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asd_workflow.R
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asd_workflow.R
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# This script crashes with 16 GB or less of RAM.
# Rsession will use 30 GB or RAM in the long-run, not sure about peaks.
# Packages and functions --------------------------------------------------------------------
library( tidyverse )
require( rje )
library( Matrix )
library( irlba )
library( uwot )
library( FNN )
library( igraph )
library( cowplot )
# rowVars for sparse matrices:
colVars_spm <- function( spm ) {
stopifnot( is( spm, "dgCMatrix" ) )
ans <- sapply( seq.int(spm@Dim[2]), function(j) {
mean <- sum( spm@x[ (spm@p[j]+1):spm@p[j+1] ] ) / spm@Dim[1]
sum( ( spm@x[ (spm@p[j]+1):spm@p[j+1] ] - mean )^2 ) +
mean^2 * ( spm@Dim[1] - ( spm@p[j+1] - spm@p[j] ) ) } ) / ( spm@Dim[1] - 1 )
names(ans) <- spm@Dimnames[[2]]
ans
}
rowVars_spm <- function( spm ) {
colVars_spm( t(spm) )
}
# define scale_color_sqrt (and functions it requires):
power_trans <- function(power){
# returns transformation object that can be used in ggplot's scale_*_continuous
scales::trans_new(
name = "tmp",
trans = function(x) x^(power),
inverse = function(x) x^(1/power),
breaks = function(lims, p) power_breaks(lims, p=power) )
}
power_breaks <- function(lims, power, n_breaks=5){
# Return vector of breaks that span the lims range evenly _after_ power transformation:
lims[1] <- max(0, lims[1]) # non-integer exponents are not defined for negative values
x <- seq(lims[1]^power, lims[2]^(power), length.out = n_breaks)^(1/power)
# make human-readable by rounding to the closest integer power of 2. Smallest
# and largest ticks are not strictly rounded - instead they are moved within
# the range of values, since ggplot would not display them otherwise:
x <- case_when(
x == max(x) ~ 2^(floor(log2(x))),
x == min(x) ~ 2^(ceiling(log2(x))),
TRUE ~ (2^(round(log2(x))))
)
return(x)
}
semi_scientific_formatting <- function(x) {
# takes numeric vector x and returns character vector where extremely large / small
# numbers are in scientific notation (e.g. 1e-30) while others are untouched:
x <- case_when(
x == 0 ~ as.character(0),
abs(x) < .01 | abs(x) >= 1000 ~ scales::scientific(x, digits = 0),
TRUE ~ as.character(x))}
scale_color_sqrt <- function(...){scale_color_gradientn(
colours = rev(rje::cubeHelix(100))[5:100],
trans = power_trans(1/2),
labels = semi_scientific_formatting,
...)}
# Load data ---------------------------------------------------------------
path <- "~/sds/sd17l002/p/ASD/"
cellinfo <- read.delim( file.path( path, "rawMatrix", "meta.txt" ), stringsAsFactors=FALSE )
counts <- readMM( file.path( path, "rawMatrix", "matrix.mtx" ) )
# make gene symbols unique (by concatenating ensembleID where necessary):
gene_info <- read.delim( file.path( path, "rawMatrix", "genes.tsv" ), header=FALSE, as.is=TRUE ) %>%
mutate(unique = case_when(
duplicated(V2) | duplicated(V2, fromLast=T) ~ paste(V2, V1, sep="_"),
TRUE ~ V2))
rownames(counts) <- gene_info$unique
colnames(counts) <- readLines( file.path( path, "rawMatrix", "barcodes.tsv" ) )
sampleTable <-
cellinfo %>% select( sample : RNA.Integrity.Number ) %>% unique
sampleTable
# extracting gene expression is much faster in column-sparse format:
Tcounts <- as(t(counts), "dgCMatrix") # fast: Tcounts[, "SYN1"]
Ccounts <- as(counts, "dgCMatrix") # fast: Ccounts[, 1337] and colSums(Ccounts)
# Preprocessing -----------------------------------------------------------
# load (or re-execute everything in this section):
sfs <- colSums(Ccounts)
norm_counts <- t(t(Ccounts) / sfs)
rownames(norm_counts) <- rownames(Ccounts)
load(file.path(path, "savepoint", "pca_40pcs_scaling_2311genes.RData"))
load(file.path(path, "savepoint", "umap_euc_spread10.RData"))
# informative genes, PCA, UMAP:
poisson_vmr <- mean(1/sfs)
gene_means <- rowMeans( norm_counts )
gene_vars <- rowVars_spm( norm_counts )
cells_expressing <- colSums( Tcounts != 0 )
is_informative <- gene_vars/gene_means > 1.5 * poisson_vmr & cells_expressing > 100
plot(gene_means, gene_vars/gene_means, pch=".", log = "xy")
points(gene_means[is_informative], (gene_vars/gene_means)[is_informative], pch=".", col = "red" )
pca <- irlba::prcomp_irlba( x = sqrt(t(norm_counts[is_informative,])),
n = 40,
scale. = TRUE)
umap_euc <- uwot::umap( pca$x, spread = 10, n_threads = 40) # euc: euclidean distance
# save(pca,
# file = file.path(path, "savepoint", "pca_40pcs_scaling_2311genes.RData"))
# save(umap_euc,
# file = file.path(path, "savepoint", "umap_euc_spread10.RData"))
# Clusters ---------------------------------------------------
# load (or re-execute everything in this section):
load(file.path(path, "savepoint", "clusters.RData"))
# find NN for each cell:
library( RcppAnnoy )
featureMatrix <- pca$x; k_nn <- 50
annoy <- new( AnnoyEuclidean, ncol(featureMatrix) )
for( i in 1:nrow(featureMatrix) )
annoy$addItem( i-1, featureMatrix[i,] )
annoy$build( 50 ) # builds a forest of n_trees trees. More trees gives higher precision when querying.
