Updated Heatmaps and Dendrograms
Last updated: 2021-02-11
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Intro
This page contains the updated code for generating the joint figures of heatmaps with the dendrogram. The update was needed to fix how the OTUs were subset based on average relative abundance. Prior to each heatmap will be a table of the OTUs that meet the given criteria.
I figured out that I originally subset based on the OTU relative abundance for each individual and sample, meaning that I subset according the just the raw Abundance irrespective of any OTU average abundance. This mistake is corrected in this document.
Heatmaps and Dendrograms
Species Level - NCI 16S Data
Relative Abudance Cutoff: 0.05
analysis.dat <- dat.16s # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.05 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.250 | Streptococcus_dentisani:Streptococcus_infantis:Streptococcus_mitis:Streptococcus_oligofermentans:Streptococcus_oralis:Streptococcus_pneumoniae:Streptococcus_pseudopneumoniae:Streptococcus_sanguinis |
| 0.076 | Pseudomonas_rhodesiae |
| 0.058 | Prevotella_melaninogenica |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(accession.number)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
plot.dat$OTU[plot.dat$OTU == "Streptococcus_dentisani:Streptococcus_infantis:Streptococcus_mitis:Streptococcus_oligofermentans:Streptococcus_oralis:Streptococcus_pneumoniae:Streptococcus_pseudopneumoniae:Streptococcus_sanguinis"] <- "Streptoccus spp.*"
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# by parts
hmd <- filter(heatmap_data, sample_type == "Barretts Only")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "Barretts Only")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "BO", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.2, 0.2, 1, 1),
guides="collect"
) +
plot_annotation(
title="NCI-16s Data showing average relative abundance of species level by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%; *S. dentisani, S. infantis, S. mitis, S. oligofermentans, S. oralis, S. pneumoniae, S. pseudopneumoniae, S. sanguinis")
)
p
if(save.plots == T){
ggsave(paste0("output/nci-species-lvl-heatmap-05-",save.Date,".pdf"), plot=p, units="in", width=20, height=5)
ggsave(paste0("output/nci-species-lvl-heatmap-05-",save.Date,".png"), plot=p, units="in", width=20, height=5)
}Relative Abudance Cutoff: 0.01
analysis.dat <- dat.16s # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.01 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="400px")| AverageRelativeAbundance | OTU |
|---|---|
| 0.250 | Streptococcus_dentisani:Streptococcus_infantis:Streptococcus_mitis:Streptococcus_oligofermentans:Streptococcus_oralis:Streptococcus_pneumoniae:Streptococcus_pseudopneumoniae:Streptococcus_sanguinis |
| 0.076 | Pseudomonas_rhodesiae |
| 0.058 | Prevotella_melaninogenica |
| 0.046 | Stenotrophomonas_maltophilia |
| 0.042 | Lactobacillus_gasseri:Lactobacillus_johnsonii |
| 0.041 | Veillonella_dispar |
| 0.034 | Acinetobacter_guillouiae |
| 0.031 | Fusobacterium_nucleatum |
| 0.025 | Rothia_mucilaginosa |
| 0.024 | Staphylococcus_epidermidis:Staphylococcus_hominis |
| 0.022 | Gemella_haemolysans |
| 0.018 | Selenomonas_sputigena |
| 0.017 | Granulicatella_adiacens:Granulicatella_paraadiacens |
| 0.017 | Haemophilus_parainfluenzae |
| 0.016 | otu19913:Actinobacillus_minor:Actinobacillus_porcinus:Actinobacillus_rossii:Haemophilus_paraphrohaemolyticus |
| 0.012 | otu16698:Tannerella_forsythia |
| 0.011 | Actinomyces_odontolyticus |
| 0.010 | Clostridium_perfringens:Clostridium_thermophilus |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(accession.number)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
plot.dat$OTU[plot.dat$OTU == "Streptococcus_dentisani:Streptococcus_infantis:Streptococcus_mitis:Streptococcus_oligofermentans:Streptococcus_oralis:Streptococcus_pneumoniae:Streptococcus_pseudopneumoniae:Streptococcus_sanguinis"] <- "Streptoccus spp.*"
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# by parts
hmd <- filter(heatmap_data, sample_type == "Barretts Only")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "Barretts Only")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "BO", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.5, 0.2, 1, 1),
guides="collect"
) +
plot_annotation(
title="NCI-16s Data showing average relative abundance of genera by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/nci-species-lvl-heatmap-01-",save.Date,".pdf"), plot=p, units="in", width=25, height=7)
ggsave(paste0("output/nci-species-lvl-heatmap-01-",save.Date,".png"), plot=p, units="in", width=25, height=7)
}Specific OTUs
p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+ plt_hmap4+
plot_layout(
nrow=1, widths = c(0.2, 0.2, 1, 1, 1),
guides="collect"
) +
plot_annotation(
title="NCI-16s Data showing average relative abundance of specific OTUs by individual"
)
p
if(save.plots == T){
ggsave(paste0("output/nci-specific-otus-heatmap-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/nci-specific-otus-heatmap-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}Species Level - TCGA RNAseq Data
The heatmaps for slide 6 focus on the TCGA RNAseq data.
