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Low_dosage_MDF_trial.Rmd
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---
title: "Low inclusion of beta-mannnan trial"
author: "Shashank Gupta"
date: "`r format(Sys.time(), '%B %d, %Y')`"
output:
html_document:
toc: true # table of content true
toc_depth: 4 # upto three depths of headings (specified by #, ## and ###)
number_sections: true ## if you want number sections at each table header
theme: united # many options for theme, this one is my favorite.
highlight: tango # specifies the syntax highlighting style
---
```{r, include=FALSE}
options(tinytex.verbose = TRUE)
```
```{r setup, include=FALSE}
knitr::opts_chunk$set(warning = FALSE, message = FALSE)
```
Load all the packages
```{r message=FALSE, warning=FALSE}
library("ranacapa")
library("phyloseq")
library("ggplot2")
library("stringr")
library("plyr")
library("reshape2")
library("reshape")
library("dplyr")
library("tidyr")
library("doBy")
library("plyr")
library("microbiome")
library("ggpubr")
library("vegan")
library("tidyverse")
library("magrittr")
library("cowplot")
library("dendextend")
library("WGCNA")
library("metagenomeSeq")
library("decontam")
library("RColorBrewer")
library("ampvis2")
library("ggpubr")
library("formatR")
library("DT")
library("taxcleanr") #devtools::install_github("shashank-KU/taxcleanr")
library("pheatmap")
library("DESeq2")
library("microbiomeMarker")
```
# Overview of the multi-omics
# Sampling for low inclusion of beta-mannnan trial
A total of 30 salmon were sampled at the first time point (T0), followed by the sampling of 120 salmon individuals (30 for each diet) at each subsequent time point (T1 – 5 weeks after the trial start; T2 – 10 weeks after the trial start; T3 – 15 weeks after the trial start). The distal gut section was dissected using sterile scalpels and tweezers, and approximately 100 mg of gut content was collected from each fish using inoculation loops.
# Phenotypic scoring
```{r}
annotation_colors <- list(
Timepoint = c(`T0`="gray" ,`T1` = "#FF7F00", `T2` = "#FFD92F", `T3`="#F781BF"),
Diet = c(`ext-ctrl`="#FDC086", `CTR`="#e41a1cff", `MC1`="#377EB8", `MC2`="#4DAF4A", `MN3`="#984EA3"))
# Specify the path to your CSV file on GitHub
csv_file <- "Phenotypic/Metrics_low_dosage.csv"
csv_url <- paste0("https://raw.githubusercontent.com/shashank-KU/ImprovaFish-MDF-Effects/main/", csv_file)
# Read the CSV file
fish_data <- read.csv(url(csv_url), row.names = 1)
fish_data$Diet <- factor(fish_data$Diet, levels=c('start', 'CTR', 'MC1', 'MC2', 'MN3'))
plot2 <- ggboxplot(fish_data, x = "Diet", y = "BW",
color = "black", palette = "jco", legend = "none", outlier.shape = NA)+
#stat_compare_means(comparisons = my_comparisons, hide.ns = F) +
#stat_compare_means() +
ggplot2::geom_jitter(
mapping = aes_string(fill="Diet"),
position = ggplot2::position_jitter(seed=123),
shape = 21,
color = "black",
size = 2,
alpha = 0.8
) +
theme_bw() + ylab("Body weigth") +
theme(legend.position="none",axis.title.x=element_blank()) +
scale_fill_manual(values = annotation_colors$Diet) +
scale_colour_manual( values = annotation_colors$Diet) +
facet_grid(. ~ Timepoint, scales="free_x", space = "free_x")
```
```{r, warning=FALSE}
# fish_data %>%
# complete(Timepoint, Diet, fill = list(BW = NA, Length = NA)) %>%
# group_by(Timepoint, Diet) %>%
# summarise(across(c(Heart_Score, Fat_Score,cataract_V,cataract_H ), mean, na.rm = TRUE), .groups = 'drop') %>%
# mutate(across(where(is.numeric), round, 2)) %>%
# DT::datatable()
library(dplyr)
library(tidyr)
fish_data %>%
complete(Timepoint, Diet, fill = list(BW = NA, Length = NA, GW = NA, CF = NA, HSI = NA, CSI = NA)) %>%
group_by(Timepoint, Diet) %>%
summarise(
Mean_BW = mean(BW, na.rm = TRUE),
Mean_Length = mean(Length, na.rm = TRUE),
Mean_GW = mean(GW, na.rm = TRUE),
Mean_CF = mean(CF, na.rm = TRUE),
Mean_HSI = mean(HSI, na.rm = TRUE),
Mean_CSI = mean(CSI, na.rm = TRUE),
Mean_Heart_W = mean(Heart_W, na.rm = TRUE),
Mean_Liver_W = mean(Liver_W, na.rm = TRUE),
SEM_BW = sd(BW, na.rm = TRUE) / sqrt(sum(!is.na(BW))),
SEM_Length = sd(Length, na.rm = TRUE) / sqrt(sum(!is.na(Length))),
SEM_GW = sd(GW, na.rm = TRUE) / sqrt(sum(!is.na(GW))),
SEM_CF = sd(CF, na.rm = TRUE) / sqrt(sum(!is.na(CF))),
SEM_HSI = sd(HSI, na.rm = TRUE) / sqrt(sum(!is.na(HSI))),
SEM_CSI = sd(CSI, na.rm = TRUE) / sqrt(sum(!is.na(CSI))),
