Testing the sampling depth is a crucial step for community ecologists. We need to be sure that our sampling covers (nearly) the whole diversity per sample, so unexplained variance is not due to low sequencing depth. To do so, we use rarefaction curves.
Here, we use the iNEXT Package by Anne Chao. It performs interpolation (rarefaction) as well as extrapolation. First we load the packages and data:
rm(list = ls())
library(iNEXT)
library(ggplot2)
library(RColorBrewer)
library(ggpubr)
library(viridis)
setwd("../04_Alpha_Diversity/")
OTU_Table = as.data.frame(read.csv("../00_Data/Oomycota/05_OwnSamples_OTU_Table_min-freq-9588_transposed_withMetadata.tsv",
header = T,
sep = "\t",
stringsAsFactors = T))
SampleMetadata = OTU_Table[,1:5]
rownames(OTU_Table) = SampleMetadata$SampleID
rownames(SampleMetadata) = SampleMetadata$SampleID
species = OTU_Table[,6:ncol(OTU_Table)]
species_mat = as.matrix(species)The OTU Table contains several replicates in terms of microhabitat and tree species. We want to test the rarefaction for the microhabitats, so now we aggregate the OTU table:
species_mat_aggregated = aggregate(species_mat, by = list(SampleMetadata$Microhabitat), FUN = "sum")
rownames(species_mat_aggregated) = levels(list(SampleMetadata$Microhabitat)[[1]])
species_mat_aggregated = species_mat_aggregated[,-1]Now we perform the rarefaction with iNEXT. The function requires the species in rows and the samples in the columns, so we need to transpose the table. Then, we specify several parameters (more details in the manual):
qmeans the diversity measure. We set it to 0 because we want the species richness (i.e. the number of OTUs)nbootis the number of bootstrap replicates for the extrapolationconfis the confidence interval for the extrapolationknotsis the number of calculations for the rarefaction curve. The more knots the smoother the curve (but the more time it requires)endpointmeans in this case the final number of sequences for the extrapolation
The calculation might take a long time, so it makes sense to save the result and load it afterwards for the plotting
# Uncomment the following lines when you run this script for the first time
# After that, just load the Output file
#Oomycota_out = iNEXT(t(species_mat_aggregated), q = 0, #q = 0 means species richness
# datatype = "abundance", nboot = 99, conf = 0.97, knots = 250, endpoint = 450000)
#save(Oomycota_out, file = "Oomycota_out.RData")
load("Oomycota_out.RData")The resulting file can be directly loaded into ggiNEXT, which converts it into a ggplot object. With type you can specify the type of plot. In this case, it is the number of sequences plotted against the number of OTUs (because we specified q = 0, the species richness). The default x-axis label would be "Number of Individuals", but as we are dealing with Barcoding data we override the label with "Number of Sequences".
g = ggiNEXT(Oomycota_out, type = 1, color.var = "site") +
scale_fill_manual(name = "Microhabitat",
values = c(viridis(7, direction = -1),
"#8e8878", "#524640"),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
scale_color_manual(name = "Microhabitat",
values = c(viridis(7, direction = -1),
"#8e8878", "#524640"),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
scale_shape_manual(name = "Microhabitat",
values = rep(16, 9),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
theme_minimal() +
labs(title = "Rarefaction - Oomycota",
x = "Number of sequences", y = "Number of OTUs") +
scale_x_continuous(labels = scales::comma) +
theme(legend.text = element_text(size = 12),
legend.title = element_text(size = 14, face = "bold"),
legend.position = "right",
legend.direction = "vertical",
plot.title = element_text(size = 18, face = "bold", hjust = 0.5, vjust = 0.5),
legend.title.align = 0.5,
axis.title = element_text(size = 14, face = "bold"),
axis.text = element_text(size = 12)) +
guides(fill = guide_legend(title="Microhabitat"),
color = guide_legend(title="Microhabitat"),
shape = guide_legend(title="Microhabitat"))
# In the default plot, brighter colors are hard to see.
# So here we rearrange the layers to make them more visible
g$layers = c(g$layers[[3]], g$layers[[2]], g$layers[[1]])
g
In all microhabitats, the rarefaction curves approach a plateau. This means our sequencing effort was enough to uncover most of the diversity. Increasing the sampling depth would only yield slightly more OTUs. That's great!
