Zeya Xue 5/9/2019
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Written by Zhengyao "Zeya" Xue, ORCID
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The data files and R scripts can be found in this GitHub repo
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Thumbnail of expected Heatmap
- Thumbnail of expected bar plot
- P.S. The demonstration shown here is using data from this paper
The starting point of the workflow is + A count table, normalized or not. Looks like this:
## CDS_ID Low_A Low_B High_A High_B
## 1 CDS_100 10.980509 8.364332 28.15585 0.000000
## 2 CDS_10011 2.284764 1.522854 10.25240 0.000000
## 3 CDS_10013 9.833839 6.554506 0.00000 0.000000
## 4 CDS_10015 7.194288 2.501831 0.00000 0.000000
## 5 CDS_10016 0.000000 0.000000 44.98916 0.000000
## 6 CDS_10017 18.738610 6.516398 0.00000 7.725487
- A annotation or taxonomy table
## CDS_ID KO_ID Domain Phylum Class Order
## 1 CDS_2383 <NA> Archaea Euryarchaeota Methanococci Methanococcales
## 2 CDS_4184 <NA> Archaea Euryarchaeota Methanomicrobia Methanosarcinales
## 3 CDS_6601 <NA> Archaea Euryarchaeota Methanomicrobia Methanosarcinales
## 4 CDS_6904 <NA> Archaea Euryarchaeota Methanococci Methanococcales
## 5 CDS_7927 <NA> Archaea Euryarchaeota Halobacteria Natrialbales
## 6 CDS_15355 <NA> Archaea Euryarchaeota Methanomicrobia Methanomicrobiales
## Family Genus Species
## 1 Methanocaldococcaceae Methanocaldococcus Methanocaldococcus_infernus
## 2 Methanosarcinaceae Methanosarcina Methanosarcina_barkeri
## 3 Methanosarcinaceae Methanosarcina Methanosarcina_barkeri
## 4 Methanococcaceae Methanococcus Methanococcus_vannielii
## 5 Natrialbaceae Haloterrigena Haloterrigena_jeotgali
## 6 Methanomicrobiaceae Methanolacinia Methanolacinia_petrolearia
- A sample metainfo table
## SampleID Place Group
## 1 High_A High A
## 2 High_B High B
## 3 Low_A Low A
## 4 Low_B Low B
library(phyloseq);packageVersion("phyloseq")## [1] '1.22.3'
library(DESeq2);packageVersion("DESeq2")## [1] '1.18.1'
library(ggplot2)
library(reshape2)
library(superheat)
library(plyr)
library(dplyr)
library(RColorBrewer)# The otu table slot of phyloseq object
TabTPM <- read.table(file.path("example_data/sample_TPM.tsv"),
header = TRUE, sep = "\t")
row.names(TabTPM) <- TabTPM$CDS_ID
TabTPM <- TabTPM[,-1]
TabTPM <- as.matrix.data.frame(TabTPM)
# The tax table slot of phyloseq object
Tabanno <- read.table(file.path("example_data/sample_annotation_classifications.tsv"),
header = TRUE, sep = "\t", na.strings = "<NA>")
rownames(Tabanno) <- Tabanno$CDS_ID
Tabanno <- Tabanno[,c(-1,-2)] # remove CDS_ID and KOID
Tabanno <- as.matrix.data.frame(Tabanno)
# The sample data slot of phyloseq object
samdf <- read.csv(file.path("example_data/Samdf.csv"))## Warning in read.table(file = file, header = header, sep = sep, quote =
## quote, : incomplete final line found by readTableHeader on 'example_data/
## Samdf.csv'
rownames(samdf) <- samdf$SampleID
ps <- phyloseq(otu_table(TabTPM, taxa_are_rows = TRUE),
tax_table(Tabanno), sample_data(samdf))
ps # 20000 taxa and 4 samples## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 20000 taxa and 4 samples ]
## sample_data() Sample Data: [ 4 samples by 3 sample variables ]
## tax_table() Taxonomy Table: [ 20000 taxa by 7 taxonomic ranks ]
# Define function to get the deepest taxa assignment level
RECps <- function(ps) {
TaxTab2 <- as.data.frame(ps@tax_table)
list.s = as.character(TaxTab2$Species)
list.g = as.character(TaxTab2$Genus)
list.f = as.character(TaxTab2$Family)
list.o = as.character(TaxTab2$Order)
list.c = as.character(TaxTab2$Class)
list.p = as.character(TaxTab2$Phylum)
list.k = as.character(TaxTab2$Kingdom)
list.d = as.character(TaxTab2$Domain)
list.REC = character(length(as.character(TaxTab2$Domain)))
for(i in 1:dim(TaxTab2)[1]){
S = which(TaxTab2$Species[i] == "" | is.na(TaxTab2$Species[i]))
G = which(TaxTab2$Genus[i] == "" | is.na(TaxTab2$Genus[i]))
Fa = which(TaxTab2$Family[i] == "" | is.na(TaxTab2$Family[i]))
O = which(TaxTab2$Order[i] == "" | is.na(TaxTab2$Order[i]))
C = which(TaxTab2$Class[i] == "" | is.na(TaxTab2$Class[i]))
P = which(TaxTab2$Phylum[i] == "" | is.na(TaxTab2$Phylum[i]))
K = which(TaxTab2$Kingdom[i] == "" | is.na(TaxTab2$Kingdom[i]))
D = which(TaxTab2$Domain[i] == "" | is.na(TaxTab2$Domain[i]))
if(length(S) == 0){
list.REC[i] <- list.s[i]
} else if(length(G) == 0){
list.REC[i] <- list.g[i]