SEM_Heart_W = sd(Heart_W, na.rm = TRUE) / sqrt(sum(!is.na(Heart_W))),
SEM_Liver_W = sd(Liver_W, na.rm = TRUE) / sqrt(sum(!is.na(Liver_W))),
.groups = 'drop'
) %>%
mutate(
across(where(is.numeric), ~round(., 2)),
across(starts_with("Mean_"), ~paste(., "±", get(paste0("SEM_", sub("Mean_", "", cur_column())))))
) %>%
filter(complete.cases(.)) %>%
select(Timepoint, Diet, starts_with("Mean_"), -starts_with("SEM_")) %>%
rename_with(~sub("Mean_", "", .), starts_with("Mean_")) %>%
t() %>%
DT::datatable()
```
# Metagenomics
Import data and clean the taxonomy
```{r, warning=FALSE, message=FALSE}
base_url <- "https://raw.githubusercontent.com/shashank-KU/ImprovaFish-MDF-Effects/main/"
# Specify the path to your RDS file on GitHub
rds_file <- "Metagenomics/low_dosage_trial/metagenomics_low_MDF.rdata"
rds_url <- paste0(base_url, rds_file)
# Read the RDS file
all <- readRDS(url(rds_url))
tax <- data.frame(tax_table(all), stringsAsFactors = FALSE)
tax <- tax[,1:7] # No info in col 8-15
# Set informative colnames
colnames(tax) <- c("Kingdom", "Phylum","Class","Order","Family","Genus", "Species")
tax.clean <- data.frame(row.names = row.names(tax),
Kingdom = str_replace(tax[,1], "d__",""),
Phylum = str_replace(tax[,2], "p__",""),
Class = str_replace(tax[,3], "c__",""),
Order = str_replace(tax[,4], "o__",""),
Family = str_replace(tax[,5], "f__",""),
Genus = str_replace(tax[,6], "g__",""),
Species = str_replace(tax[,7], "s__",""),
stringsAsFactors = FALSE)
tax.clean[is.na(tax.clean)] <- ""
# - Clean rank by rank
# Kingdom - Remove the unassigned completely
# Phylum
# Class
# Remove extra info about origin from some bacteria
# Remove all fields that contain "uncultured", "Unknown" or "Ambigious"
bad <- c("Ambiguous_taxa","uncultured", "Subgroup_21")
tax.clean[tax.clean$Class %in% bad,3:7] <- ""
# Order
bad <- c("0319-6G20","1-20","11-24", "ADurb.Bin180","D8A-2", "Group_1.1c", "JGI_0000069-P22","Marine_Group_II",
"Pla3_lineage","Run-SP154",
"Ambiguous_taxa", "uncultured", "UBA10353_marine_group",
"Subgroup_17", "SAR86_clade", "SAR11_clade", "SAR202_clade")
tax.clean[tax.clean$Order %in% bad,4:7] <- ""
# Family
bad <- c("Ambiguous_taxa","11-24","67-14", "uncultured", "SAR116_clade", "Run-SP154",
"Marine_Group_II", "env.OPS_17", "SAR116_clade", "S085", "S-70", "NS9_marine_group", "Mitochondria")
tax.clean[tax.clean$Family %in% bad,5:7] <- ""
# Genus
bad <- c("Ambiguous_taxa","Unknown_Family","uncultured","Subgroup_10", "1174-901-12", "67-14")
tax.clean[tax.clean$Genus %in% bad,6:7] <- ""
# Species
bad <- c("Ambiguous_taxa","marine_metagenome","low_GC","wastewater_metagenome","unidentified",
"uncultured_synthetic", "uncultured_organism")
tax.clean[tax.clean$Species %in% bad,6:7] <- ""
#tax.clean[grepl("uncultured", tax.clean$Species),"Species"] <- ""
#tax.clean[grepl("unidentified", tax.clean$Species),"Species"] <- ""
# Remove remove ".", change "-" and " " to "_"
for (i in 1:ncol(tax.clean)){
tax.clean[,i] <- str_replace_all(tax.clean[,i], "[.]","")
tax.clean[,i] <- str_replace_all(tax.clean[,i], "[(]","")
tax.clean[,i] <- str_replace_all(tax.clean[,i], "[)]","")
tax.clean[,i] <- str_replace_all(tax.clean[,i], "-","_")
tax.clean[,i] <- str_replace_all(tax.clean[,i], " ","_")
}
for (i in 1:7){ tax.clean[,i] <- as.character(tax.clean[,i])}
# File holes in the tax table
for (i in 1:nrow(tax.clean)){
# Fill in missing taxonomy
if (tax.clean[i,2] == ""){
kingdom <- paste("Kingdom_", tax.clean[i,1], sep = "")
tax.clean[i, 2:7] <- kingdom
} else if (tax.clean[i,3] == ""){
phylum <- paste("Phylum_", tax.clean[i,2], sep = "")
tax.clean[i, 3:7] <- phylum
} else if (tax.clean[i,4] == ""){
class <- paste("Class_", tax.clean[i,3], sep = "")
tax.clean[i, 4:7] <- class
} else if (tax.clean[i,5] == ""){
order <- paste("Order_", tax.clean[i,4], sep = "")
tax.clean[i, 5:7] <- order
} else if (tax.clean[i,6] == ""){
family <- paste("Family_", tax.clean[i,5], sep = "")
tax.clean[i, 6:7] <- family
} else if (tax.clean[i,7] == ""){
tax.clean$Species[i] <- paste("Genus_",tax.clean$Genus[i], sep = "_")
}
}
rm(bad, class, family, i, kingdom,new,order,phylum,uncul)
tax_table(all) <- as.matrix(tax.clean)
all
all.clean <- subset_taxa(all,
Kingdom != "Unassigned" &
Kingdom != "Archaea" &
Kingdom != "Eukaryota" &
Family != "Mitochondria" &
Order != "Chloroplast" &
Genus != "Ralstonia"
) %>%
prune_taxa(taxa_sums(.) > 0, .)
