Cluster analysis
Katharina Hembach
10/16/2020
Last updated: 2021-07-14
Checks: 7 0
Knit directory: neural_scRNAseq/
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|---|---|---|---|---|
| Rmd | d2fc383 | khembach | 2021-07-14 | include UNC13A in heatmap and feature plot |
| html | ecba9c2 | khembach | 2021-04-20 | Build site. |
| Rmd | 459907c | khembach | 2021-04-20 | include CSNK1E in list of marker genes; new heatmap colors |
| html | a983fc3 | khembach | 2021-01-21 | Build site. |
| html | 32f4cd3 | khembach | 2021-01-21 | Build site. |
| Rmd | 6c9f7ba | khembach | 2021-01-21 | heatmap with virus cell tropism markers |
| html | c56d284 | khembach | 2020-11-12 | Build site. |
| Rmd | ca4b784 | khembach | 2020-11-12 | save marker genes |
| html | ac0f11a | khembach | 2020-11-11 | Build site. |
| Rmd | d96dd7c | khembach | 2020-11-11 | fix cluster number and gene names for heatmap |
| html | 090b450 | khembach | 2020-11-11 | Build site. |
| html | a31892e | khembach | 2020-11-11 | Build site. |
| Rmd | 6a0642f | khembach | 2020-11-11 | subset to only glial clusters for reactive astrocyte heatmap |
| html | 4622291 | khembach | 2020-11-11 | Build site. |
| Rmd | 209f182 | khembach | 2020-11-11 | analyse expression of reactive astrocyte markers |
| html | ecbde99 | khembach | 2020-10-16 | Build site. |
| Rmd | 87ac379 | khembach | 2020-10-16 | DR with TDP expression |
| html | 3a0cb5c | khembach | 2020-10-16 | Build site. |
| Rmd | a528e83 | khembach | 2020-10-16 | cluster analysis TDP experiment |
Load packages
library(ComplexHeatmap)
library(cowplot)
library(ggplot2)
library(dplyr)
library(muscat)
library(purrr)
library(RColorBrewer)
library(viridis)
library(scran)
library(Seurat)
library(SingleCellExperiment)
library(stringr)
library(RCurl)
library(BiocParallel)Load data & convert to SCE
so <- readRDS(file.path("output", "so_TDP_05_plasmid_expression.rds"))
sce <- as.SingleCellExperiment(so, assay = "RNA")
colData(sce) <- as.data.frame(colData(sce)) %>%
mutate_if(is.character, as.factor) %>%
DataFrame(row.names = colnames(sce))Number of clusters by resolution
cluster_cols <- grep("res.[0-9]", colnames(colData(sce)), value = TRUE)
sapply(colData(sce)[cluster_cols], nlevels)RNA_snn_res.0.2 RNA_snn_res.0.4 RNA_snn_res.0.8 RNA_snn_res.1
11 17 24 25 Cluster-sample counts
# set cluster IDs to resolution 0.4 clustering
so <- SetIdent(so, value = "RNA_snn_res.0.4")
so@meta.data$cluster_id <- Idents(so)
sce$cluster_id <- Idents(so)
(n_cells <- table(sce$cluster_id, sce$sample_id))
TDP2wON TDP4wOFF TDP4wONa TDP4wONb
0 1188 1015 1599 1384
1 1168 938 1621 943
2 907 811 1066 1091
3 725 619 925 775
4 616 560 996 771
5 672 594 846 802
6 576 411 477 243
7 375 348 530 446
8 450 307 444 467
9 254 176 396 304
10 207 174 307 251
11 63 64 231 143
12 97 3 88 36
13 49 14 42 32
14 37 17 23 24
15 12 25 32 9
16 10 1 42 1Relative cluster-abundances
fqs <- prop.table(n_cells, margin = 2)
mat <- round(as.matrix(unclass(fqs))*100, 2)
colfunc <- colorRampPalette(c("ghostwhite", "deepskyblue4"))
Heatmap(mat,
# col = rev(brewer.pal(11, "RdGy")[-6]),
col = colfunc(10),
name = "Percentage\nof cells",
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
row_title = "cluster ID",
column_title = "sample ID",
column_title_side = "bottom",
rect_gp = gpar(col = "white"),
cell_fun = function(i, j, x, y, width, height, fill)
grid.text(mat[j, i], x = x, y = y,
gp = gpar(col = "black", fontsize = 10)))
Cell cycle scoring with Seurat
We assign each cell a cell cycle scores and visualize them in the DR plots. We use known G2/M and S phase markers that come with the Seurat package. The markers are anticorrelated and cells that to not express the markers should be in G1 phase.
