Commit 5a4e2bde authored by Olivier Rue's avatar Olivier Rue
Browse files

real

parent 9c3c6d25
Pipeline #42602 passed with stage
in 14 seconds
...@@ -38,6 +38,8 @@ library(tidyverse) ...@@ -38,6 +38,8 @@ library(tidyverse)
library(ComplexHeatmap) library(ComplexHeatmap)
library(phyloseqCompanion) library(phyloseqCompanion)
library(circlize) library(circlize)
library(ggVennDiagram)
library(ggplot2)
``` ```
...@@ -49,89 +51,6 @@ options(DT.options = list(pageLength = 10, ...@@ -49,89 +51,6 @@ options(DT.options = list(pageLength = 10,
buttons = c('copy', 'csv', 'excel', 'pdf', 'print'))) buttons = c('copy', 'csv', 'excel', 'pdf', 'print')))
``` ```
# MEAT
```{bash, eval=F}
# Genologin
cd /work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/scripts
sh dada2_frogs.sh
```
```{bash, eval=F}
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/frogs.biom MEAT/ITS1.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/frogs.biom MEAT/ITS2.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/frogs.biom MEAT/D1D2.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/frogs.biom MEAT/RPB2.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/frogs.tsv MEAT/ITS1.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/frogs.tsv MEAT/ITS2.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/frogs.tsv MEAT/D1D2.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/frogs.tsv MEAT/RPB2.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/multiaff.tsv MEAT/ITS1_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/multiaff.tsv MEAT/ITS2_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/multiaff.tsv MEAT/D1D2_multiaff.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/multiaff.tsv MEAT/RPB2_multiaff.tsv
```
## Preparation of final BIOM files {.tabset}
### ITS1
### ITS2
### D1D2
### RPB2
## Analysis
```{r, eval=F}
if (!file.exists("REAL/ITS1.rds")) {
biomfile <- "REAL/ITS1.biom"
physeq_its1 <- import_frogs(biomfile, taxMethod = "blast")
metadata <- read.table("REAL/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its1) <- metadata
sample_data(physeq_its1)$Marker <- "ITS1"
saveRDS(physeq_its1,"REAL/ITS1.rds")
} else {
physeq_its1 <- readRDS("REAL/ITS1.rds")
}
if (!file.exists("REAL/ITS2.rds")) {
biomfile <- "REAL/ITS2.biom"
physeq_its2 <- import_frogs(biomfile, taxMethod = "blast")
metadata <- read.table("REAL/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its2) <- metadata
sample_data(physeq_its2)$Marker <- "ITS2"
saveRDS(physeq_its2,"REAL/ITS2.rds")
} else {
physeq_its2 <- readRDS("REAL/ITS2.rds")
}
if (!file.exists("REAL/D1D2.rds")) {
biomfile <- "REAL/D1D2.biom"
physeq_d1d2 <- import_frogs(biomfile, taxMethod = "blast")
metadata <- read.table("REAL/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_d1d2) <- metadata
sample_data(physeq_d1d2)$Marker <- "D1D2"
saveRDS(physeq_d1d2,"REAL/D1D2.rds")
} else {
physeq_d1d2 <- readRDS("REAL/D1D2.rds")
}
if (!file.exists("REAL/RPB2.rds")) {
biomfile <- "REAL/RPB2.biom"
physeq_rpb2 <- import_frogs(biomfile, taxMethod = "blast")
metadata <- read.table("REAL/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_rpb2) <- metadata
sample_data(physeq_rpb2)$Marker <- "RPB2"
saveRDS(physeq_rpb2,"REAL/RPB2.rds")
} else {
physeq_rpb2 <- readRDS("REAL/RPB2.rds")
}
```
```{r, eval=T} ```{r, eval=T}
create_frogs_object <- function(path, marker, type, file, multifile){ create_frogs_object <- function(path, marker, type, file, multifile){
...@@ -183,40 +102,125 @@ keep_only_interest_species <- function(physeq, true_species){ ...@@ -183,40 +102,125 @@ keep_only_interest_species <- function(physeq, true_species){
} }
``` ```
# MEAT
```{r, eval=T} ```{bash, eval=F}
rpb2 <- create_frogs_object("REAL","RPB2", "DNA", "RPB2.biom", "RPB2_multiaff.tsv") # Genologin
tax_table(rpb2)[, colnames(tax_table(rpb2))] <- gsub(tax_table(rpb2)[, colnames(tax_table(rpb2))], pattern = "[a-z]:", replacement = "") cd /work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/scripts
d1d2 <- create_frogs_object("REAL","D1D2", "DNA", "D1D2.biom", "D1D2_multiaff.tsv") sh dada2_frogs.sh