all.clean
```
Rarefaction plot
```{r eecho=T, results='hide'}
sample_data(all.clean)$New_Diet <- factor(sample_data(all.clean)$New_Diet, levels=c('ext-ctrl', 'CTR', 'MC1', 'MC2', 'MN3'))
annotation_colors <- list(
samplingTime = c(`T0`="gray" ,`T1` = "#FF7F00", `T2` = "#FFD92F", `T3`="#F781BF"),
New_Diet = c(`ext-ctrl`="#FDC086", `CTR`="#e41a1cff", `MC1`="#377EB8", `MC2`="#4DAF4A", `MN3`="#984EA3"))
p <- ggrare(all.clean, step = 1000,
color = "samplingTime",
se = F,
parallel = TRUE,
plot = FALSE)
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
p <- p + theme_bw() +
scale_fill_manual(values =annotation_colors$samplingTime) +
scale_colour_manual( values = annotation_colors$samplingTime) +
facet_wrap(~New_Diet) +
guides(color = guide_legend(title = "Life stage"))
```
Alpha and Beta diversity
```{r, message=FALSE, warning=FALSE, fig.align='center', fig.width=14, fig.height=10}
shannon.div <- estimate_richness(all.clean, measures = c("Shannon", "Observed"))
sampledata1<- data.frame(sample_data(all.clean))
row.names(shannon.div) <- gsub("[.]","-", row.names(shannon.div))
sampleData <- merge(sampledata1, shannon.div, by = 0 , all = TRUE)
my_comparisons <- list( c("ext-ctrl", "MC1"), c("ext-ctrl", "MC2"), c("ext-ctrl", "MN3"), c("CTR", "MC1"), c("CTR", "MC2"), c("CTR", "MN3"))
p1 <- ggboxplot(sampleData, x = "New_Diet", y = "Observed",
color = "black", palette = "jco", legend = "none", outlier.shape = NA)+
stat_compare_means(comparisons = my_comparisons) +
stat_compare_means(label.y = 350) +
ggplot2::geom_jitter(
mapping = aes_string(fill="New_Diet"),
position = ggplot2::position_jitter(seed=123),
shape = 21,
color = "black",
size = 2,
alpha = 0.8
) +
theme_bw() +
theme(legend.position="none",axis.title.x=element_blank()) +
scale_fill_manual(values = annotation_colors$New_Diet) +
scale_colour_manual( values = annotation_colors$New_Diet) +
labs(y= "Observed richness (ASVs)")
set.seed(1)
PCoA_bray <- ordinate(physeq = all.clean, method = "PCoA", distance = "bray")
PCoA_bray_plot<- plot_ordination(
physeq = all.clean,
ordination = PCoA_bray,
color = "New_Diet"
) +
geom_point(aes(fill ="New_Diet" ), size =2) +
geom_point(shape = 1, size = 2,colour = "black") +
theme_bw() +
xlab("PCoA 1 [18.2 %]") + ylab("PCoA 2 [8.6 %]") +
stat_ellipse() +
scale_fill_manual(values = annotation_colors$New_Diet) +
scale_colour_manual( values = annotation_colors$New_Diet) +
guides(color = guide_legend(title = "Diet"))
bottom_row <- plot_grid(p1, PCoA_bray_plot, labels = c('B', 'C'), align = 'h', rel_widths = c(1, 1.3))
```
Supplementary Fig. S4
```{r, message=FALSE, warning=FALSE, fig.align='center', fig.width=14, fig.height=10}
plot_grid(p, bottom_row, labels = c('A', ''), ncol = 1, rel_heights = c(1, 1.2))
```
Computes the Bray-Curtis distance between all the samples in the dataset "all.clean" and tests for differences between samples in the variable "New_Diet" using the Adonis2 function. The Adonis2 function was run with 9999 permutations.
```{r}
sampledf <- data.frame(sample_data(all.clean))
bcdist <- phyloseq::distance(all.clean, method="bray",normalized=TRUE)
adonis2(bcdist ~ New_Diet,
data = sampledf, permutations = 9999)
```
Contamination removal
```{r, message=FALSE}
df <- as.data.frame(sample_data(all.clean)) # Put sample_data into a ggplot-friendly data.frame
df$Sample_or_Control <- ifelse( df$New_Diet %in% c("ext-ctrl"), "Control_Sample", "True_Sample")
sample_data(all.clean) <- df
df$LibrarySize <- sample_sums(all.clean)
df <- df[order(df$LibrarySize),]
df$Index <- seq(nrow(df))
sample_data(all.clean)$is.neg <- sample_data(all.clean)$Sample_or_Control == "Control_Sample"
contamdf.prev05 <- isContaminant(all.clean, method="prevalence", neg="is.neg", threshold=0.5)
paste("The number of contamination found is",table(contamdf.prev05$contaminant)[2])
all.noncontam <- prune_taxa(!contamdf.prev05$contaminant, all.clean)
psdata <- subset_samples(all.noncontam, Sample_or_Control=="True_Sample")
psdata <- prune_taxa(taxa_sums(psdata) > 0, psdata)
psdata
```
Inspect the number of reads per sample and compare to rarefaction curves
```{r}
psdata.p <- prune_taxa(taxa_sums(psdata) >0, psdata)
psdata.p
```
Taxonomic classification- Phylum level taxonomic distribution. Bars report the mean abundance for each individual sample.
```{r warning=FALSE, message=FALSE, fig.align='center'}
psdata.r<- transform_sample_counts(psdata.p, function(x) x / sum(x) )
Final.RNA <- aggregate_rare(psdata.r, level = "Phylum", detection = 1/100, prevalence = 20/100)
getPalette = colorRampPalette(brewer.pal(10, "Dark2"))
PhylaPalette = getPalette(10)
Final.RNA_phylum_plot<- plot_composition(Final.RNA, sample.sort = "Proteobacteria",otu.sort = "abundance", verbose = TRUE)
Final.RNA_phylum_plot <- Final.RNA_phylum_plot +
theme_bw() +
theme(axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
scale_fill_manual(values = PhylaPalette)
Final.RNA_phylum_plot
```
```{r, message=FALSE}
#Bacterial Community Composition for Manuscript
Final.seq.melt.RNA <- psmelt(tax_glom(psdata.r, "Species"))
tax_ranks <- c("Phylum", "Class", "Order", "Family", "Genus", "Species")
for (rank in tax_ranks) {
n_unique <- length(unique(Final.seq.melt.RNA[[rank]]))
message(paste(rank, ": ", n_unique, sep = ""))
}
```
```{r}
paste("number of unique Phylum is", table(grepl("Kingdom", unique(Final.seq.melt.RNA$Phylum)))[1])
paste("number of unique Class is", table(grepl("Kingdom|Phylum", unique(Final.seq.melt.RNA$Class)))[1])
paste("number of unique Order is", table(grepl("Kingdom|Class|Phylum", unique(Final.seq.melt.RNA$Order)))[1])
paste("number of unique Family is", table(grepl("Kingdom|Order|Class|Phylum", unique(Final.seq.melt.RNA$Family)))[1])
paste("number of unique Genus is", table(grepl("Kingdom|Family|Order|Class|Phylum", unique(Final.seq.melt.RNA$Genus)))[1])
paste("number of unique Species is", table(grepl("Kingdom|Family|Order|Class|Phylum|Genus", unique(Final.seq.melt.RNA$Species)))[1])
```
```{r, echo=FALSE}
aggregate_top_taxa <- function (x, top, level) {
.Deprecated("aggregate_rare",
"The microbiome::aggregate_top_taxa function is deprecated.")
