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JH12_Figure_1_alpha.R
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#############################################################
#
# Ref to the ARTICLE
#
# Code to generate the Alfa diversity Fig. 1 used in Maver et al., manuscript
# Revision 07/21
# mauro.maver@unibz.it
# d.bulgarelli@dundee.ac.uk
#
#############################################################
#############################################################
# Clean-up the memory and start a new session
#############################################################
rm(list=ls())
dev.off()
#############################################################
# Libraries required
#############################################################
#required packages
library("phyloseq")
library("DESeq2")
library("ggplot2")
library("vegan")
library("grid")
library("gridExtra")
library("PMCMR")
writeLines(capture.output(sessionInfo()), "sessionInfo.txt")
colorblind_Palette <- c("#E69F00", "#56B4E9", "#009E73", "#F0E442", "#0072B2", "#D55E00", "#CC79A7", "#000000")
#import the Phyloseq object 2 file
JH12_alfa_div <- readRDS(file = "JH12_data_phyloseq_genus_0221_Silva138.rds")
JH12_alfa_div
###
#import the rarefied OTU counts (note file name counts2.txt this file has been generated in excel and includes the #OTU ID as header of the first column)
dat_count_rare <- read.delim("JH12_data_phyloseq_rare_table_counts_140219.txt", sep = "\t", header=T, row.names= 1, blank.lines.skip = FALSE)
###
#Index calculations
JH12_alpha_rare <- estimate_richness(JH12_alfa_div, measures = c("Observed", "Shannon", "Chao1"))
JH12_alpha_rare
#design file
design <- read.delim("Map_JH12_2.txt", sep = "\t", header=TRUE, row.names=1)
design
design_treatment <- as.data.frame(design[, 5])
rownames(design_treatment) <- rownames(design)
colnames(design_treatment) <- c("treatment")
design_treatment
#description
design_description <- as.data.frame(design[, 6])
rownames(design_description) <- rownames(design)
colnames(design_description) <- c("description")
design_description
#data frame Genotype_Description
design_TD <- cbind(design_treatment, design_description)
#remove nursery sample
design_TD <- design_TD[colnames(dat_count_rare), ]
#
##
###
#### OBSERVED Genera
#Observed Genera
JH12_alpha_rare_Observed <- as.data.frame(JH12_alpha_rare[ ,1])
rownames(JH12_alpha_rare_Observed) <- rownames(JH12_alpha_rare)
colnames(JH12_alpha_rare_Observed) <- c("Observed")
#Combine the dataset sample description and Observed Genera
JH12_alpha_rare_Observed_TD <- cbind(design_TD, JH12_alpha_rare_Observed)
JH12_alpha_rare_Observed_TD <- as.data.frame(JH12_alpha_rare_Observed_TD)
JH12_alpha_rare_Observed_TD$treatment
JH12_alpha_rare_Observed_TD$description
#Order the levels according to a defined order
JH12_alpha_rare_Observed_TD$treatment <- ordered(JH12_alpha_rare_Observed_TD$treatment, levels=c("G0", "G24", "G46"))
JH12_alpha_rare_Observed_TD$description <- ordered(JH12_alpha_rare_Observed_TD$description, levels=c("QUARRY", "BARKEQUARRY", "MOREXQUARRY"))
description_names <- c(
`QUARRY` = "Bulk soil",
`BARKEQUARRY` = "Barke",
`MOREXQUARRY` = "Morex")