tax_table(d1d2)[, colnames(tax_table(d1d2))] <- gsub(tax_table(d1d2)[, colnames(tax_table(d1d2))], pattern = "[a-z]:", replacement = "") ```
its1 <- create_frogs_object("REAL","ITS1", "DNA", "ITS1.biom", "ITS1_multiaff.tsv")
its2 <- create_frogs_object("REAL","ITS2", "DNA", "ITS2.biom", "ITS2_multiaff.tsv")
all <- merge_phyloseq(its1, its2, d1d2, rpb2) ```{bash, eval=F}
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/affiliation.biom REAL_MEAT/ITS1.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/affiliation.biom REAL_MEAT/ITS2.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/affiliation.biom REAL_MEAT/D1D2.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/affiliation.biom REAL_MEAT/RPB2.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/affiliation.tsv REAL_MEAT/ITS1.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/affiliation.tsv REAL_MEAT/ITS2.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/affiliation.tsv REAL_MEAT/D1D2.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/affiliation.tsv REAL_MEAT/RPB2.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS1/multiaff.tsv REAL_MEAT/ITS1_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/ITS2/multiaff.tsv REAL_MEAT/ITS2_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/D1D2/multiaff.tsv REAL_MEAT/D1D2_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/REAL/results/DADA2_FROGS/RPB2/multiaff.tsv REAL_MEAT/RPB2_multiaff.tsv
```
sample_data(all)$Marker <- factor(sample_data(all)$Marker, levels=c("ITS1","ITS2","D1D2","RPB2")) ## Analysis of raw BIOM files
Method <- get_variable(all, "method")
saveRDS(all, "REAL/real_all.rds") ```{r, eval=T}
if (!file.exists("REAL_MEAT/ITS1.rds")) {
biomfile <- "REAL_MEAT/ITS1.biom"
physeq_its1 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_its1) <- gsub(tax_table(physeq_its1), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_MEAT/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its1) <- metadata
sample_data(physeq_its1)$Marker <- "ITS1"
sample_names(physeq_its1) <- glue::glue(paste("{sample_names(physeq_its1)}","ITS1", sep="_"))
saveRDS(physeq_its1,"REAL_MEAT/ITS1.rds")
} else {
physeq_its1 <- readRDS("REAL_MEAT/ITS1.rds")
}
if (!file.exists("REAL_MEAT/ITS2.rds")) {
biomfile <- "REAL_MEAT/ITS2.biom"
physeq_its2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_its2) <- gsub(tax_table(physeq_its2), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_MEAT/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its2) <- metadata
sample_data(physeq_its2)$Marker <- "ITS2"
sample_names(physeq_its2) <- glue::glue(paste("{sample_names(physeq_its2)}","ITS2", sep="_"))
saveRDS(physeq_its2,"REAL_MEAT/ITS2.rds")
} else {
physeq_its2 <- readRDS("REAL_MEAT/ITS2.rds")
}
if (!file.exists("REAL_MEAT/D1D2.rds")) {
biomfile <- "REAL_MEAT/D1D2.biom"
physeq_d1d2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_d1d2) <- gsub(tax_table(physeq_d1d2), pattern = "[a-z]__", replacement = "")
tax_table(physeq_d1d2) <- gsub(tax_table(physeq_d1d2), pattern = "Eukaryota", replacement = "Fungi")
metadata <- read.table("REAL_MEAT/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_d1d2) <- metadata
sample_data(physeq_d1d2)$Marker <- "D1D2"
sample_names(physeq_d1d2) <- glue::glue(paste("{sample_names(physeq_d1d2)}","D1D2", sep="_"))
saveRDS(physeq_d1d2,"REAL_MEAT/D1D2.rds")
} else {
physeq_d1d2 <- readRDS("REAL_MEAT/D1D2.rds")
}
if (!file.exists("REAL_MEAT/RPB2.rds")) {
biomfile <- "REAL_MEAT/RPB2.biom"
physeq_rpb2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_rpb2) <- gsub(tax_table(physeq_rpb2), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_MEAT/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_rpb2) <- metadata
sample_data(physeq_rpb2)$Marker <- "RPB2"
sample_names(physeq_rpb2) <- glue::glue(paste("{sample_names(physeq_rpb2)}","RPB2", sep="_"))
saveRDS(physeq_rpb2,"REAL_MEAT/RPB2.rds")
} else {
physeq_rpb2 <- readRDS("REAL_MEAT/RPB2.rds")
}
``` ```
```{r, eval=T} ```{r, eval=T}