} else if(length(Fa) == 0){
list.REC[i] <- list.f[i]
} else if(length(O) == 0){
list.REC[i] <- list.o[i]
} else if(length(C) == 0){
list.REC[i] <- list.c[i]
} else if(length(P) == 0){
list.REC[i] <- list.p[i]
} else if(length(K) == 0){
list.REC[i] <- list.k[i]
} else if(length(D) == 0){
list.REC[i] <- list.d[i]
} else {
list.REC[i] <- "meow"
}
}
TaxTab2$REC <- list.REC
TaxTab2$REC <- factor(TaxTab2$REC)
phyloseq(otu_table(ps), sample_data(ps),
TaxTab2 %>% as.matrix() %>% tax_table())
}
ps.REC <- RECps(ps)
ps.REC # 20000 taxa and 4 samples ## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 20000 taxa and 4 samples ]
## sample_data() Sample Data: [ 4 samples by 3 sample variables ]
## tax_table() Taxonomy Table: [ 20000 taxa by 8 taxonomic ranks ]
# Clean up the taxonomy
ps.REC.glom <- ps.REC %>% tax_glom(taxrank = "REC", NArm = FALSE)
# Run the next line if want relative abundance
ps.REC.per <- ps.REC.glom %>% transform_sample_counts(function(x) x/sum(x) )
taxa.df <- psmelt(ps.REC.per) # melt ps object
# aggregate for REC level plot
taxa.agg <- aggregate(Abundance ~ REC + SampleID,
data = taxa.df,
mean)
taxa.cast <- dcast(taxa.agg, REC ~ SampleID, mean, value.var = "Abundance")
# Define palette
my_palette <- colorRampPalette(c("red", "yellow", "green"))(n = 299)
# defines the color breaks manually for a "skewed" color transition
col_breaks = c(seq(-1,0,length=100), # for red
seq(0.01,0.8,length=100), # for yellow
seq(0.81,1,length=100)) # for green
# only plot the top 30 most abundant taxa
# need to change results from factor to numeric because of R
row.names(taxa.cast) <- taxa.cast$REC
taxa.cast <- taxa.cast[, -1]
indx <- sapply(taxa.cast, is.factor)
taxa.cast[indx] <- lapply(taxa.cast[indx], function(x) as.numeric(as.character(x)))
taxa.cast30 <- cbind(taxa.cast, total = rowSums(taxa.cast)) # need numeric values
taxa.cast30$taxa <- rownames(taxa.cast30)
taxa.cast30 <- head(arrange(taxa.cast30,desc(total)), n = 30)## Warning: package 'bindrcpp' was built under R version 3.4.4
row.names(taxa.cast30) <- taxa.cast30$taxa
taxa.cast30 <- taxa.cast30[, -c(5,6)] # remove total and taxa name colums
superheat(taxa.cast30,
# retain original order of rows/cols
pretty.order.rows = TRUE,
pretty.order.cols = TRUE,
row.dendrogram = TRUE,
col.dendrogram = TRUE,
grid.hline = TRUE,
row.title = "Annotation",
column.title = "SampleID",
left.label.text.size = 4,
bottom.label.text.size = 5,
left.label.size = 0.5,
# change the grid color to white (more pretty on a dark background)
grid.hline.col = "white",
grid.vline.col = "white") 
# Clean up the taxonomy
ps.REC.glom <- ps.REC %>% tax_glom(taxrank = "REC", NArm = FALSE)
# Run the next line if want relative abundance
ps.REC.per <- ps.REC.glom %>% transform_sample_counts(function(x) x/sum(x) )
taxa.df <- psmelt(ps.REC.per) # melt ps object
# aggregate for REC level plot
taxa.agg <- aggregate(Abundance ~ REC + SampleID,
data = taxa.df,
mean)
# Get the names of the most abundant 15 taxa
ps.Notop15 <- prune_taxa(names(sort(taxa_sums(ps.REC.per), TRUE)[16:nrow(ps.REC.per@tax_table)]), ps.REC.per)
taxa_names_filt <- ps.Notop15@tax_table[,8] %>% as.character() # 8 for REC level
# convert REC colum to a character vector from a factor because R
taxa.agg$REC <- as.character(taxa.agg$REC)
# change the less abundant taxa names to "Other"
taxa.agg[taxa.agg$REC %in% taxa_names_filt,]$REC <- "Other"
# Set colors for plotting
mycol = colorRampPalette(brewer.pal(12, "Paired"))(16)
# Set levels of taxon for pretty plots
## I do not know this beforehand, modified after 1st generating plot to know the
## taxa names
taxa.agg$REC = factor(taxa.agg$REC, levels = c("Alteromonas_macleodii",
"Anaerophaga_thermohalophila",
"Aureispira_sp._CCB-QB1",
"Bacteroides_fragilis",
"Dyadobacter_alkalitolerans",
"Escherichia_coli",
"Haliscomenobacter_hydrossis",
"Lacinutrix_himadriensis",
"Lewinella_cohaerens",
"Nitrosomonas_communis",
"Phaeodactylibacter_xiamenensis",
"Salinibacter_ruber",
"Saprospira_grandis",
"Synechococcus_sp._BL107",
"Synechococcus_sp._CC9605",
"Other"))
ggplot(taxa.agg, aes(x = SampleID, y = Abundance, fill = REC)) +
geom_bar(stat = "identity") + #position = "fill" is for making the bar 100%
scale_fill_manual(values = mycol)+
theme(axis.title.x = element_blank()) + # Remove x axis title
guides(fill = guide_legend(reverse = FALSE, keywidth = 1, keyheight = 1)) +
ylab("Relative Abundance \n")