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15- Shared and unique ASVs.Rmd
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```{r}
library(Rcpp)
library(dada2)
library(phyloseq)
library(permute)
library(lattice)
library(vegan)
library(ggplot2)
library(tidyverse)
library(ggstatsplot)
library(dplyr)
library(microbiome)
library(microbiomeutilities)
library(knitr)
library(RColorBrewer)
library(DT)
library(gt)
library(cowplot)
library(PerformanceAnalytics)
```
```{r}
ps.prev <- readRDS("phyobjects/STEP 14/ps.prev.f.rds")
ps_tss_1 <- transform_sample_counts(ps.prev, function(x){x / sum(x)})
mtd <- data.frame(sample_data(ps.prev), check.names = FALSE)
```
```{r}
# compute OTU prevalence
prvl <- list()
for (i in c("M", "V", "MM", "VM", "MMV", "VMV",
"water.M", "water.V", "water.MM", "water.VM", "water.MMV", "water.VMV")) {
prvl[[i]] <- subset_samples(ps_tss_1, sample.name.1 == i) %>%
prevalence(detection = 0.0005, count = TRUE, include.lowest = TRUE)
}
for (i in c("feed.M", "feed.V")) {
prvl[[i]] <- subset_samples(ps_tss_1, sample.name.1 == i) %>%
prevalence(detection = 0.0005, count = TRUE, include.lowest = TRUE)
}
```
Compute ASV overlap
```{r}
ovrl <- bind_cols(prvl) %>%
pivot_longer(M:VMV,
names_to = "SAMPLE",
values_to = "Intestine") %>%
# compute OTU overlap between water/feed and intestinal samples
mutate(
WaterOverlapM = case_when(
water.M > 0 & Intestine > 0 ~ "Shared",
water.M > 0 & Intestine == 0 ~ "Unique to water",
water.M == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
WaterOverlapV = case_when(
water.V > 0 & Intestine > 0 ~ "Shared",
water.V > 0 & Intestine == 0 ~ "Unique to water",
water.V == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
WaterOverlapMM = case_when(
water.MM > 0 & Intestine > 0 ~ "Shared",
water.MM > 0 & Intestine == 0 ~ "Unique to water",
water.MM == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
WaterOverlapVM = case_when(
water.VM > 0 & Intestine > 0 ~ "Shared",
water.VM > 0 & Intestine == 0 ~ "Unique to water",
water.VM == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
WaterOverlapMMV = case_when(
water.MMV > 0 & Intestine > 0 ~ "Shared",
water.MMV > 0 & Intestine == 0 ~ "Unique to water",
water.MMV == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
WaterOverlapVMV = case_when(
water.VMV > 0 & Intestine > 0 ~ "Shared",
water.VMV > 0 & Intestine == 0 ~ "Unique to water",
water.VMV == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapM = case_when(
feed.M > 0 & Intestine > 0 ~ "Shared",
feed.M > 0 & Intestine == 0 ~ "Unique to feed",
feed.M == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapV = case_when(
feed.V > 0 & Intestine > 0 ~ "Shared",
feed.V > 0 & Intestine == 0 ~ "Unique to feed",
feed.V == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapMM = case_when(
feed.M > 0 & Intestine > 0 ~ "Shared",
feed.M > 0 & Intestine == 0 ~ "Unique to feed",
feed.M == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapVM = case_when(
feed.M > 0 & Intestine > 0 ~ "Shared",
feed.M > 0 & Intestine == 0 ~ "Unique to feed",
feed.M == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapMMV = case_when(
feed.V > 0 & Intestine > 0 ~ "Shared",
feed.V > 0 & Intestine == 0 ~ "Unique to feed",
feed.V == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent"),
FeedOverlapVMV = case_when(
feed.V > 0 & Intestine > 0 ~ "Shared",
feed.V > 0 & Intestine == 0 ~ "Unique to feed",
feed.V == 0 & Intestine > 0 ~ "Unique to intestine",
TRUE ~ "Absent")) %>%
pivot_longer(WaterOverlapM:FeedOverlapVMV,
names_to = "OverlapType",
values_to = "Category") %>%
count(OverlapType, SAMPLE, Category) %>%
mutate(
Category = factor(
Category,
levels = c("Unique to water", "Unique to feed",
"Shared", "Unique to intestine", "Absent")),
#Diet = ifelse(grepl("FM", Sample), "FM", "ICJ", "ACJ", "IWA", "AWA", "SBM"),
Sample_Regime = factor(SAMPLE, levels = c("M", "V", "MM", "VM", "MMV", "VMV")))
```
Plotting
Plot microbial overlap between feed and intestinal samples.