Relative Abudance Cutoff: 0.05
analysis.dat <- dat.rna %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.05 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.426 | Pseudomonas fluorescens group |
| 0.320 | Pseudomonas sp. UW4 |
| 0.075 | Arthrobacter phenanthrenivorans |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.2, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA RNAseq Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-05-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-05-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}Relative Abudance Cutoff: 0.01
analysis.dat <- dat.rna %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.01 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.426 | Pseudomonas fluorescens group |
| 0.320 | Pseudomonas sp. UW4 |
| 0.075 | Arthrobacter phenanthrenivorans |
| 0.041 | Pseudomonas sp. UK4 |
| 0.029 | Bradyrhizobium sp. BTAi1 |
| 0.027 | Pseudomonas putida group |
| 0.011 | Bacillus cereus group |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.2, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA RNAseq Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-01-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-01-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}Relative Abudance Cutoff: 0.001
analysis.dat <- dat.rna %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.001 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.426 | Pseudomonas fluorescens group |
| 0.320 | Pseudomonas sp. UW4 |
| 0.075 | Arthrobacter phenanthrenivorans |
| 0.041 | Pseudomonas sp. UK4 |
| 0.029 | Bradyrhizobium sp. BTAi1 |
| 0.027 | Pseudomonas putida group |
| 0.011 | Bacillus cereus group |
| 0.009 | Propionibacterium acnes |
| 0.006 | Escherichia coli |
| 0.004 | Arthrobacter sp. FB24 |
| 0.004 | Bacillus megaterium |
| 0.003 | Haemophilus influenzae |
| 0.003 | Pseudomonas aeruginosa group |
| 0.003 | Pseudomonas brassicacearum |
| 0.002 | Enterobacter cloacae complex |
| 0.001 | Psychrobacter sp. PRwf-1 |
| 0.001 | Enterococcus faecalis |
| 0.001 | Arthrobacter chlorophenolicus |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.5, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA RNAseq Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-001-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/tcga-rna-species-lvl-heatmap-001-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}Specific OTUs
The heatmaps for slide 9 focus on the TCGA RNAseq data. The difference here is we focus on 4 OTUs.
p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.2, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA RNAseq Data showing average relative abundance of specific OTUs by individual"
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-rna-specific-otus-heatmap-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/tcga-rna-specific-otus-heatmap-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}Sepcies Level - TCGA WGS Data
The heatmaps for slide7 focus on the TCGA WGS data.
Relative Abudance Cutoff: 0.05
analysis.dat <- dat.wgs %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.05 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.465 | Coprobacillus sp. D7 |
| 0.106 | Propionibacterium acnes |
| 0.074 | Haemophilus influenzae |
| 0.057 | Prevotella melaninogenica |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.3, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA WGS Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-05-",save.Date,".pdf"), plot=p, units="in", width=20, height=5)
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-05-",save.Date,".png"), plot=p, units="in", width=20, height=5)
}Relative Abudance Cutoff: 0.01