We compute cell cycle phase:
# A list of cell cycle markers, from Tirosh et al, 2015
cc_file <- getURL("https://raw.githubusercontent.com/hbc/tinyatlas/master/cell_cycle/Homo_sapiens.csv")
cc_genes <- read.csv(text = cc_file)
# match the marker genes to the features
m <- match(cc_genes$geneID[cc_genes$phase == "S"],
str_split(rownames(GetAssayData(so)),
pattern = "\\.", simplify = TRUE)[,1])
s_genes <- rownames(GetAssayData(so))[m]
(s_genes <- s_genes[!is.na(s_genes)]) [1] "ENSG00000012963.UBR7" "ENSG00000049541.RFC2"
[3] "ENSG00000051180.RAD51" "ENSG00000073111.MCM2"
[5] "ENSG00000075131.TIPIN" "ENSG00000076003.MCM6"
[7] "ENSG00000076248.UNG" "ENSG00000077514.POLD3"
[9] "ENSG00000092470.WDR76" "ENSG00000092853.CLSPN"
[11] "ENSG00000093009.CDC45" "ENSG00000094804.CDC6"
[13] "ENSG00000095002.MSH2" "ENSG00000100297.MCM5"
[15] "ENSG00000101868.POLA1" "ENSG00000104738.MCM4"
[17] "ENSG00000111247.RAD51AP1" "ENSG00000112312.GMNN"
[19] "ENSG00000117748.RPA2" "ENSG00000118412.CASP8AP2"
[21] "ENSG00000119969.HELLS" "ENSG00000131153.GINS2"
[23] "ENSG00000132646.PCNA" "ENSG00000132780.NASP"
[25] "ENSG00000136492.BRIP1" "ENSG00000136982.DSCC1"
[27] "ENSG00000143476.DTL" "ENSG00000144354.CDCA7"
[29] "ENSG00000151725.CENPU" "ENSG00000156802.ATAD2"
[31] "ENSG00000159259.CHAF1B" "ENSG00000162607.USP1"
[33] "ENSG00000163950.SLBP" "ENSG00000167325.RRM1"
[35] "ENSG00000168496.FEN1" "ENSG00000171848.RRM2"
[37] "ENSG00000175305.CCNE2" "ENSG00000176890.TYMS"
[39] "ENSG00000197299.BLM" "ENSG00000198056.PRIM1"
[41] "ENSG00000276043.UHRF1" m <- match(cc_genes$geneID[cc_genes$phase == "G2/M"],
str_split(rownames(GetAssayData(so)),
pattern = "\\.", simplify = TRUE)[,1])
g2m_genes <- rownames(GetAssayData(so))[m]
(g2m_genes <- g2m_genes[!is.na(g2m_genes)]) [1] "ENSG00000010292.NCAPD2" "ENSG00000011426.ANLN"
[3] "ENSG00000013810.TACC3" "ENSG00000072571.HMMR"
[5] "ENSG00000075218.GTSE1" "ENSG00000080986.NDC80"
[7] "ENSG00000087586.AURKA" "ENSG00000088325.TPX2"
[9] "ENSG00000089685.BIRC5" "ENSG00000092140.G2E3"
[11] "ENSG00000094916.CBX5" "ENSG00000100401.RANGAP1"
[13] "ENSG00000102974.CTCF" "ENSG00000111665.CDCA3"
[15] "ENSG00000112742.TTK" "ENSG00000113810.SMC4"
[17] "ENSG00000114346.ECT2" "ENSG00000115163.CENPA"
[19] "ENSG00000117399.CDC20" "ENSG00000117724.CENPF"
[21] "ENSG00000120802.TMPO" "ENSG00000123975.CKS2"
[23] "ENSG00000126787.DLGAP5" "ENSG00000129195.PIMREG"
