────────────────────────────────────────
## setting value
## version R version 4.5.3 (2026-03-11 ucrt)
## os Windows 11 x64 (build 26200)
## system x86_64, mingw32
## ui RTerm
## language (EN)
## collate Danish_Denmark.utf8
## ctype Danish_Denmark.utf8
## tz Europe/Copenhagen
## date 2026-08-25
## pandoc 3.8.3 @ C:/Program Files/RStudio/resources/app/bin/quarto/bin/tools/ (via rmarkdown)
## quarto 1.9.38 @ C:\\PROGRA~1\\RStudio\\RESOUR~1\\app\\bin\\quarto\\bin\\quarto.exe
##
## ─ Packages ───────────────────────────────────────────────────────────────────
## package * version date (UTC) lib source
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##
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## * ── Packages attached to the search path.
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