analysis.dat <- dat.wgs %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.01 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.465 | Coprobacillus sp. D7 |
| 0.106 | Propionibacterium acnes |
| 0.074 | Haemophilus influenzae |
| 0.057 | Prevotella melaninogenica |
| 0.028 | Cyanothece sp. CCY0110 |
| 0.027 | Fusobacterium nucleatum |
| 0.022 | Campylobacter concisus |
| 0.019 | Bradyrhizobium sp. BTAi1 |
| 0.016 | Bradyrhizobium diazoefficiens |
| 0.014 | Streptococcus pneumoniae |
| 0.012 | Bradyrhizobium japonicum |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.3, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA WGS Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-01-",save.Date,".pdf"), plot=p, units="in", width=20, height=5)
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-01-",save.Date,".png"), plot=p, units="in", width=20, height=5)
}Relative Abudance Cutoff: 0.001
analysis.dat <- dat.wgs %>%
dplyr::mutate(OTU = otu2) # insert dataset to be used in analysis
avgRelAbundCutoff <- 0.001 # minimum average relative abundance for OTUs
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))%>%
dplyr::filter(AverageRelativeAbundance>=avgRelAbundCutoff) %>%
dplyr::arrange(desc(AverageRelativeAbundance))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.465 | Coprobacillus sp. D7 |
| 0.106 | Propionibacterium acnes |
| 0.074 | Haemophilus influenzae |
| 0.057 | Prevotella melaninogenica |
| 0.028 | Cyanothece sp. CCY0110 |
| 0.027 | Fusobacterium nucleatum |
| 0.022 | Campylobacter concisus |
| 0.019 | Bradyrhizobium sp. BTAi1 |
| 0.016 | Bradyrhizobium diazoefficiens |
| 0.014 | Streptococcus pneumoniae |
| 0.012 | Bradyrhizobium japonicum |
| 0.010 | Bradyrhizobium sp. S23321 |
| 0.008 | candidate division TM7 single-cell isolate TM7a |
| 0.008 | Veillonella parvula |
| 0.008 | Streptococcus parasanguinis |
| 0.007 | Rhodopseudomonas palustris |
| 0.006 | Pseudomonas fluorescens group |
| 0.006 | Bradyrhizobium sp. ORS 278 |
| 0.006 | Streptococcus mitis |
| 0.005 | Beggiatoa sp. PS |
| 0.004 | Staphylococcus epidermidis |
| 0.004 | Corynebacterium tuberculostearicum |
| 0.004 | Streptococcus suis |
| 0.003 | Haemophilus parainfluenzae |
| 0.003 | Streptococcus pseudopneumoniae |
| 0.003 | Delftia sp. Cs1-4 |
| 0.003 | Lactobacillus gasseri |
| 0.003 | [Eubacterium] hallii |
| 0.002 | Delftia acidovorans |
| 0.002 | Streptococcus oralis |
| 0.002 | Pseudomonas putida group |
| 0.002 | Streptococcus thermophilus |
| 0.002 | Pseudomonas aeruginosa group |
| 0.002 | Atopobium rimae |
| 0.002 | Prevotella intermedia |
| 0.001 | candidate division TM7 single-cell isolate TM7c |
| 0.001 | Acidovorax delafieldii |
| 0.001 | Staphylococcus capitis |
| 0.001 | Escherichia coli |
| 0.001 | Oligotropha carboxidovorans |
| 0.001 | Acinetobacter junii |
| 0.001 | Acidovorax ebreus |
| 0.001 | Lactococcus garvieae |
| 0.001 | Gemmata obscuriglobus |
| 0.001 | Lactobacillus crispatus |
| 0.001 | Streptococcus gordonii |
| 0.001 | Xanthomonas campestris |
| 0.001 | Bacteroides sp. 3_1_33FAA |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::filter(aveAbund>=avgRelAbundCutoff) %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.3, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA WGS Data showing average relative abundance of species by individual",
subtitle=paste0("Subset to OTU average relative abundance > ",prntRelAbund,"%")
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-001-",save.Date,".pdf"), plot=p, units="in", width=20, height=10)
ggsave(paste0("output/tcga-wgs-species-lvl-heatmap-001-",save.Date,".png"), plot=p, units="in", width=20, height=10)
}Specific OTUs
The heatmaps for slide76 focus on the TCGA WGS data.