[25] "ENSG00000131747.TOP2A" "ENSG00000134222.PSRC1"
[27] "ENSG00000134690.CDCA8" "ENSG00000136108.CKAP2"
[29] "ENSG00000137804.NUSAP1" "ENSG00000137807.KIF23"
[31] "ENSG00000138160.KIF11" "ENSG00000138182.KIF20B"
[33] "ENSG00000138778.CENPE" "ENSG00000139354.GAS2L3"
[35] "ENSG00000143228.NUF2" "ENSG00000143401.ANP32E"
[37] "ENSG00000143815.LBR" "ENSG00000148773.MKI67"
[39] "ENSG00000157456.CCNB2" "ENSG00000164104.HMGB2"
[41] "ENSG00000169607.CKAP2L" "ENSG00000169679.BUB1"
[43] "ENSG00000170312.CDK1" "ENSG00000173207.CKS1B"
[45] "ENSG00000175063.UBE2C" "ENSG00000175216.CKAP5"
[47] "ENSG00000184661.CDCA2" "ENSG00000188229.TUBB4B"
[49] "ENSG00000189159.JPT1" so <- CellCycleScoring(so, s.features = s_genes, g2m.features = g2m_genes,
set.ident = TRUE)DR colored by cluster ID
cs <- sample(colnames(so), 5e3)
.plot_dr <- function(so, dr, id)
DimPlot(so, cells = cs, group.by = id, reduction = dr, pt.size = 0.4) +
guides(col = guide_legend(nrow = 11,
override.aes = list(size = 3, alpha = 1))) +
theme_void() + theme(aspect.ratio = 1)
ids <- c("cluster_id", "sample_id", "Phase")
for (id in ids) {
cat("## ", id, "\n")
p1 <- .plot_dr(so, "tsne", id)
lgd <- get_legend(p1)
p1 <- p1 + theme(legend.position = "none")
p2 <- .plot_dr(so, "umap", id) + theme(legend.position = "none")
ps <- plot_grid(plotlist = list(p1, p2), nrow = 1)
p <- plot_grid(ps, lgd, nrow = 1, rel_widths = c(1, 0.2))
print(p)
cat("\n\n")
}cluster_id
sample_id
Find markers using scran
Markers
We identify candidate marker genes for each cluster that enable a separation of that group from any other group. The null hypothesis is that the log FC between a cluster and the compared cluster is 2.
scran_markers <- findMarkers(sce,
groups = sce$cluster_id, block = sce$sample_id,
direction = "up", lfc = 2, full.stats = TRUE, log.p = FALSE)We write tables with the top marker genes per cluster.
gs2 <- lapply(scran_markers, function(u) u[u$Top %in% 1:2,])
for (i in seq_along(gs2)) {
write.table(x = gs2[[i]] %>% as.data.frame %>%
dplyr::mutate(gene = rownames(gs2[[i]])) %>%
dplyr::relocate(gene),
file = file.path("output",
paste0("TDP-06-no_integration_cluster", i-1, "_marker_genes.txt")),
sep = "\t", quote = FALSE, row.names = FALSE)
}Heatmap of mean marker-exprs. by cluster
We aggregate the cells to pseudobulks and plot the average expression of the condidate marker genes in each of the clusters.