x <- aggregate_taxa(x, level)
tops <- top_taxa(x, top)
tax <- tax_table(x)
inds <- which(!rownames(tax) %in% tops)
tax[inds, level] <- "Other"
tax_table(x) <- tax
tt <- tax_table(x)[, level]
tax_table(x) <- tax_table(tt)
aggregate_taxa(x, level)
}
```
Supplementary table for the manuscript
```{r, message=FALSE, warning=FALSE}
Phylum_df <- summaryBy(Abundance~Phylum, data=Final.seq.melt.RNA, FUN=sum)
Phylum_df$Percent <- round(Phylum_df$Abundance.sum/sum(Phylum_df$Abundance.sum)*100, 4)
Phylum_df <- plyr::arrange(Phylum_df, plyr::desc(Percent))
Phylum_df$PercentageRound <- round(Phylum_df$Percent, digits = 2)
DT::datatable(Phylum_df)
class_df <- summaryBy(Abundance~Class, data=Final.seq.melt.RNA, FUN=sum)
class_df$Percent <- round(class_df$Abundance.sum/sum(class_df$Abundance.sum)*100, 4)
class_df <- plyr::arrange(class_df, plyr::desc(Percent))
class_df$PercentageRound <- round(class_df$Percent, digits = 2)
DT::datatable(class_df)
order_df <- summaryBy(Abundance~Order, data=Final.seq.melt.RNA, FUN=sum)
order_df$Percent <- round(order_df$Abundance.sum/sum(order_df$Abundance.sum)*100, 4)
order_df <- plyr::arrange(order_df, plyr::desc(Percent))
order_df$PercentageRound <- round(order_df$Percent, digits = 2)
DT::datatable(order_df)
family_df <- summaryBy(Abundance~Family, data=Final.seq.melt.RNA, FUN=sum)
family_df$Percent <- round(family_df$Abundance.sum/sum(family_df$Abundance.sum)*100, 4)
family_df <- plyr::arrange(family_df, plyr::desc(Percent))
family_df$PercentageRound <- round(family_df$Percent, digits = 2)
DT::datatable(family_df)
genus_df <- summaryBy(Abundance~Genus, data=Final.seq.melt.RNA, FUN=sum)
genus_df$Percent <- round(genus_df$Abundance.sum/sum(genus_df$Abundance.sum)*100, 4)
genus_df <- plyr::arrange(genus_df, plyr::desc(Percent))
genus_df$Round <- round(genus_df$Percent, digits = 2)
DT::datatable(genus_df)
```
Supplementary Figure
```{r, message=FALSE, warning=FALSE, fig.align='center', fig.height=12, fig.width=18}
FigureS4_1 <- transform_sample_counts(psdata.r, function(x) x / sum(x) )
FigureS4_1 <- aggregate_top_taxa(FigureS4_1, level = "Phylum", top = 10)
getPalette = colorRampPalette(brewer.pal(10, "Dark2"))
PhylaPalette = getPalette(10)
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
FigureS4_1_plot<- plot_composition(FigureS4_1, sample.sort = "Proteobacteria",otu.sort = "abundance", verbose = TRUE)
FigureS4_1_plot <- FigureS4_1_plot +
theme_bw() +
#theme(axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
scale_fill_manual(values = cols) + theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))+ ylab("Relative abundance")+ labs(fill = "Taxa") +
theme(axis.text.x=element_blank(),
axis.ticks.x=element_blank())
FigureS4_1 <- transform_sample_counts(psdata.r, function(x) x / sum(x) )
FigureS4_2 <- aggregate_top_taxa(FigureS4_1, level = "Class", top = 10)
getPalette = colorRampPalette(brewer.pal(10, "Dark2"))
PhylaPalette = getPalette(10)
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
FigureS4_2_plot<- plot_composition(FigureS4_2, sample.sort = "Gammaproteobacteria",otu.sort = "abundance", verbose = TRUE)
FigureS4_2_plot <- FigureS4_2_plot +
theme_bw() +
#theme(axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
scale_fill_manual(values = cols) + theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))+ ylab("Relative abundance")+ labs(fill = "Taxa") +
theme(axis.text.x=element_blank(),
axis.ticks.x=element_blank())
FigureS4_1 <- transform_sample_counts(psdata.r, function(x) x / sum(x) )
FigureS4_3 <- aggregate_top_taxa(FigureS4_1, level = "Family", top = 10)
getPalette = colorRampPalette(brewer.pal(10, "Dark2"))
PhylaPalette = getPalette(10)
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
FigureS4_3_plot<- plot_composition(FigureS4_3, sample.sort = "Other",otu.sort = "abundance", verbose = TRUE)
FigureS4_3_plot <- FigureS4_3_plot +
theme_bw() +
#theme(axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
scale_fill_manual(values = cols) + theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) + ylab("Relative abundance")+ labs(fill = "Taxa") +
theme(axis.text.x=element_blank(),
axis.ticks.x=element_blank())
FigureS4_1 <- transform_sample_counts(psdata.r, function(x) x / sum(x) )
FigureS4_4 <- aggregate_top_taxa(FigureS4_1, level = "Genus", top = 10)
getPalette = colorRampPalette(brewer.pal(10, "Dark2"))
PhylaPalette = getPalette(10)
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
FigureS4_4_plot<- plot_composition(FigureS4_4, sample.sort = "Other",otu.sort = "abundance", verbose = TRUE)
FigureS4_4_plot <- FigureS4_4_plot +
theme_bw() +
#theme(axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
scale_fill_manual(values = cols) + theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))+ ylab("Relative abundance")+ labs(fill = "Taxa") +
theme(axis.text.x=element_blank(),
axis.ticks.x=element_blank())