analysis.dat <- dat.wgs.s # insert dataset to be used in analysis
otu.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::summarise(AverageRelativeAbundance=mean(Abundance, na.rm=T))
kable(otu.dat[,c(2,1)], format="html", digits=3) %>%
kable_styling(full_width = T)%>%
scroll_box(width="100%", height="100%")| AverageRelativeAbundance | OTU |
|---|---|
| 0.027 | Fusobacterium nucleatum |
| 0.005 | Streptococcus spp. |
| 0.022 | Campylobacter concisus |
| 0.057 | Prevotella |
plot.dat <- analysis.dat %>% filter(sample_type != "0") %>%
dplyr::group_by(OTU) %>%
dplyr::mutate(aveAbund=mean(Abundance, na.rm=T)) %>%
dplyr::ungroup() %>%
dplyr::mutate(ID = as.factor(Patient_ID),
Abundance = ifelse(is.na(Abundance), 0, Abundance)) %>%
dplyr::select(sample_type, OTU, ID, Abundance, aveAbund)
# widen plot.dat for dendro
dat.wide <- plot.dat %>%
dplyr::mutate(
ID = paste0(ID, "_",sample_type)
) %>%
dplyr::select(ID, OTU, Abundance) %>%
dplyr::group_by(ID, OTU) %>%
dplyr::summarise(
Abundance = mean(Abundance)
) %>%
tidyr::pivot_wider(
id_cols = OTU,
names_from = ID,
values_from = Abundance,
values_fill = 0
)
rn <- dat.wide$OTU
mat <- as.matrix(dat.wide[,-1])
rownames(mat) <- rn
sample_names <- colnames(mat)
# Obtain the dendrogram
dend <- as.dendrogram(hclust(dist(mat)))
dend_data <- dendro_data(dend)
# Setup the data, so that the layout is inverted (this is more
# "clear" than simply using coord_flip())
segment_data <- with(
segment(dend_data),
data.frame(x = y, y = x, xend = yend, yend = xend))
# Use the dendrogram label data to position the gene labels
gene_pos_table <- with(
dend_data$labels,
data.frame(y_center = x, gene = as.character(label), height = 1))
# Table to position the samples
sample_pos_table <- data.frame(sample = sample_names) %>%
dplyr::mutate(x_center = (1:n()),
width = 1)
# Neglecting the gap parameters
heatmap_data <- mat %>%
reshape2::melt(value.name = "expr", varnames = c("gene", "sample")) %>%
left_join(gene_pos_table) %>%
left_join(sample_pos_table)
# extract and rejoin sample IDs and sample_type names for plotting
# first for the heatmap data.frame
A <- str_split(heatmap_data$sample, "_")
heatmap_data$ID <- heatmap_data$sample_type <- "0"
for(i in 1:nrow(heatmap_data)){
heatmap_data$ID[i] <- A[[i]][1]
heatmap_data$sample_type[i] <- A[[i]][2]
}
# second for the sample position dataframe (dendo)
A <- str_split(sample_pos_table$sample, "_")
sample_pos_table$ID <- sample_pos_table$sample_type <- "0"
for(i in 1:nrow(sample_pos_table)){
sample_pos_table$ID[i] <- A[[i]][1]
sample_pos_table$sample_type[i] <- A[[i]][2]
}
# Limits for the vertical axes
gene_axis_limits <- with(
gene_pos_table,
c(min(y_center - 0.5 * height), max(y_center + 0.5 * height))
) +
0.1 * c(-1, 1) # extra spacing: 0.1
## Build Heatmap Pieces
# EAC w/ Barrets
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap1 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
# margin: top, right, bottom, and left
#axis.ticks.y = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid = element_blank(),
legend.position = "none")
# Part 2: "EAC-adjacent tissue w/ Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC-adjacent tissue w/ Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC-adjacent tissue w/ Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap2 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("expr",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.1, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC-adj. w/ Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.5,vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank(),
legend.position = "none")
# Part 3: "EAC tissues w/o Barretts History"
hmd <- filter(heatmap_data, sample_type == "EAC tissues w/o Barretts History")
hmd$x_center <- as.numeric(as.factor(hmd$x_center))
spd <- filter(sample_pos_table, sample_type == "EAC tissues w/o Barretts History")
spd$x_center <- as.numeric(as.factor(spd$x_center))
plt_hmap3 <- ggplot(hmd,
aes(x = x_center, y = y_center, fill = expr,
height = height, width = width)) +
geom_tile() +