nn_cells <- t( sapply( 1:annoy$getNItems(), function(i) annoy$getNNsByItem( i-1, k_nn) + 1 ) )
nndists_cells <- sapply( 1:ncol(nn_cells), function(j) sqrt( rowSums( ( featureMatrix - featureMatrix[ nn_cells[,j], ] )^2 ) ) )
rm(featureMatrix, annoy)
# cluster on nearest neighbor graph (Louvain):
adj <- Matrix(0, nrow = nrow(pca$x), ncol = nrow(pca$x)) # has to be sparse, otherwise takes 80 GB of RAM
for(i in 1:ncol(nn_cells))
adj[ cbind(1:nrow(pca$x), nn_cells[, i]) ] <- 1
for(i in 1:ncol(nn_cells))
adj[ cbind(nn_cells[, i], 1:nrow(pca$x)) ] <- 1
cl_louvain <- cluster_louvain( graph_from_adjacency_matrix(adj, mode = "undirected") )
# merge clusters that are separated by patient heterogeneity:
tmp_clusters <- cl_louvain$membership
tmp_clusters <- case_when(tmp_clusters %in% c(5, 6, 8, 1, 10, 20, 2, 16) ~ 5, # excitatory Neurons
tmp_clusters %in% c(11, 15, 19) ~ 11, # astrocytes
tmp_clusters %in% c(3, 9, 18) ~ 3, # OPCs
tmp_clusters %in% c(22, 17) ~ 22, # endothelial and/or pericytes
TRUE ~ tmp_clusters)
anno_clusters = c(
"3" = "OPC",
"4" = "Oligodendrocyte",
"5" = "neurons_excit",
"7" = "IN_PV",
"11"= "Astrocyte",
"12"= "IN_SV2C",
"13"= "Microglia",
"14"= "IN_VIP",
"21"= "neurons_NRGN",
"22"= "endothelial_and_pericytes",
"23"= "IN_SST"
)
celltypes <- factor(anno_clusters[as.character(tmp_clusters)],
levels= anno_clusters[as.character(sort(unique(tmp_clusters)))])
# Louvain clusters
p_louv <- ggplot()+ coord_fixed() +
geom_point(data = data.frame(umap_euc, cl=factor(tmp_clusters)),
aes(X1, X2, col = cl), size = .1) +
geom_label(data = group_by(data.frame(umap_euc, cl=factor(tmp_clusters)), cl) %>%summarise(X1=mean(X1), X2=mean(X2)),
aes(X1, X2, label = cl))
p_louv
# clusters from paper
p_paper <- ggplot()+ coord_fixed()+
geom_point(data =data.frame(cell = colnames(counts), umap_euc) %>%
left_join(select(cellinfo, cell, cluster), by="cell"),
aes(X1, X2, col = cluster), size = .1) +
geom_label(data = data.frame(cell = colnames(counts), umap_euc) %>%
left_join(select(cellinfo, cell, cluster), by = "cell") %>% group_by(cluster) %>%
summarise(X1=mean(X1), X2=mean(X2)),
aes(X1, X2, label = cluster))
p_paper
# save(list = c("cl_louvain", "tmp_clusters", "celltypes", "anno_clusters",
# "nn_cells", "nn_inothercluster"),
# file = file.path(path, "savepoint", "clusters.RData"))
# Doublets and ambiguous cells ----------------------------------
# load (or re-execute everything in this section):
load(file.path(path, "savepoint", "doublets.RData"))
# number of NN from different cluster:
nn_inothercluster <- colSums(
matrix(tmp_clusters[ t(nn_cells) ],
ncol = nrow(nn_cells)) !=
matrix(rep(tmp_clusters, each = ncol(nn_cells)),
ncol = nrow(nn_cells)) )
# in silico doublets: randomly draw cells from different clusters and pool their UMIs to form a "synthetic" doublet:
cellsA <- sample(1:ncol(counts), 50000)
cellsB <- rep(NA, 50000)
smpA <- cellinfo$sample[cellsA]
clA <- tmp_clusters[cellsA]
tmp <- data.frame(smpA, clA) %>% group_by(smpA, clA) %>% tally
for(i in 1:nrow(tmp)) {
is_smp <- cellinfo$sample[cellsA] == tmp$smpA[i]
is_cl <- tmp_clusters[cellsA] == tmp$clA[i]
# sample amongst cells from same sample and different cluster:
cellsB[ is_smp & is_cl ] <- base::sample(
x = which(cellinfo$sample == tmp$smpA[i] & !tmp_clusters == tmp$clA[i]),
size = tmp$n[i],
replace = T) # in case one cluster is larger than all others combined
}
doublet_raw <- Ccounts[, cellsA] + Ccounts[, cellsB]
doublet_pcs <- predict(pca,
newdata = sqrt( (t(doublet_raw) / colSums(doublet_raw))[, is_informative] ))
# Alternative 1 (clearer):
a <- FNN::get.knn(rbind(pca$x, doublet_pcs), k = 50)
nn_doublets <- a$nn.index
nndists_doublets <- a$nn.dist
# Alternative 2 (faster):
library( RcppAnnoy )
featureMatrix <- rbind(pca$x, doublet_pcs); k_nn <- 50
annoy <- new( AnnoyEuclidean, ncol(featureMatrix) )
for( i in 1:nrow(featureMatrix) )
annoy$addItem( i-1, featureMatrix[i,] )
annoy$build( 50 ) # builds a forest of n_trees trees. More trees gives higher precision when querying.