## including marker genes of rank 1 and 2
gs <- lapply(scran_markers, function(u) rownames(u)[u$Top %in% 1:2])
## candidate cluster markers
lapply(gs, function(x) {
y <- str_split(x, pattern = "\\.", simplify = TRUE)[,2]
y[which(y == "")] <- x[which(y == "")]
y
})$`0`
[1] "SAMD11" "SCG2" "SCRG1" "FABP7" "TAC1" "STMN2" "RTN1"
[8] "SEZ6L2" "MT-ND4L" "NOC2L" "UTS2" "IGFBP5" "GAP43" "VGF"
[15] "MT-ND5"
$`1`
[1] "C1orf61" "FABP5" "VIM" "GFAP" "MT-CO1" "MT-ND4L" "FGFBP2"
[8] "SPARCL1" "SPP1" "METRN" "S100B" "MT-ND1" "MT-ND4"
$`2`
[1] "UTS2" "IGFBP2" "SCRG1" "PNOC" "STMN2" "RTN1" "SEZ6L2"
[8] "SAMD11" "GAP43" "VGF" "LAPTM4B" "PCP4" "MT-ND4L"
$`3`
[1] "S100A10" "IGFBP5" "FABP7" "TAC1" "STMN2" "UTS2" "VGF"
[8] "MT-ND5"
$`4`
[1] "UTS2" "TRH" "CRH" "STMN2" "NTS" "TAC1" "CSRP2" "RTN1"
[9] "MT-ND5"
$`5`
[1] "S100A10" "VGF" "PNOC" "STMN2" "SAMD11" "IGFBP5" "SCRG1"
[8] "TAC1" "MT-RNR2" "MT-ND5"
$`6`
[1] "PTGDS" "DLK1" "METRN" "TTYH1" "MT-CO1" "C1orf61" "CLU"
[8] "LY6H" "VIM" "MT-CO3"
$`7`
[1] "UTS2" "CRH" "STMN2" "PCP4" "MT-ND5"
$`8`
[1] "IGFBP5" "NEFM" "PNOC" "STMN2" "RTN1" "MT-ND4L" "SAMD11"
[8] "GAP43" "SPP1" "VGF" "LAPTM4B" "MT-ND5"
$`9`
[1] "S100A10" "TAC1" "CRH" "STMN2" "LYPD1" "IGFBP5" "VGF"
[8] "NTS" "RTN1" "HOXB5"
$`10`
[1] "S100A10" "PCSK1" "TAC1" "VGF" "STMN2" "TRH" "DLK1"
$`11`
[1] "WDPCP" "IGFBP5" "NRN1" "TAC1" "CRH" "STMN2" "SNCG"
[8] "MT-ND5" "CHL1" "GPX1" "ATP2B1" "RTN1" "CALB2" "MT-ND4L"
$`12`
[1] "NPTX2" "STMN2" "TDP43-HA" "FGF18" "MEF2A" "MT-ND4"
$`13`
[1] "C1orf61" "VIM" "CKB" "PCLAF" "METRN" "GFAP" "MT-ND4"
[8] "SPP1" "TYMS" "MT-CO1" "MT-ND4L"
$`14`
[1] "SPP1" "VIM" "CRYAB" "FTL" "DDIT3"
$`15`
[1] "S100A11" "COL1A1" "LGALS1" "COL3A1"
$`16`
[1] "UTS2" "C1orf61" "CRH" "STMN2" "CLU" "VIM" "METRN" sub <- sce[unique(unlist(gs)), ]
pbs <- aggregateData(sub, assay = "logcounts", by = "cluster_id", fun = "mean")
mat <- t(muscat:::.scale(assay(pbs)))
## remove the Ensembl ID from the gene names
cnames <- colnames(mat)
colnames(mat) <- str_split(cnames, pattern = "\\.", simplify = TRUE)[,2]
colnames(mat)[which(colnames(mat) == "")] <- cnames[which(colnames(mat) == "")]
Heatmap(mat,
name = "scaled avg.\nexpression",
col = viridis(10),
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
row_title = "cluster_id",
rect_gp = gpar(col = "white"))
Known marker genes
Apart from the usual marker genes, we also want to analyse the expression of Casein Kinase 1 Epsilon (CSNK1E).
