dev/benchmark/TCGA_workflow_ttBulk_for_comparison.R

In stemangiola/ttBulk: Brings transcriptomics to the tidyverse

library(tidyverse)
library(tidybulk)
library(pasilla)
library(tictoc)

options(tidybulk_do_validate = FALSE) 


tt = readRDS("dev/PRAD_TCGA_counts.rds") %>% tidybulk(patient, transcript, count) %>% aggregate_duplicates() %>% filter(transcript %>% is.na %>% `!`)

time_df = tibble(step = "", time = list(), lines = NA, assignments = NA)

# START WORKFLOW
plot_densities = function(){
	# Normalise
tt_scaled = tt %>% identify_abundant(factor_of_interest = PFI.2 ) %>% scale_abundance()
# Plot densities
 p = tt_scaled %>%
	pivot_longer(	values_to = "count",	names_to = "Normalisation",	cols = c(count, `count_scaled`)	) %>%
	ggplot(aes(count + 1, group = patient, color = PFI.2)) +
	facet_grid( ~ Normalisation) +
	geom_density() +
	scale_x_log10()

	tt_scaled
}
plot_MDS = function(){
	tt_mds = tt_scaled %>% 	reduce_dimensions(method = "MDS", .dims = 3)
# Visualise MDS for cell types
p = tt_mds %>%
	select(contains("Dim"), patient, PFI.2) %>%
	distinct() %>%
	GGally::ggpairs(	columns = 1:3,	ggplot2::aes(colour = PFI.2),	upper = list(continuous = GGally::wrap("cor", size = 3))	)

	tt_mds
}
plot_adjusted_MDS = function(){
	# Adjust abundance for type, and check MDS densities
	p = tt_mds %>%
		
		# Add fake batch
		nest(data = -patient) %>%
		mutate(batch = sample(0:1, size = n(), replace = TRUE)) %>%
		unnest(data) %>%
		
		adjust_abundance( ~  PFI.2 + batch) %>%
		filter(`count_scaled_adjusted` %>% is.na %>% `!`) %>%
		reduce_dimensions(.abundance = `count_scaled_adjusted`,	method = "MDS",	.dims = 3) %>%
		
		# Plot
		select(contains("Dim"), patient, PFI.2,  batch) %>%
		distinct() %>%
		pivot_longer(names_to = "Dim", values_to = ".value", cols = contains("Dim")) %>%
		separate(Dim, c("Dim", "Adj"), sep = "\\.") %>%
		pivot_longer(names_to = "variation",	values_to = "Type of variation",	cols = c(PFI.2, batch)	) %>%
		ggplot(aes(y = .value, x = variation, fill = `Type of variation`)) +
		geom_boxplot() +
		facet_grid(Adj ~ Dim)
	
}

test_abundance = function(){
	# DE analyses
 tt_scaled %>% test_differential_abundance( ~ PFI.2)
}

plot_MA = function(){
	# MA plot
	p = tt_test %>%
		keep_abundant() %>%
		mutate(de = FDR < 0.05 & abs(logFC) >= 2) %>%
		distinct(transcript, logCPM, logFC, de) %>%
		mutate(transcript = ifelse(de &	abs(logFC) > 3, as.character(transcript), NA)) %>%
		ggplot(aes(x = logCPM, y = logFC, label = transcript)) +
		geom_point(aes(	color = de,	size = de,	alpha = de)) +
		ggrepel::geom_text_repel()
}
plot_DE_comparative = function(){
	# Plot top genes for raw, scaled and ajusted counts
	p = tt_test %>%
		inner_join((.) %>% distinct(transcript, PValue) %>% arrange(PValue) %>% head(6)) %>%
		pivot_longer(names_to = "Stage",	 values_to = "count",	 cols = starts_with("count")) %>%
		ggplot(aes(x = Stage, y = count + 1, fill = PFI.2)) +
		facet_wrap( ~ transcript) +
		scale_y_log10()
}
plot_heatmap = function(){
	p = tt_test %>%
		filter(FDR < 0.05 & abs(logFC) > 2) %>%
		tidyHeatmap::heatmap(	 patient,		 transcript,		 count, transform = log1p 	) 
}

tic()
tt_scaled = plot_densities()
time_df = time_df %>% bind_rows(tibble(step = "Normalisation", time = list(toc()), lines = 7, assignments = 1))

tic()
tt_mds = plot_MDS()
time_df = time_df %>% bind_rows(tibble(step = "Reduce dimensionality", time = list(toc()), lines = 5, assignments = 1))

tic()
plot_adjusted_MDS()
time_df = time_df %>% bind_rows(tibble(step = "Removal unwanted variation", time = list(toc()), lines = 12, assignments = 0))

tic()
tt_test = test_abundance()
time_df = time_df %>% bind_rows(tibble(step = "Test differential abundance", time = list(toc()), lines = 1, assignments = 0))

tic()
plot_MA()
time_df = time_df %>% bind_rows(tibble(step = "Plot MA", time = list(toc()), lines = 8, assignments = 0))

tic()
plot_DE_comparative()
time_df = time_df %>% bind_rows(tibble(step = "Plot results across stages", time = list(toc()), lines = 7, assignments = 0))

tic()
plot_heatmap()
time_df = time_df %>% bind_rows(tibble(step = "Plot heatmap", time = list(toc()), lines = 3, assignments = 0))



time_df  %>% mutate(step = factor(step, levels = unique(step))) %>%  saveRDS("dev/stats_TCGA_ttBulk.rds")


# > time_df = time_df %>% bind_rows(tibble(step = "Normalisation", time = list(toc()), lines = 7, assignments = 1))
# 38.097 sec elapsed
# > 
# 	> tic()
# > tt_mds = plot_MDS()
# tidybulk says: to access the raw results do `attr(..., "internals")$MDS`
# > time_df = time_df %>% bind_rows(tibble(step = "Reduce dimensionality", time = list(toc()), lines = 5, assignments = 1))
# 177.665 sec elapsed
# > 
# 	> tic()
# > tt_test = test_abundance()
# tidybulk says: The design column names are "(Intercept), PFI.2"
# tidybulk says: to access the raw results (fitted GLM) do `attr(..., "internals")$edgeR`
# > time_df = time_df %>% bind_rows(tibble(step = "Test differential abundance", time = list(toc()), lines = 1, assignments = 0))
# 109.862 sec elapsed
# >