p1<-ggplot(JH12_alpha_rare_Observed_TD, aes(x=treatment, y=Observed, fill=treatment))+
geom_boxplot(outlier.colour = "red", outlier.shape = 1, outlier.size = 4.5)+
#geom_jitter(size = 3, shape = 16)+
geom_point(size=3, shape = 16)+
scale_fill_manual(name = "Concentration", labels = c("G0", "G24", "G46"), values = colorblind_Palette)+
facet_wrap(~ description, labeller = as_labeller(description_names))+
theme_bw()+
ylab("Observed Genera")+
theme(axis.title.y = element_text(color="Black", size=16),
axis.title.x = element_blank(),
strip.text = element_text(size=18),
legend.key.size = unit(2,"line"),
legend.text = element_text(size = 14),
axis.text.y = element_text(size = 12),
axis.text.x = element_blank(),
legend.position="none",
legend.title = element_text(size = 16))
# STATS
shapiro.test(JH12_alpha_rare_Observed_TD$Observed)
hist(JH12_alpha_rare_Observed_TD$Observed)
qqnorm(JH12_alpha_rare_Observed_TD$Observed)
qqline(JH12_alpha_rare_Observed_TD$Observed)
kruskal.test(Observed ~ treatment, data = JH12_alpha_rare_Observed_TD)
kruskal.test(Observed ~ description, data = JH12_alpha_rare_Observed_TD)
dunn.test.control(JH12_alpha_rare_Observed_TD$Observed,JH12_alpha_rare_Observed_TD$description, "bonferroni")
posthoc.kruskal.dunn.test(Observed ~ description, data = JH12_alpha_rare_Observed_TD, p.adjust="bonf")
pairwise.wilcox.test(JH12_alpha_rare_Observed_TD$Observed, JH12_alpha_rare_Observed_TD$description, p.adjust.method = "BH")
#
##
###
#### CHAO1
#Chao1 Genera
JH12_alpha_rare_Chao1 <- as.data.frame(JH12_alpha_rare[ ,2])
rownames(JH12_alpha_rare_Chao1) <- rownames(JH12_alpha_rare)
colnames(JH12_alpha_rare_Chao1) <- c("Chao1")
#Combine the dataset sample description and Chao1 Genera
JH12_alpha_rare_Chao1_TD <- cbind(design_TD, JH12_alpha_rare_Chao1)
JH12_alpha_rare_Chao1_TD <- as.data.frame(JH12_alpha_rare_Chao1_TD)
JH12_alpha_rare_Chao1_TD$treatment
JH12_alpha_rare_Chao1_TD$description
#Order the levels according to a defined order
JH12_alpha_rare_Chao1_TD$treatment <- ordered(JH12_alpha_rare_Chao1_TD$treatment, levels=c("G0", "G24", "G46"))
JH12_alpha_rare_Chao1_TD$description <- ordered(JH12_alpha_rare_Chao1_TD$description, levels=c("QUARRY", "BARKEQUARRY", "MOREXQUARRY"))
p2<-ggplot(JH12_alpha_rare_Chao1_TD, aes(x=treatment, y=Chao1, fill=treatment))+
geom_boxplot(outlier.colour = "red", outlier.shape = 1, outlier.size = 4.5)+
#geom_jitter(size = 3, shape = 16)+
geom_point(size=3, shape = 16)+
scale_fill_manual(name = "Concentration", labels = c("G0", "G24", "G46"), values = colorblind_Palette)+
facet_wrap(~ description, labeller = as_labeller(description_names))+
theme_bw()+
ylab("Chao1")+
theme(axis.title.y = element_text(color="Black", size=16),
axis.title.x = element_blank(),
strip.text = element_text(size=18),
legend.key.size = unit(2,"line"),
legend.text = element_text(size = 14),
axis.text.y = element_text(size = 12),
axis.text.x = element_blank(),
legend.position="none",
legend.title = element_text(size = 16))
# STATS
shapiro.test(JH12_alpha_rare_Chao1_TD$Chao1)
hist(JH12_alpha_rare_Chao1_TD$Chao1)
qqnorm(JH12_alpha_rare_Chao1_TD$Chao1)
qqline(JH12_alpha_rare_Chao1_TD$Chao1)
kruskal.test(Chao1 ~ treatment, data = JH12_alpha_rare_Chao1_TD)