plot_grid(FigureS4_1_plot, FigureS4_3_plot, FigureS4_2_plot, FigureS4_4_plot, labels = 'AUTO', ncol = 2, rel_widths = c(1,1.1,1,1.1))
```
Alpha and Beta diversity
```{r, message=FALSE, warning=FALSE}
shannon.div <- estimate_richness(psdata.p, measures = c("Shannon", "Observed"))
sampledata1<- data.frame(sample_data(psdata.p))
row.names(shannon.div) <- gsub("[.]","-", row.names(shannon.div))
sampleData <- merge(sampledata1, shannon.div, by = 0 , all = TRUE)
sampleData <- sampleData %>%
mutate(New_Diet = case_when(
samplingTime == "T0" ~ "start",
TRUE ~ New_Diet
))
sampleData$New_Diet <- factor(sampleData$New_Diet, levels=c( 'CTR', 'MC1', 'MC2', 'MN3'))
sampleData$samplingTime <- factor(sampleData$samplingTime, levels=c('T0', 'T1', 'T2', 'T3'))
my_comparisons <- list( c("CTR", "MC1"), c("CTR", "MC2"), c("CTR", "MN3"),
c("MC1", "MC2"), c("MC1", "MN3"), c("MC2", "MN3") )
plot1 <- ggboxplot(sampleData, x = "New_Diet", y = "Shannon",
color = "black", palette = "jco", legend = "none", outlier.shape = NA)+
geom_pwc(comparisons = my_comparisons, hide.ns = TRUE) +
#stat_compare_means(label.y = 5) +
ggplot2::geom_jitter(
mapping = aes_string(fill="New_Diet"),
position = ggplot2::position_jitter(seed=123),
shape = 21,
color = "black",
size = 2,
alpha = 0.8
) +
theme_bw() + scale_y_continuous("Shannon index") +
theme(legend.position="none",axis.title.x=element_blank()) +
scale_fill_manual(values = annotation_colors$New_Diet) +
scale_colour_manual( values = annotation_colors$New_Diet) +
facet_grid(. ~ samplingTime, scales="free_x", space = "free_x")
my_comparisons <- list(c("T0", "T3"), c("T1", "T3"), c("T2", "T3"))
# Assuming your dataframe is named sampleData
sampleData$New_Diet[sampleData$New_Diet == "start"] <- "CTR"
plot2_x <- ggboxplot(sampleData, x = "samplingTime", y = "Shannon",
color = "black", palette = "jco", legend = "none", outlier.shape = NA)+
stat_compare_means(comparisons = my_comparisons) +
#stat_compare_means(label.y = 5) +
ggplot2::geom_jitter(
mapping = aes_string(fill="samplingTime"),
position = ggplot2::position_jitter(seed=123),
shape = 21,
color = "black",
size = 2,
alpha = 0.8
) +
theme_bw() +
theme(legend.position="none",axis.title.x=element_blank()) +
scale_fill_manual(values = annotation_colors$samplingTime) +
scale_colour_manual( values = annotation_colors$samplingTime) +
facet_grid(. ~ New_Diet) +
labs(y= "Shannon index") + ylim(c(1.5, 5.2))
```
```{r, message=FALSE, warning=FALSE, fig.align='center', fig.width=14, fig.height=10}
set.seed(1)
PCoA_bray <- ordinate(physeq = psdata.p, method = "PCoA", distance = "bray")
plot3 <- plot_ordination(
physeq = psdata.p,
ordination = PCoA_bray,
color = "samplingTime"
) +
geom_point(aes(fill ="samplingTime" ), size =2) +
geom_point(shape = 1, size = 2,colour = "black") +
theme_bw() + ggtitle("PCoA Plot - Bray") +
theme(plot.title = element_text(hjust = 0.5)) +
ggtitle("Hindgut: Microbiome") +
xlab("PCoA 1 [21.1 %]") + ylab("PCoA 2 [7.7 %]") +
stat_ellipse() +
scale_fill_manual(values = annotation_colors$samplingTime) +
scale_colour_manual( values = annotation_colors$samplingTime) +
guides(color = guide_legend(title = "Time"))
# Run adonis test
sampledf <- data.frame(sample_data(psdata.p))
bcdist <- phyloseq::distance(psdata.p, method="bray",normalized=TRUE)
adonis2(bcdist ~ samplingTime, data = sampledf, permutations = 9999)
set.seed(1)
plot4 <- plot_ordination(
physeq = psdata.p,
ordination = PCoA_bray,
color = "New_Diet"
) +
geom_point(aes(fill ="New_Diet" ), size =2) +
geom_point(shape = 1, size = 2,colour = "black") +
theme_bw() + ggtitle("PCoA Plot - Bray") +
theme(plot.title = element_text(hjust = 0.5)) +
ggtitle("Hindgut: Microbiome") +
xlab("PCoA 1 [21.1 %]") + ylab("PCoA 2 [7.7 %]") +
stat_ellipse() +
scale_fill_manual(values = annotation_colors$New_Diet) +
scale_colour_manual( values = annotation_colors$New_Diet) +
guides(color = guide_legend(title = "Diet"))
# Run adonis test
sampledf <- data.frame(sample_data(psdata.p))
adonis2(bcdist ~ New_Diet, data = sampledf, permutations = 9999)
# plot5 <- plot_grid(plot1, plot2, labels = c('', ''), label_size = 12, ncol = 1, align = "hv")
# plot6 <- plot_grid(plot3, plot4, labels = c('', ''), label_size = 12, ncol = 1, align = "hv")
# plot_grid(plot5, plot6, ncol = 2, rel_widths = c(1.5, 1))
```
```{r, echo=FALSE }
#' Relative Abundance Plot
#'
#' For creating nice microbiome plots
#'
#' @param phylo_ob Phyloseq object with metadata in sample_data.
#' @param predictor Predictor of interest for statistics/plotting in sample_data.
#' @param type Taxonomic rank from tax_table, case insensitive; default is "genus".
#' @param relative_abun Use relative abundances, else absolute; default is TRUE.
#' @param id Define id variable for mixed models.
#' @param xlabs X-axis label
#' @param ylabs Y-axis label
#' @param main Title of plot
#' @param violin Use geom_violin for plotting, else boxplot; default is TRUE.