```{r}
ovrl_bar_feed <- ovrl %>%
filter(OverlapType == "FeedOverlapM" & Sample_Regime == "M" |
OverlapType == "FeedOverlapV" & Sample_Regime == "V" |
OverlapType == "FeedOverlapMM" & Sample_Regime == "MM"|
OverlapType == "FeedOverlapVM" & Sample_Regime == "VM" |
OverlapType == "FeedOverlapMMV" & Sample_Regime == "MMV"|
OverlapType == "FeedOverlapVMV" & Sample_Regime == "VMV") %>%
filter(Category != "Absent") %>%
ggplot(aes(x = SAMPLE, c("M", "V", "MM", "VM", "MMV", "VMV"),
y = n, fill = Category, label = n)) +
geom_bar(stat = "identity") +
geom_text(position = position_stack(vjust = 0.5), color = "white", size = 2.5) +
labs(x = "", y = "Number of ASVs", size = 10) +
scale_y_continuous(expand = expansion(mult = c(0, 0.1))) +
scale_fill_manual(values = c("#B2DF8A", "grey50", "#33A02C")) +
#facet_nested(~ Diet + Site, scales = "free_x", nest_line = TRUE) +
scale_x_discrete(limit=c("M", "V", "MM", "VM", "MMV", "VMV")) +
theme_bw() +
theme(axis.text.x = element_text (size = 9),
strip.background = element_blank(),
legend.title = element_blank(),
legend.margin = margin(0, 0, 0, 0),
legend.box.margin = margin(0, 0, 0, -5))
```
Plot microbial overlap between water and intestinal samples.
```{r}
ovrl_bar_water <- ovrl %>%
filter(OverlapType == "WaterOverlapM" & Sample_Regime == "M" |
OverlapType == "WaterOverlapV" & Sample_Regime == "V" |
OverlapType == "WaterOverlapMM" & Sample_Regime == "MM"|
OverlapType == "WaterOverlapVM" & Sample_Regime == "VM" |
OverlapType == "WaterOverlapMMV" & Sample_Regime == "MMV"|
OverlapType == "WaterOverlapVMV" & Sample_Regime == "VMV") %>%
filter(Category != "Absent") %>%
ggplot(aes(x = SAMPLE, c("M", "V", "MM", "VM", "MMV", "VMV"),
y = n, fill = Category, label = n)) +
geom_bar(stat = "identity") +
geom_text(position = position_stack(vjust = 0.5), color = "white", size = 2.5) +
labs(x = "", y = "Number of ASVs", size = 10) +
scale_y_continuous(expand = expansion(mult = c(0, 0.1))) +
scale_fill_manual(values = c("#A6CEE3", "grey50", "#1F78B4")) +
#facet_nested(~ Diet + Site, scales = "free_x", nest_line = TRUE) +
scale_x_discrete(limit=c("M", "V", "MM", "VM", "MMV", "VMV")) +
theme_bw() +
theme(axis.text.x = element_text (size = 9),
strip.background = element_blank(),
legend.title = element_blank(),
legend.margin = margin(0, 0, 0, 0),
legend.box.margin = margin(0, 0, 0, -5))
```
Assemble plots
```{r}
ovrl_bar_feed + ovrl_bar_water
```
Abundance of ASVs shared
Compute shared ASV abundance
Compute the abundance of shared ASVs between feed and intestinal samples.