## source file with list of known marker genes
source(file.path("data", "known_cell_type_markers.R"))
fs[["kinase"]] <- "CSNK1E"
fs[["ALS-related"]] <- "UNC13A"
fs <- lapply(fs, sapply, function(g)
grep(pattern = paste0("\\.", g, "$"), rownames(sce), value = TRUE)
)
fs <- lapply(fs, function(x) unlist(x[lengths(x) !=0]) )
gs <- gsub(".*\\.", "", unlist(fs))
ns <- vapply(fs, length, numeric(1))
ks <- rep.int(names(fs), ns)
labs <- lapply(fs, function(x) gsub(".*\\.", "",x))Heatmap of mean marker-exprs. by cluster
# split cells by cluster
cs_by_k <- split(colnames(sce), sce$cluster_id)
# compute cluster-marker means
ms_by_cluster <- lapply(fs, function(gs) vapply(cs_by_k, function(i)
Matrix::rowMeans(logcounts(sce)[gs, i, drop = FALSE]),
numeric(length(gs))))
# prep. for plotting & scale b/w 0 and 1
mat <- do.call("rbind", ms_by_cluster)
mat <- muscat:::.scale(mat)
rownames(mat) <- gs
cols <- muscat:::.cluster_colors[seq_along(fs)]
cols <- setNames(cols, names(fs))
row_anno <- rowAnnotation(
df = data.frame(label = factor(ks, levels = names(fs))),
col = list(label = cols), gp = gpar(col = "white"))
# percentage of cells from each of the samples per cluster
sample_props <- prop.table(n_cells, margin = 1)
col_mat <- as.matrix(unclass(sample_props))
sample_cols <- c("#882255", "#11588A", "#117733", "#44AA99")
sample_cols <- setNames(sample_cols, colnames(col_mat))
col_anno <- HeatmapAnnotation(
perc_sample = anno_barplot(col_mat, gp = gpar(fill = sample_cols),
height = unit(2, "cm"),
border = FALSE),
annotation_label = "fraction of sample\nin cluster",
gap = unit(10, "points"))
col_lgd <- Legend(labels = names(sample_cols),
title = "sample",
legend_gp = gpar(fill = sample_cols))
hm <- Heatmap(mat,
name = "scaled avg.\nexpression",
col = viridis(10),
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
column_title = "cluster_id",
column_title_side = "bottom",
column_names_side = "bottom",
column_names_rot = 0,
column_names_centered = TRUE,
rect_gp = gpar(col = "white"),
left_annotation = row_anno,
top_annotation = col_anno)
draw(hm, annotation_legend_list = list(col_lgd))
DR colored by marker expression
# downsample to 5000 cells
cs <- sample(colnames(sce), 5e3)
sub <- subset(so, cells = cs)
# UMAPs colored by marker-expression
for (m in seq_along(fs)) {
cat("## ", names(fs)[m], "\n")
ps <- lapply(seq_along(fs[[m]]), function(i) {
if (!fs[[m]][i] %in% rownames(so)) return(NULL)
FeaturePlot(sub, features = fs[[m]][i], reduction = "umap",
pt.size = 0.4) +
theme(aspect.ratio = 1, legend.position = "none") +
ggtitle(labs[[m]][i]) + theme_void() + theme(aspect.ratio = 1)
})
# arrange plots in grid
ps <- ps[!vapply(ps, is.null, logical(1))]
p <- plot_grid(plotlist = ps, ncol = 4, label_size = 10)
print(p)
cat("\n\n")
}NSC
proliferating
neuronal
mature_astrocytes
glial_astrocytic
radial_glia
oligodendrocyte
GABAergic_neurons
glycinergic_neurons
glutaminergic_neurons
dopaminergic_neurons
apoptotic
kinase
ALS-related
| Version | Author | Date |
|---|---|---|
| ecba9c2 | khembach | 2021-04-20 |
## plot the expression of the endogenous TDP-43 and TDP-HA
tdp <- c("ENSG00000120948.TARDBP", "ENSG00000120948.TARDBP-alevin", "TDP43-HA")
names(tdp) <- c("TARDBP", "TARDBP-alevin", "TDP-HA")
cat("## TDP-43\n")TDP-43
ps <- lapply(seq_along(tdp), function(i) {