nn_doublets <- t( sapply( 1:annoy$getNItems(), function(i) annoy$getNNsByItem( i-1, k_nn) + 1 ) )
nndists_doublets <- sapply( 1:ncol(nn_doublets), function(j) sqrt( rowSums( ( featureMatrix - featureMatrix[ nn_doublets[,j], ] )^2 ) ) )
rm(featureMatrix, annoy)
# percentage of synthetic doublets in neighborhood for each cell:
dblts_perc <- rowMeans( nn_doublets > ncol(counts) )[ 1:ncol(counts) ]
# Run UMAP with Annoy's output
ump2 <- uwot::umap( NULL, nn_method = list( idx=nn_doublets, dist=nndists_doublets),
n_threads=40, spread = 15, verbose=TRUE )
is_synth <- 1:nrow(ump2) > nrow(pca$x)
# save(list = c("nn_doublets", "nndists_doublets", "cellsA", "cellsB",
# "dblts_perc", "is_synth", "ump2"),
# file = file.path(path, "savepoint", "doublets.RData"))
# DESeq -------------------------------------------------------------------
library(DESeq2)
library(BiocParallel)
# visualize dirty cells we clean away:
tmp <- data.frame(umap_euc,
diagnosis = cellinfo$diagnosis,
clean = dblts_perc < 3/50 & nn_inothercluster < 1,
Gene = Tcounts[, "TTF2"] / sfs/mean(1/sfs),
cl = factor(celltypes))
ggplot() + coord_fixed()+
geom_point(data=filter(tmp, clean), aes(X1, X2, col = cl), size=.1) +
geom_point(data=filter(tmp, !clean), aes(X1, X2), col = "black", size=.1) +
geom_label(data=group_by(tmp, cl) %>% summarise(X1=mean(X1), X2=mean(X2)), aes(X1, X2, label=cl))
tmp <- as.matrix(table(sample=cellinfo$sample, clean = dblts_perc < 3/50 & nn_inothercluster < 1))
data.frame(sample = rownames(tmp), dirtyProportion = tmp[,1] / (tmp[,1] + tmp[,2])) %>% left_join(sampleTable, by="sample") %>% ggplot(aes(sample, dirtyProportion, col = diagnosis))+geom_point()
# compute for a single cluster
sel <- celltypes == "neurons_excit" & dblts_perc < 3/50 & nn_inothercluster < 1
pseudobulks <- as.matrix(t( fac2sparse(cellinfo$sample[sel]) %*% t(Ccounts[, sel]) ))
coldat <- filter(sampleTable, sample %in% colnames(pseudobulks)) %>%
mutate(individual = factor(individual),
diagnosis = factor(diagnosis, levels = c("Control", "ASD")),
region = factor(region))
rownames(coldat) <- coldat$sample
dds <- DESeq2::DESeqDataSetFromMatrix( pseudobulks,
coldat[colnames(pseudobulks), ],
design = ~ sex + region + age + diagnosis )
# For cluster 5, I tested that we do not need interactions between sex, region and diagnosis. I used
# DESeq's LTR for this (see mail to Simon at mid-September 2019).
dds <- DESeq2::DESeq(dds,
parallel=TRUE, BPPARAM=BiocParallel::MulticoreParam(20))
res_df <- DESeq2::results(dds, name = "diagnosis_ASD_vs_Control") %>% as.data.frame() %>% rownames_to_column("Gene")
data.frame(umap_euc, Gene = Tcounts[, "ZNF770"], sfs=sfs, diagnosis=cellinfo$diagnosis) %>%
ggplot(aes(X1, X2, col=Gene/sfs/mean(1/sfs)))+geom_point(size=.1) +
scale_color_sqrt(name="ZNF770") +
facet_wrap(~ diagnosis) + coord_fixed()