#' @param violin_scale Scale option for geom_violin; default is "width".
#' @param legend_title Legend title; default is name of predictor.
#' @param N_taxa Number of taxa to be plotted; default is 15.
#' @param By_median Order plot by median abundances, else mean abundances; default is TRUE.
#' @param no_other_type Taxa in lower abundances than top N_taxa, are grouped as "other", this will remove this group from the plot; default is FALSE.
#' @param legend_names Define variable names for legend text.
#' @param Time Time variable name for longitudinal datasets.
#' @param Timepoint Value in variable @Time to select.
#' @param Strata Name of variable for stratification;
#' @param Strata_val Value in variable @Strata to keep; default is 1.
#' @param no_legends Removes legend; default is FALSE.
#' @param no_names Removes taxa names; default is FALSE.
#' @param italic_names Taxa names will be in italic e.g. usable for family, genus, species levels; default is TRUE
#' @param Only_sig Only keep significant taxa; default is FALSE.
#' @param log Present plot on a log scale; default is TRUE.
#' @param log_max Maximum value of log-axis options:1, 10, 100; default is 100.
#' @param stat_out Outputs a data.frame with statistics to Global environment; default is FALSE.
#' @param p_val Displays p-values on plot; default is TRUE.
#' @param p_stars Shows stars instead of p-values; default is FALSE.
#' @param stats Select type of statistical test; options: "non-parametric", "parametric", "mixed", "mgs_feature"; default is "non-parametric".
#' @param p_adjust adjust p-values; default is "FALSE.
#' @param p_adjust_method options: "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr"; default is "fdr".
#' @param p_adjust_full correction applied for all taxa in the dataset; default is FALSE.
#' @param colors define list of colors for plot. If not color brewer will be used; default is NULL.
#' @param color_by define taxonomic rank to color by; default is NULL.
#' @param order Order by abundance, else alphabetically; default is TRUE.
#' @param reverse Flip taxa order; default is FALSE.
#' @param list_taxa A list of specific taxa names to be analyzed; default is NULL.
#' @param select_taxa Choose all taxa from one or more taxonomic variables, eg. "Staphylococcus" or "Staph" or "coccus" or c("staph",bifido"); default is NULL.
#' @param select_type Taxonomic rank of the @select_taxa; default is "genus".
#' @param bar_chart Choose to make bar chart; default is FALSE.
#' @param bar_chart_stacked Produce stacked bar chart; default is FALSE
#' @param percent Print percentages on bar chart; default is FALSE.
#' @param facet_wrap Facet wrap chart by variable; eg. Time; default is NULL.
#' @param facet_label Facet wrap labels; default is NULL.
#' @param facet_n Show n for each facet; default is TRUE.
#' @param order_by Choose variable to order the selected taxa by; eg. Time; default is Time.
#' @param order_val Choose value for @order_by; default is NULL.
#' @param text_angle_x Choose value for rotation of axis-text; default is 0.
#'
#' @import ggplot2 phyloseq metagenomeSeq dplyr tidyr RColorBrewer lmerTest
#' @return A ggplot
#' @export
rabutable <- function(phylo_ob,
predictor="none",
type="genus",
relative_abun=TRUE,
id=NULL,
xlabs = "Relative abundance (%)",
ylabs = "Average relative abundance",
main = "Relative abundance plot",
violin=TRUE,
violin_scale = "width",
legend_title=predictor,
N_taxa=NULL,
By_median=TRUE,
no_other_type=FALSE,
legend_names=NULL,
Time="Time",
Timepoint=NULL,
Strata=NULL,
Strata_val="1",
no_legends = FALSE,
no_names=FALSE,
italic_names=TRUE,
Only_sig=FALSE,
log=TRUE,
log_max=100,
stat_out=FALSE,
p_val = TRUE,
p_stars=FALSE,
stats="non-parametric",
p_adjust=FALSE,
p_adjust_method="fdr",
p_adjust_full=FALSE,
colors=NULL,
color_by=NULL,
order=TRUE,
reverse=FALSE,
list_taxa=NULL,
select_taxa=NULL,
select_type="genus",
bar_chart=FALSE,
bar_chart_stacked=FALSE,
facet_wrap=NULL,
facet_label=NULL,
facet_n=TRUE,
percent=FALSE,
order_by="Time",
order_val=NULL,
text_angle_x=0)
{
if(!is.null(list_taxa) & is.null(N_taxa)) N_taxa = length(list_taxa)
if(is.null(N_taxa) & is.null(list_taxa)) N_taxa=15
options(dplyr.summarise.inform = FALSE)
if(bar_chart_stacked==TRUE) {
bar_chart=TRUE
p_val=FALSE
}
if(predictor=="none") {
sample_data(phylo_ob)$none <- "All samples"
p_val=FALSE
if(bar_chart_stacked==FALSE & is.null(color_by)) no_legends = TRUE
}
phylo_ob <- prune_samples(sample_sums(phylo_ob)>0,phylo_ob) #removes empty samples;
otu_mat <- as(otu_table(phylo_ob), "matrix")
if(taxa_are_rows(phylo_ob)) otu_mat <- t(otu_mat)
if(!is.null(facet_wrap)) index <- !is.na(get_variable(phylo_ob, predictor)) & !is.na(get_variable(phylo_ob, facet_wrap))
else index <- !is.na(get_variable(phylo_ob, predictor))
if(length(unique(index)) !=1) message(paste(length(which(index==F)), "samples have been removed from full dataset (predictor/facet_wrap NAs)"))
otu_mat <- otu_mat[index,]
otu_mat <- otu_mat[,colSums(otu_mat)>0] #removes empty OTUs;