if (!tdp[i] %in% rownames(so)) return(NULL)
FeaturePlot(sub, features = tdp[i], reduction = "umap", pt.size = 0.4) +
theme(aspect.ratio = 1, legend.position = "none") +
ggtitle(names(tdp)[i]) + theme_void() + theme(aspect.ratio = 1)
})
# arrange plots in grid
ps <- ps[!vapply(ps, is.null, logical(1))]
p <- plot_grid(plotlist = ps, ncol = 4, label_size = 10)
print(p)
cat("\n\n")## UNC13A
cat("## UNC13A\n")UNC13A
FeaturePlot(sub, features = "ENSG00000130477.UNC13A", reduction = "umap",
pt.size = 0.4) +
theme(aspect.ratio = 1, legend.position = "none") +
ggtitle("UNC13A") + theme_void() + theme(aspect.ratio = 1)
cat("\n\n")Reactive astrocyte markers
## source file with list of known marker genes
source(file.path("data", "reactive_astrocyte_markers.R"))
fs <- lapply(fs, sapply, function(g)
grep(pattern = paste0("\\.", g, "$"), rownames(sce), value = TRUE)
)
fs <- lapply(fs, function(x) unlist(x[lengths(x) !=0]) )
gs <- gsub(".*\\.", "", unlist(fs))
ns <- vapply(fs, length, numeric(1))
ks <- rep.int(names(fs), ns)
labs <- lapply(fs, function(x) gsub(".*\\.", "",x))Heatmap of reactive astrocyte markers
# compute cluster-marker means
ms_by_cluster <- lapply(fs, function(gs) vapply(cs_by_k, function(i)
Matrix::rowMeans(logcounts(sce)[gs, i, drop = FALSE]),
numeric(length(gs))))
# prep. for plotting & scale b/w 0 and 1
mat <- do.call("rbind", ms_by_cluster)
mat <- muscat:::.scale(mat)
rownames(mat) <- gs
cols <- muscat:::.cluster_colors[seq_along(fs)]
cols <- setNames(cols, names(fs))
row_anno <- rowAnnotation(
df = data.frame(label = factor(ks, levels = names(fs))),
col = list(label = cols), gp = gpar(col = "white"))
# percentage of cells from each of the samples per cluster
sample_props <- prop.table(n_cells, margin = 1)
col_mat <- as.matrix(unclass(sample_props))
sample_cols <- c("#882255", "#11588A", "#117733", "#44AA99")
sample_cols <- setNames(sample_cols, colnames(col_mat))
col_anno <- HeatmapAnnotation(
perc_sample = anno_barplot(col_mat, gp = gpar(fill = sample_cols),
height = unit(2, "cm"),
border = FALSE),
annotation_label = "fraction of sample\nin cluster",
gap = unit(10, "points"))
col_lgd <- Legend(labels = names(sample_cols),
title = "sample",
legend_gp = gpar(fill = sample_cols))
hm <- Heatmap(mat,
name = "scaled avg.\nexpression",
col = viridis(10),
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
column_title = "cluster_id",
column_title_side = "bottom",
column_names_side = "bottom",
column_names_rot = 0,
column_names_centered = TRUE,
rect_gp = gpar(col = "white"),
left_annotation = row_anno,
top_annotation = col_anno)
draw(hm, annotation_legend_list = list(col_lgd))
Subset to only the glial cell clusters
## subset to glial clusters
subs <- c("1", "6", "13", "14", "15", "16")
cs_by_k_sub <- cs_by_k[subs]
# compute cluster-marker means
ms_by_cluster <- lapply(fs, function(gs) vapply(cs_by_k_sub, function(i)
Matrix::rowMeans(logcounts(sce)[gs, i, drop = FALSE]),
numeric(length(gs))))
# prep. for plotting & scale b/w 0 and 1
mat <- do.call("rbind", ms_by_cluster)
mat <- muscat:::.scale(mat)
rownames(mat) <- gs
cols <- muscat:::.cluster_colors[seq_along(fs)]
cols <- setNames(cols, names(fs))
row_anno <- rowAnnotation(
df = data.frame(label = factor(ks, levels = names(fs))),
col = list(label = cols), gp = gpar(col = "white"))