#facet_wrap(.~sample_type)+
scale_fill_gradient2("Abundance",trans="sqrt", high = "darkred", low = "darkblue", breaks=c(0, 0.10, 0.30, 0.50, 0.80)) +
scale_x_continuous(breaks = spd$x_center,
labels = spd$ID,
expand = c(0, 0)) +
# For the y axis, alternatively set the labels as: gene_position_table$gene
scale_y_continuous(breaks = gene_pos_table[, "y_center"],
labels = rep("", nrow(gene_pos_table)),
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "EAC w/o Barretts", y = NULL) +
theme_classic() +
theme(axis.text.x = element_text(size = rel(1), hjust = 0.75, vjust=0.5, angle = 90),
axis.ticks.y = element_blank(),
# margin: top, right, bottom, and left
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"),
panel.grid.minor = element_blank())
# Dendrogram plot
plt_dendr <- ggplot(segment_data) +
geom_segment(aes(x = x, y = y, xend = xend, yend = yend)) +
scale_x_reverse(expand = c(0, 0.5)) +
scale_y_continuous(breaks = gene_pos_table$y_center,
labels = gene_pos_table$gene,
limits = gene_axis_limits,
expand = c(0, 0)) +
labs(x = "", y = "", colour = "", size = "") +
theme_classic() +
theme(panel.grid = element_blank(),
axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
plot.margin = unit(c(1, 0.01, 0.01, -0.7), "cm"))
prntRelAbund <- avgRelAbundCutoff*100p <- plt_dendr+plt_hmap1+plt_hmap2+plt_hmap3+
plot_layout(
nrow=1, widths = c(0.5, 0.4, 0.15, 1),
guides="collect"
) +
plot_annotation(
title="TCGA WGS Data showing average relative abundance of specific OTUs by individual"
)
p
if(save.plots == T){
ggsave(paste0("output/tcga-wgs-specific-otus-heatmap-",save.Date,".pdf"), plot=p, units="in", width=25, height=5)
ggsave(paste0("output/tcga-wgs-specific-otus-heatmap-",save.Date,".png"), plot=p, units="in", width=25, height=5)
}
sessionInfo()R version 4.0.3 (2020-10-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)
Matrix products: default
locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.1252
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] cowplot_1.1.1 dendextend_1.14.0 ggdendro_0.1.22 reshape2_1.4.4
[5] car_3.0-10 carData_3.0-4 gvlma_1.0.0.3 patchwork_1.1.1
[9] viridis_0.5.1 viridisLite_0.3.0 gridExtra_2.3 xtable_1.8-4
[13] kableExtra_1.3.1 data.table_1.13.6 readxl_1.3.1 forcats_0.5.1
[17] stringr_1.4.0 dplyr_1.0.3 purrr_0.3.4 readr_1.4.0
[21] tidyr_1.1.2 tibble_3.0.6 ggplot2_3.3.3 tidyverse_1.3.0
[25] lmerTest_3.1-3 lme4_1.1-26 Matrix_1.2-18 vegan_2.5-7
[29] lattice_0.20-41 permute_0.9-5 phyloseq_1.34.0 workflowr_1.6.2
loaded via a namespace (and not attached):
[1] minqa_1.2.4 colorspace_2.0-0 rio_0.5.16
[4] ellipsis_0.3.1 rprojroot_2.0.2 XVector_0.30.0
[7] fs_1.5.0 rstudioapi_0.13 farver_2.0.3
[10] lubridate_1.7.9.2 xml2_1.3.2 codetools_0.2-16
[13] splines_4.0.3 knitr_1.31 ade4_1.7-16
[16] jsonlite_1.7.2 nloptr_1.2.2.2 broom_0.7.4
[19] cluster_2.1.0 dbplyr_2.1.0 BiocManager_1.30.10
[22] compiler_4.0.3 httr_1.4.2 backports_1.2.1
[25] assertthat_0.2.1 cli_2.3.0 later_1.1.0.1
[28] htmltools_0.5.1.1 prettyunits_1.1.1 tools_4.0.3
[31] igraph_1.2.6 gtable_0.3.0 glue_1.4.2
[34] Rcpp_1.0.6 Biobase_2.50.0 cellranger_1.1.0
[37] vctrs_0.3.6 Biostrings_2.58.0 rhdf5filters_1.2.0
[40] multtest_2.46.0 ape_5.4-1 nlme_3.1-149
[43] iterators_1.0.13 xfun_0.20 ps_1.5.0
[46] openxlsx_4.2.3 rvest_0.3.6 lifecycle_0.2.0
[49] statmod_1.4.35 zlibbioc_1.36.0 MASS_7.3-53
[52] scales_1.1.1 hms_1.0.0 promises_1.1.1
[55] parallel_4.0.3 biomformat_1.18.0 rhdf5_2.34.0
[58] curl_4.3 yaml_2.2.1 stringi_1.5.3
[61] highr_0.8 S4Vectors_0.28.1 foreach_1.5.1
[64] BiocGenerics_0.36.0 zip_2.1.1 boot_1.3-25
[67] rlang_0.4.10 pkgconfig_2.0.3 evaluate_0.14
[70] Rhdf5lib_1.12.1 labeling_0.4.2 tidyselect_1.1.0
[73] plyr_1.8.6 magrittr_2.0.1 R6_2.5.0
[76] IRanges_2.24.1 generics_0.1.0 DBI_1.1.1
[79] foreign_0.8-80 pillar_1.4.7 haven_2.3.1
[82] withr_2.4.1 mgcv_1.8-33 abind_1.4-5
[85] survival_3.2-7 modelr_0.1.8 crayon_1.4.1
[88] rmarkdown_2.6 progress_1.2.2 grid_4.0.3
[91] git2r_0.28.0 reprex_1.0.0 digest_0.6.27
[94] webshot_0.5.2 httpuv_1.5.5 numDeriv_2016.8-1.1
[97] stats4_4.0.3 munsell_0.5.0