OTU_index <- colnames(otu_mat)
tax <- as(tax_table(phylo_ob), "matrix") %>% data.frame(stringsAsFactors=FALSE)
tax <- tax[rownames(tax) %in% OTU_index,]
tax[is.na(tax)] <- "unclassified"
tax[tax==""] <- "unclassified"
names(tax) <- tolower(names(tax))
type <- tolower(type)
if(!is.null(select_type)) select_type <- tolower(select_type)
tax$OTU <- rownames(tax)
samp <- data.frame(sample_data(phylo_ob), stringsAsFactors=TRUE)
samp <- samp[index,]
if(is.null(facet_wrap)) samp$wrap <- ""
if(!is.null(facet_wrap)) samp$wrap <- samp[,facet_wrap]
if(!is.null(Timepoint)){
index <- rownames(samp[(samp[,Time] ==Timepoint),])
otu_mat <- otu_mat[rownames(otu_mat) %in% index,]
otu_mat <- otu_mat[,colSums(otu_mat)>0] #removes empty OTUs;
OTU_index <- colnames(otu_mat)
tax <- tax[rownames(tax) %in% OTU_index,]
samp <- samp[rownames(samp) %in% index,]
}
list <-as.character(tax[,type])
unique_tax <- unique(list)
abund <- as.data.frame(matrix(rep(0,(length(unique_tax)*nrow(otu_mat))),ncol=length(unique_tax)))
row.names(abund) <- row.names(otu_mat)
names(abund) <- unique_tax
for(i in names(abund)){
if(is.array(otu_mat[,list==i])) abund[,i] <- rowSums(otu_mat[,list== i])
else abund[,i] <- otu_mat[,list== i]
}
abund_org <- abund
if(relative_abun==TRUE) abund <- apply(abund,1,function(x) x/sum(x)) %>% t %>% as.data.frame()
abund_all <- abund
if (is.null(list_taxa) & !is.null(select_taxa)) {
list_taxa <- NULL
for(i in 1:length(select_taxa)){
list_taxa <- c(list_taxa,(as.character(unique(tax[grep(select_taxa[[i]],tax[,select_type],ignore.case=TRUE),type]))))
}
}
if (!is.null(list_taxa)) {
if (is.null(N_taxa)) N_taxa <- length(list_taxa)
abund <- abund[,colnames(abund) %in% list_taxa, drop = FALSE]
unique_tax <- names(abund)
}
if(length(abund)>1){
index <- !is.na(rownames(samp))
if (!is.null(order_val)) index <- samp[,order_by] ==order_val
abund <- abund[,order(-colSums(abund[index,]))]
if (By_median) abund <- abund[,order(-apply(abund[index,], 2, median))]
if("unclassified" %in% unique_tax) abund <- abund[c(setdiff(names(abund), "unclassified"),"unclassified")] #Move unclassified to end
if(N_taxa<length(unique_tax)) abund <- abund[-(length(unique_tax)-(length(unique_tax)-N_taxa)+1):-length(unique_tax)]
if(no_other_type==FALSE) abund[, paste("Other",type)] <- rowSums(abund_all[,!names(abund_all) %in% names(abund)])
}
index <- !is.na(rownames(samp))
if(!is.null(Strata)) index <- samp[,Strata]==Strata_val
samp2 <- samp %>% filter(index)
if(p_val==TRUE & (bar_chart==FALSE | (bar_chart==TRUE & bar_chart_stacked==FALSE))){
if(p_adjust_full ==TRUE | stats=="mgs_feature"){
abund2 <- abund_org %>% filter(index)
if(relative_abun==TRUE & stats!="mgs_feature") abund2 <- apply(abund2,1,function(x) x/sum(x)) %>% t %>% as.data.frame()
}
else abund2 <- abund %>% filter(index)
if(stats=="mgs_feature" & length(levels(factor(samp2[,predictor])))>2){
stats="non-parametric"
message("MGS not available for >2 predictors, switching to non-parametric")
}
if(stats=="mixed" & is.null(id)){
stats="non-parametric"
message("No id variable for mixed model, switching to non-parametric")
}
if(stats=="mixed"){
message("Mixed model statistics")
pred <- samp2[,predictor]
id <- samp2[,id]
if(!is.null(facet_wrap)) {
message(paste0("Using ",facet_wrap," as mixed model group variable"))
facet <- samp2[,"wrap"]
pval <- cbind(abund2,pred,id,facet) %>% as_tibble() %>%
gather(variable, value,-"pred",-"id",-"facet") %>%
group_by(variable) %>%
summarize(pval = lmerTest::lmer(value ~ pred + factor(facet) + (1 | id)) %>% anova %>% filter(row_number()==1) %>% .$'Pr(>F)', .groups = 'drop')
}
else {
message(paste0("No group variable defined for mixed model in facet_wrap"))
pval <- cbind(abund2,pred,id) %>% as_tibble() %>%
gather(variable, value,-"pred",-"id") %>%
group_by(variable) %>%
summarize(pval = lmerTest::lmer(value ~ pred + (1 | id)) %>% anova %>% .$'Pr(>F)', .groups = 'drop')
}
pval <- pval %>% mutate(wrap="Mixed",p_adjust=p.adjust(pval, p_adjust_method))
pval$wrap <- factor(pval$wrap,levels=c(levels(factor(samp2[,facet_wrap])),"Mixed"))
}
else {
pval <- data.frame()
for (i in 1:length(unique(samp2$wrap))){
index <- samp2$wrap==unique(samp2$wrap)[[i]]
abund3 <- abund2 %>% filter(index)
pred <- samp2[index,predictor]
# test with featureModel
if(stats=="mgs_feature"){
mgs <- metagenomeSeq::newMRexperiment(counts = t(abund3))
mgsp <- metagenomeSeq::cumNormStat(mgs)
mgs <- metagenomeSeq::cumNorm(mgs, mgsp)
mod <- model.matrix(~as.numeric(pred == unique(pred)[1]))
if(length(unique(samp2$wrap))>1) message(paste0("MGS FeatureModel for facet_wrap = ",unique(samp2$wrap)[[i]]))
else message("MGS FeatureModel")
mgsfit <- metagenomeSeq::fitFeatureModel(obj=mgs,mod=mod)
pval_tmp <- data.frame(variable=mgsfit$taxa,pval=mgsfit$pvalues)
}
if(stats=="non-parametric"){ #Kruskal-Wallis
if(i==1) message("Non-parametric statistics")
pval_tmp <- cbind(abund3,pred) %>% as_tibble() %>%
gather(variable, value,-"pred") %>%
group_by(variable) %>%