OTU_Table_cerco = as.data.frame(read.csv("../00_Data/Cercozoa/05_Cercozoa_OwnSamples_OTU_Table_min-freq-15684_transposed_withMetadata.tsv",
header = T,
sep = "\t",
stringsAsFactors = T))
SampleMetadata_cerco = OTU_Table_cerco[,1:5]
rownames(OTU_Table_cerco) = SampleMetadata_cerco$SampleID
rownames(SampleMetadata_cerco) = SampleMetadata_cerco$SampleID
species_cerco = OTU_Table_cerco[,6:ncol(OTU_Table_cerco)]
species_mat_cerco = as.matrix(species_cerco)
species_mat_aggregated_cerco = aggregate(species_mat_cerco, by = list(SampleMetadata_cerco$Microhabitat), FUN = "sum")
rownames(species_mat_aggregated_cerco) = levels(list(SampleMetadata_cerco$Microhabitat)[[1]])
species_mat_aggregated_cerco = species_mat_aggregated_cerco[,-1]
#Cercozoa_out = iNEXT(t(species_mat_aggregated), q = 0, #q = 0 means species richness
# datatype = "abundance", nboot = 99, conf = 0.97, knots = 250, endpoint = 450000)
#save(Cercozoa_out, file = "Cercozoa_out.RData")
load("Cercozoa_out.RData")
g_cerco = ggiNEXT(Cercozoa_out, type = 1, color.var = "site") +
scale_fill_manual(name = "Microhabitat",
values = c(viridis(7, direction = -1),
"#8e8878", "#524640"),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
scale_color_manual(name = "Microhabitat",
values = c(viridis(7, direction = -1),
"#8e8878", "#524640"),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
scale_shape_manual(name = "Microhabitat",
values = rep(16, 9),
limits = c("Arboreal Soil", "Bark", "Deadwood",
"Fresh Leaves", "Hypnum", "Lichen",
"Orthotrichum", "Leaf Litter", "Soil")) +
theme_minimal() +
labs(x = "Number of sequences", y = "Number of OTUs",
title = "Rarefaction - Cercozoa") +
scale_x_continuous(labels = scales::comma) +
theme(legend.text = element_text(size = 12),
legend.title = element_text(size = 14, face = "bold"),
legend.position = "right",
legend.direction = "vertical",
legend.title.align = 0.5,
plot.title = element_text(size = 18, face = "bold", hjust = 0.5, vjust = 0.5),
axis.title = element_text(size = 14, face = "bold"),
axis.text = element_text(size = 12)) +
guides(fill = guide_legend(title="Microhabitat"),
color = guide_legend(title="Microhabitat"),
shape = guide_legend(title="Microhabitat"))
g_cerco$layers = c(g_cerco$layers[[3]], g_cerco$layers[[2]], g_cerco$layers[[1]])
g_cerco
Now combine the two plots:
g$labels$title = NULL
g_cerco$labels$title = NULL
combi = ggarrange(g_cerco, g,
labels = c("A", "B"),
ncol = 2, nrow = 1,
common.legend = T, legend = "right",
align = "h", vjust = 2.5) #%>%
#annotate_figure(fig.lab = "Figure X", fig.lab.face = "bold",
# fig.lab.size = 18,
# top = text_grob("Rarefaction",
# face = "bold", size = 20))
#ggsave("RarefactionCombined.tif", plot = combi,
# device = "tiff", dpi = 600, width = 24, height = 12,
# units = "cm")
ggsave("RarefactionCombined.png", plot = combi,
device = "png", dpi = 600, width = 24, height = 12,
units = "cm")
ggsave("RarefactionCombined.jpeg", plot = combi,
device = "jpeg", dpi = 600, width = 24, height = 12,
units = "cm")
ggsave("RarefactionCombined.pdf", plot = combi,
device = "pdf", dpi = 600, width = 24, height = 12,
units = "cm")
combi