kruskal.test(Chao1 ~ description, data = JH12_alpha_rare_Chao1_TD)
dunn.test.control(JH12_alpha_rare_Chao1_TD$Chao1,JH12_alpha_rare_Chao1_TD$description, "bonferroni")
posthoc.kruskal.dunn.test(Chao1 ~ description, data = JH12_alpha_rare_Chao1_TD, p.adjust="bonf")
dunn.test.control(JH12_alpha_rare_Chao1_TD$Chao1,JH12_alpha_rare_Chao1_TD$treatment, "bonferroni")
posthoc.kruskal.dunn.test(Chao1 ~ treatment, data = JH12_alpha_rare_Chao1_TD, p.adjust="bonf")
pairwise.wilcox.test(JH12_alpha_rare_Chao1_TD$Chao1, JH12_alpha_rare_Chao1_TD$description, p.adjust.method = "BH")
#
##
###
#### SHANNON
#Shannon Genera
JH12_alpha_rare_Shannon <- as.data.frame(JH12_alpha_rare[ ,4])
rownames(JH12_alpha_rare_Shannon) <- rownames(JH12_alpha_rare)
colnames(JH12_alpha_rare_Shannon) <- c("Shannon")
#Combine the dataset sample description and Shannon Genera
JH12_alpha_rare_Shannon_TD <- cbind(design_TD, JH12_alpha_rare_Shannon)
JH12_alpha_rare_Shannon_TD <- as.data.frame(JH12_alpha_rare_Shannon_TD)
JH12_alpha_rare_Shannon_TD$treatment
JH12_alpha_rare_Shannon_TD$description
#Order the levels according to a defined order
JH12_alpha_rare_Shannon_TD$treatment <- ordered(JH12_alpha_rare_Shannon_TD$treatment, levels=c("G0", "G24", "G46"))
JH12_alpha_rare_Shannon_TD$description <- ordered(JH12_alpha_rare_Shannon_TD$description, levels=c("QUARRY", "BARKEQUARRY", "MOREXQUARRY"))
p3<-ggplot(JH12_alpha_rare_Shannon_TD, aes(x=treatment, y=Shannon, fill=treatment))+
geom_boxplot(outlier.colour = "red", outlier.shape = 1, outlier.size = 4.5)+
#geom_jitter(size = 3, shape = 16)+
geom_point(size=3, shape = 16)+
scale_fill_manual(name = "Concentration", labels = c("G0", "G24", "G46"), values = colorblind_Palette)+
facet_wrap(~ description, labeller = as_labeller(description_names))+
theme_bw()+
ylab("Shannon")+
theme(axis.title.y = element_text(color="Black", size=16),
axis.title.x = element_blank(),
strip.text = element_text(size=18),
legend.key.size = unit(2,"line"),
legend.text = element_text(size = 14),
axis.text.y = element_text(size = 12),
axis.text.x = element_blank(),
legend.position="bottom",
legend.title = element_text(size = 16))
# STATS
shapiro.test(JH12_alpha_rare_Shannon_TD$Shannon)
hist(JH12_alpha_rare_Shannon_TD$Shannon)
qqnorm(JH12_alpha_rare_Shannon_TD$Shannon)
qqline(JH12_alpha_rare_Shannon_TD$Shannon)
kruskal.test(Shannon ~ treatment, data = JH12_alpha_rare_Shannon_TD)
kruskal.test(Shannon ~ description, data = JH12_alpha_rare_Shannon_TD)
dunn.test.control(JH12_alpha_rare_Shannon_TD$Shannon,JH12_alpha_rare_Shannon_TD$description, "bonferroni")
posthoc.kruskal.dunn.test(Shannon ~ description, data = JH12_alpha_rare_Shannon_TD, p.adjust="bonf")
dunn.test.control(JH12_alpha_rare_Shannon_TD$Shannon,JH12_alpha_rare_Shannon_TD$treatment, "bonferroni")
posthoc.kruskal.dunn.test(Shannon ~ treatment, data = JH12_alpha_rare_Shannon_TD, p.adjust="bonf")
pairwise.wilcox.test(JH12_alpha_rare_Shannon_TD$Shannon, JH12_alpha_rare_Shannon_TD$description, p.adjust.method = "BH")
# Merging the 3 plots
lay5 <- rbind(c(1),
c(2),
c(3))
grid.arrange(p1, p2, p3, layout_matrix = lay5,
top = textGrob("",gp=gpar(fontsize=14,font=2)))