summarize(pval = kruskal.test(value ~ pred)$p.value, .groups = 'drop')
}
if(stats=="parametric"){
if(i==1) message("Parametric statistics")
pval_tmp <- cbind(abund3,pred) %>% as_tibble() %>%
gather(variable, value,-"pred") %>%
group_by(variable) %>%
summarize(pval = oneway.test(value ~ pred)$p.value, .groups = 'drop')
}
pval_tmp <- pval_tmp %>%
mutate(wrap=unique(samp2$wrap)[[i]],p_adjust=p.adjust(pval, p_adjust_method))
pval <- rbind(pval,pval_tmp)
}
}
if(p_adjust) message(paste(p_adjust_method,"correction applied for",length(unique(pval$variable)),"taxa"))
}
bacteria <- rev(names(abund))
subset <- cbind(samp[!names(samp) %in% bacteria], abund) #fjerner evt eksisterende navne fra dataset og merger;
subset$predictor2 <- as.factor(subset[,predictor])
subset$ID <- rownames(subset)
if(!is.null(Strata)) subset[,Strata] <- as.factor(subset[,Strata])
if(!is.null(facet_wrap)){
subset$wrap <- as.factor(subset[,facet_wrap])
if(!is.null(Strata))
molten <- subset[,c("ID",paste(bacteria),"predictor2",Strata,"wrap")] %>% gather(variable, value,-"predictor2",-"ID",-all_of(Strata),-"wrap")
else
molten <- subset[,c("ID",paste(bacteria),"predictor2","wrap")] %>% gather(variable, value,-"predictor2",-"ID",-"wrap")
}
if(is.null(facet_wrap)){
if(!is.null(Strata))
molten <- subset[,c("ID",paste(bacteria),"predictor2",Strata)] %>% gather(variable, value,-"predictor2",-"ID",-all_of(Strata))
else
molten <- subset[,c("ID",paste(bacteria),"predictor2")] %>% gather(variable, value,-"predictor2",-"ID")
}
if(!is.null(color_by)){
molten[molten$variable != paste("Other",type),"colvar"] <- molten %>% dplyr::filter(variable != paste("Other",type)) %>% .[,"variable"] %>% match(tax[,type]) %>% tax[.,color_by] %>% as.character
molten[molten$variable == paste("Other",type),"colvar"] <- paste("Other",color_by) %>% as.character
}
molten$variable <- gsub('_',' ',molten$variable)
if(order) ordered <- unique(molten$variable) #level order
if(!order) ordered <-sort(unique(molten$variable))#level order alphabetically
molten$variable <- factor(molten$variable, levels=ordered)
if(is.null(color_by)) molten$colvar <- molten$variable
if(!is.null(Strata)) molten <- molten[which(molten[,Strata]==Strata_val), ]
if(is.null(colors)){
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
cols <- cols[1:length(levels(factor(molten$predictor2)))]
}
if(!is.null(colors)) cols <- colors
if(bar_chart==TRUE & bar_chart_stacked==FALSE & is.null(legend_names)) legend_names <- as.character(levels(factor(molten$predictor2)))
if(is.null(legend_names)) legend_names <- as.character(levels(factor(molten$predictor2)))
ordered2<- rev(unique(molten$colvar))
if(reverse){
if(bar_chart==FALSE) {
molten$predictor2 <- factor(molten$predictor2, levels=rev(levels(molten$predictor2)))#manual faceting for levels;
legend_names <- rev(legend_names)
cols <- rev(cols)
}
if(bar_chart==TRUE) {
molten$colvar <- factor(molten$colvar, levels=rev(levels(factor(molten$colvar))))#manual faceting for levels;
molten$variable <- factor(molten$variable, levels=rev(levels(factor(molten$variable))))
cols <- rev(cols)
ordered2<- rev(ordered2)
}
}
if(bar_chart){
log=FALSE
cols <- c(brewer.pal(8,"Set1"), brewer.pal(7,"Dark2"),brewer.pal(7,"Set2"),brewer.pal(12,"Set3"),brewer.pal(7,"Accent"),brewer.pal(12,"Paired"),"gray")
# ordered <- levels(factor(molten$colvar))
if(is.null(color_by) & bar_chart_stacked==FALSE) cols <- cols[1:length(levels(factor(molten$predictor2)))]
else cols <- cols[c(1:length(levels(factor(molten$colvar)))-1,length(cols))]
if(!is.null(colors)) cols <- colors
if(is.null(color_by) & reverse==FALSE) cols <- rev(cols)
if(!is.null(color_by) & reverse==TRUE) cols <- rev(cols)
if(is.null(facet_wrap)) molten$wrap <- ""
molten_mean <- molten %>%
dplyr::group_by(variable,predictor2,wrap,colvar) %>%
dplyr::summarize(value = mean(value))
molten_mean$colvar <- factor(molten_mean$colvar, levels=ordered2)
}
#Calculate pvalue for outcomes
if(p_val==TRUE & ((bar_chart==TRUE & bar_chart_stacked==FALSE) | bar_chart==FALSE) & is.null(color_by)){
if(is.null(facet_wrap)) molten$wrap <- ""
if(!is.null(facet_wrap)) {
pval <- data.frame(pval=pval[gsub('_',' ',pval$variable) %in% ordered,]$pval,p_adjust=pval[gsub('_',' ',pval$variable) %in% ordered,]$p_adjust, variable=gsub('_',' ',pval[gsub('_',' ',pval$variable) %in% ordered,]$variable),wrap=pval[gsub('_',' ',pval$variable) %in% ordered,]$wrap)
}
else {
pval <- data.frame(pval=pval[gsub('_',' ',pval$variable) %in% ordered,]$pval,p_adjust=pval[gsub('_',' ',pval$variable) %in% ordered,]$p_adjust, variable=gsub('_',' ',pval[gsub('_',' ',pval$variable) %in% ordered,]$variable))
if(length(pval$variable)-length(ordered)<0) pval <- pval[match(pval$variable,ordered[length(pval$variable)-length(ordered)]),]
}
pval$predictor2 <- molten$predictor2[1]
pval$pval <- ifelse(is.na(pval$pval),1,pval$pval)
pval$p_adjust <- ifelse(is.na(pval$p_adjust),1,pval$p_adjust)
if(Only_sig){
index <- pval[pval$pval<0.05,"variable"]
molten <- molten[molten$variable %in% index,]