p <- plot_bar(all) all <- merge_phyloseq(physeq_its1, physeq_its2, physeq_d1d2, physeq_rpb2)
sample_data(all)$Marker <- factor(sample_data(all)$Marker, levels=c("ITS1","ITS2","D1D2","RPB2"))
p <- plot_composition(physeq = all, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Species", numberOfTaxa = 22, x = "Sample")
p + facet_grid(". ~ Marker", scales = "free_x", space = "free") p + facet_grid(". ~ Marker", scales = "free_x", space = "free")
``` ```
```{r, eval=T} ```{r, eval=T}
p <- plot_composition(physeq = all, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Genus", numberOfTaxa = 22, x = "Sample") p <- plot_richness(physeq = all, x = "Marker", color = "Marker", shape = NULL, title = "Alpha diversity graphics", measures = c("Observed", "Chao1", "ACE", "Shannon", "Simpson", "InvSimpson", "Fisher"))
p + facet_grid(". ~ Marker", scales = "free_x", space = "free") p + geom_boxplot() + NULL
``` ```
```{r, eval=T} ```{r, eval=T}
p <- plot_composition(physeq = all, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Species", numberOfTaxa = 22, x = "Sample") beta.dist <- distance(all, method = "bray")
p + facet_grid(". ~ Marker", scales = "free_x", space = "free") ord <- ordinate(all, method = "MDS", distance = beta.dist)
p <- plot_ordination(physeq = all, ordination = ord, type = "samples", axes = c(1, 2), color = "Sample", shape = "Marker", label = NULL, title = "Samples ordination graphic")
p <- p + stat_ellipse(aes_string(group = "Marker"))
p + theme_bw()
```
```{r, eval=T}
x <- list(
ITS1 = as_tibble(tax_table(physeq_its1))$Genus %>% as.vector(),
ITS2 = as_tibble(tax_table(physeq_its2))$Genus %>% as.vector(),
D1D2 = as_tibble(tax_table(physeq_d1d2))$Genus %>% as.vector(),
RPB2 = as_tibble(tax_table(physeq_rpb2))$Genus %>% as.vector()
)
ggVennDiagram(x,label_alpha = 0) +
scale_fill_gradient(low = "#F4FAFE", high = "#4981BF")
``` ```
# CHEESE # CHEESE
...@@ -228,34 +232,113 @@ sh dada2_frogs.sh ...@@ -228,34 +232,113 @@ sh dada2_frogs.sh
``` ```
```{bash, eval=F} ```{bash, eval=F}
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/frogs.biom CHEESE/ITS1.biom scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/affiliation.biom REAL_CHEESE/ITS1.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/frogs.biom CHEESE/ITS2.biom scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/frogs.biom REAL_CHEESE/ITS2.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/frogs.biom CHEESE/D1D2.biom scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/affiliation.biom REAL_CHEESE/D1D2.biom
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/frogs.biom CHEESE/RPB2.biom scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/affiliation.biom REAL_CHEESE/RPB2.biom
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/frogs.tsv CHEESE/ITS1.tsv scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/affiliation.tsv REAL_CHEESE/ITS1.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/frogs.tsv CHEESE/ITS2.tsv scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/affiliation.tsv REAL_CHEESE/ITS2.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/frogs.tsv CHEESE/D1D2.tsv scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/affiliation.tsv REAL_CHEESE/D1D2.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/frogs.tsv CHEESE/RPB2.tsv scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/affiliation.tsv REAL_CHEESE/RPB2.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/multiaff.tsv CHEESE/ITS1_multiaff.tsv #scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS1/multiaff.tsv REAL_CHEESE/ITS1_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/multiaff.tsv CHEESE/ITS2_multiaff.tsv #scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/ITS2/multiaff.tsv REAL_CHEESE/ITS2_multiaff.tsv
#scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/multiaff.tsv CHEESE/D1D2_multiaff.tsv #scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/D1D2/multiaff.tsv REAL_CHEESE/D1D2_multiaff.tsv
scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/multiaff.tsv CHEESE/RPB2_multiaff.tsv #scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/multiaff.tsv REAL_CHEESE/RPB2_multiaff.tsv
``` ```
## Preparation of final BIOM files {.tabset} ## Analysis of raw BIOM files {.tabset}
### ITS1 ```{r, eval=T}
if (!file.exists("REAL_CHEESE/ITS1.rds")) {
biomfile <- "REAL_CHEESE/ITS1.biom"
physeq_its1 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_its1) <- gsub(tax_table(physeq_its1), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_CHEESE/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its1) <- metadata
sample_data(physeq_its1)$Marker <- "ITS1"
sample_names(physeq_its1) <- glue::glue(paste("{sample_names(physeq_its1)}","ITS1", sep="_"))
saveRDS(physeq_its1,"REAL_CHEESE/ITS1.rds")
} else {
physeq_its1 <- readRDS("REAL_CHEESE/ITS1.rds")
}
### ITS2 if (!file.exists("REAL_CHEESE/ITS2.rds")) {
biomfile <- "REAL_CHEESE/ITS2.biom"
physeq_its2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_its2) <- gsub(tax_table(physeq_its2), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_CHEESE/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_its2) <- metadata
sample_data(physeq_its2)$Marker <- "ITS2"
sample_names(physeq_its2) <- glue::glue(paste("{sample_names(physeq_its2)}","ITS2", sep="_"))
saveRDS(physeq_its2,"REAL_CHEESE/ITS2.rds")
} else {
physeq_its2 <- readRDS("REAL_CHEESE/ITS2.rds")
}
if (!file.exists("REAL_CHEESE/D1D2.rds")) {
biomfile <- "REAL_CHEESE/D1D2.biom"
physeq_d1d2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_d1d2) <- gsub(tax_table(physeq_d1d2), pattern = "[a-z]__", replacement = "")
tax_table(physeq_d1d2) <- gsub(tax_table(physeq_d1d2), pattern = "Eukaryota", replacement = "Fungi")
metadata <- read.table("REAL_CHEESE/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_d1d2) <- metadata
sample_data(physeq_d1d2)$Marker <- "D1D2"
sample_names(physeq_d1d2) <- glue::glue(paste("{sample_names(physeq_d1d2)}","D1D2", sep="_"))
saveRDS(physeq_d1d2,"REAL_CHEESE/D1D2.rds")
} else {
physeq_d1d2 <- readRDS("REAL_CHEESE/D1D2.rds")
}
if (!file.exists("REAL_CHEESE/RPB2.rds")) {
biomfile <- "REAL_CHEESE/RPB2.biom"
physeq_rpb2 <- import_frogs(biomfile, taxMethod = "blast")
tax_table(physeq_rpb2) <- gsub(tax_table(physeq_rpb2), pattern = "[a-z]__", replacement = "")
metadata <- read.table("REAL_CHEESE/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
sample_data(physeq_rpb2) <- metadata
sample_data(physeq_rpb2)$Marker <- "RPB2"
sample_names(physeq_rpb2) <- glue::glue(paste("{sample_names(physeq_rpb2)}","RPB2", sep="_"))
saveRDS(physeq_rpb2,"REAL_CHEESE/RPB2.rds")
} else {
physeq_rpb2 <- readRDS("REAL_CHEESE/RPB2.rds")
}
```
### D1D2 ```{r, eval=T}
all <- merge_phyloseq(physeq_its1, physeq_its2, physeq_d1d2, physeq_rpb2)
sample_data(all)$Marker <- factor(sample_data(all)$Marker, levels=c("ITS1","ITS2","D1D2","RPB2"))
p <- plot_composition(physeq = all, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Species", numberOfTaxa = 22, x = "Sample")
p + facet_grid(". ~ Marker", scales = "free_x", space = "free")
```
### RPB2 ```{r, eval=T}
p <- plot_richness(physeq = all, x = "Marker", color = "Marker", shape = NULL, title = "Alpha diversity graphics", measures = c("Observed", "Chao1", "ACE", "Shannon", "Simpson", "InvSimpson", "Fisher"))
p + geom_boxplot() + NULL
```
```{r, eval=T}
beta.dist <- distance(all, method = "bray")
ord <- ordinate(all, method = "MDS", distance = beta.dist)
p <- plot_ordination(physeq = all, ordination = ord, type = "samples", axes = c(1, 2), color = "Sample", shape = "Marker", label = NULL, title = "Samples ordination graphic")
p <- p + stat_ellipse(aes_string(group = "Marker"))
p + theme_bw()
```
```{r, eval=T}
x <- list(
ITS1 = as_tibble(tax_table(physeq_its1))$Genus %>% as.vector(),
ITS2 = as_tibble(tax_table(physeq_its2))$Genus %>% as.vector(),
D1D2 = as_tibble(tax_table(physeq_d1d2))$Genus %>% as.vector(),
RPB2 = as_tibble(tax_table(physeq_rpb2))$Genus %>% as.vector()
)
ggVennDiagram(x, show_intersect = T)
## Analysis ```
......
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