```{r}
shared_feed <- map(c("feed.M", "feed.V", "feed.M", "feed.M", "feed.V", "feed.V"), function(x) {
prune_taxa(names(which(prvl[[x]] > 0)), ps_tss_1) %>% sample_sums()
}
)
names(shared_feed) <- c("M", "V", "MM", "VM", "MMV", "VMV")
# names(prvl)
shared_feed <- map(seq_along(shared_feed), function(x){
data.frame(Abundance = shared_feed[[x]]) %>%
rownames_to_column("sample_name") %>%
inner_join(rownames_to_column(mtd, "sample_name"), by = "sample_name") %>%
filter(Regime == names(shared_feed)[x] & !sample %in% c("feed", "water")) %>%
#mutate(Segment = ifelse(Segment == "PI", "PIM", "DIM"),
#Segment = factor(Segment, levels = c("PIM", "DIM"))) %>%
mutate(Abundance = round(100 * Abundance, 1))}) %>%
bind_rows()
```
Compute the abundance of shared OTUs between water and intestinal samples.
```{r}
shared_water <- map(c("water.M", "water.V", "water.MM", "water.VM", "water.MMV", "water.VMV"), function(x) {
prune_taxa(names(which(prvl[[x]] > 0)), ps_tss_1) %>% sample_sums()
}
)
names(shared_water) <- c("M", "V", "MM", "VM", "MMV", "VMV")
# names(prvl)
shared_water <- map(seq_along(shared_water), function(x){
data.frame(Abundance = shared_water[[x]]) %>%
rownames_to_column("sample_name") %>%
inner_join(rownames_to_column(mtd, "sample_name"), by = "sample_name") %>%
filter(Regime == names(shared_water)[x] & !sample %in% c("feed", "water")) %>%
#mutate(Segment = ifelse(Segment == "PI", "PIM", "DIM"),
#Segment = factor(Segment, levels = c("PIM", "DIM"))) %>%
mutate(Abundance = round(100 * Abundance, 1))}) %>%
bind_rows()
```
Plotting
Abundance of shared OTUs between feed and intestinal samples.
```{r}
bp_shared_feed <- ggplot(shared_feed, aes(x = Regime, y = Abundance)) +
geom_boxplot(fill = "grey50", width = 0.5) +
geom_point() +
#facet_nested(~ Diet + Segment, scales = "free_x", nest_line = TRUE) +
labs(x = "", y = "Relative abundance of shared ASVs (%)", size = 10) +
scale_x_discrete(limit=c("M", "V", "MM", "VM", "MMV", "VMV")) +
scale_y_continuous(
limits = c(0, 100), breaks = 0:10*10, expand = expansion(c(0, 0))
) +
theme_bw() +
theme(axis.text.x = element_text (size = 9),
strip.background = element_blank(),
legend.position = "none")
```
Abundance of shared OTUs between water and intestinal samples.
```{r}
bp_shared_water <- shared_water %>%
ggplot(aes(x = Regime, y = Abundance)) +
geom_boxplot(fill = "grey50", width = 0.5) +
geom_point() +
#facet_nested(~ Diet + Segment, scales = "free_x", nest_line = TRUE) +
labs(x = "", y = "Relative abundance of shared ASVs (%)", size = 10) +
scale_y_continuous(
limits = c(0, 100), breaks = 0:10*10, expand = expansion(c(0, 0))
) +
scale_x_discrete(limit=c("M", "V", "MM", "VM", "MMV", "VMV")) +
theme_bw() +
theme(axis.text.x = element_text (size = 9),
strip.background = element_blank(),
legend.position = "none")
```
Assemble plots
```{r}
bp_shared_feed + bp_shared_water
```
Figure 7
```{r}
library(patchwork)
fig7a <- ovrl_bar_feed + bp_shared_feed +
plot_layout(guides = "collect") +
plot_annotation(tag_levels = list(c("A", ""))) &
theme(
legend.position = "top",
legend.margin = margin(0, 0, 0, 0),
legend.box.margin = margin(10, 10, 10, 10))
fig7b <- ovrl_bar_water + bp_shared_water +
plot_layout(guides = "collect") +
plot_annotation(tag_levels = list(c("B", ""))) &
theme(
legend.position = "top",
legend.margin = margin(0, 0, 0, 0),
legend.box.margin = margin(10, 10, 10, 10))
wrap_elements(full = fig7a) | wrap_elements(full = fig7b)
# export plot
ggsave(("figures/sharedASVs.RA.tiff"), width = 12, height = 5,
units = "in", dpi = 300, compression = "lzw")
```