# percentage of cells from each of the samples per cluster
sample_props <- prop.table(n_cells, margin = 1)
col_mat <- as.matrix(unclass(sample_props[subs,]))
sample_cols <- c("#882255", "#11588A", "#117733", "#44AA99")
sample_cols <- setNames(sample_cols, colnames(col_mat))
col_anno <- HeatmapAnnotation(
perc_sample = anno_barplot(col_mat, gp = gpar(fill = sample_cols),
height = unit(2, "cm"),
border = FALSE),
annotation_label = "fraction of sample\nin cluster",
gap = unit(10, "points"))
col_lgd <- Legend(labels = names(sample_cols),
title = "sample",
legend_gp = gpar(fill = sample_cols))
hm <- Heatmap(mat,
name = "scaled avg.\nexpression",
col = viridis(10),
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
column_title = "cluster_id",
column_title_side = "bottom",
column_names_side = "bottom",
column_names_rot = 0,
column_names_centered = TRUE,
rect_gp = gpar(col = "white"),
left_annotation = row_anno,
top_annotation = col_anno)
draw(hm, annotation_legend_list = list(col_lgd))
DR colored by reactive astrocyte marker expression
# UMAPs colored by marker-expression
for (m in seq_along(fs)) {
cat("### ", names(fs)[m], "\n")
ps <- lapply(seq_along(fs[[m]]), function(i) {
if (!fs[[m]][i] %in% rownames(so)) return(NULL)
FeaturePlot(sub, features = fs[[m]][i], reduction = "umap",
pt.size = 0.4) +
theme(aspect.ratio = 1, legend.position = "none") +
ggtitle(labs[[m]][i]) + theme_void() + theme(aspect.ratio = 1)
})
# arrange plots in grid
ps <- ps[!vapply(ps, is.null, logical(1))]
p <- plot_grid(plotlist = ps, ncol = 4, label_size = 10)
print(p)
cat("\n\n")
}panreactive_genes
A1
A2
senescence_down
Marker genes for virus cell tropism
## source file with list of known marker genes
source(file.path("data", "virus_cell_tropism_markers.R"))
fs <- lapply(fs, sapply, function(g)
grep(pattern = paste0("\\.", g, "$"), rownames(sce), value = TRUE)
)
fs <- lapply(fs, function(x) unlist(x[lengths(x) !=0]) )
gs <- gsub(".*\\.", "", unlist(fs))
ns <- vapply(fs, length, numeric(1))
ks <- rep.int(names(fs), ns)
labs <- lapply(fs, function(x) gsub(".*\\.", "",x))Heatmap of virus cell tropism markers
# compute cluster-marker means
ms_by_cluster <- lapply(fs, function(gs) vapply(cs_by_k, function(i)
Matrix::rowMeans(logcounts(sce)[gs, i, drop = FALSE]),
numeric(length(gs))))
# prep. for plotting & scale b/w 0 and 1
mat <- do.call("rbind", ms_by_cluster)
mat <- muscat:::.scale(mat)
rownames(mat) <- gs
cols <- muscat:::.cluster_colors[seq_along(fs)]
cols <- setNames(cols, names(fs))
row_anno <- rowAnnotation(
df = data.frame(label = factor(ks, levels = names(fs))),
col = list(label = cols), gp = gpar(col = "white"))
# percentage of cells from each of the samples per cluster
sample_props <- prop.table(n_cells, margin = 1)
col_mat <- as.matrix(unclass(sample_props))
sample_cols <- c("#882255", "#11588A", "#117733", "#44AA99")
sample_cols <- setNames(sample_cols, colnames(col_mat))
col_anno <- HeatmapAnnotation(
perc_sample = anno_barplot(col_mat, gp = gpar(fill = sample_cols),
height = unit(2, "cm"),
border = FALSE),
annotation_label = "fraction of sample\nin cluster",
gap = unit(10, "points"))
col_lgd <- Legend(labels = names(sample_cols),
title = "sample",
legend_gp = gpar(fill = sample_cols))
hm <- Heatmap(mat,
name = "scaled avg.\nexpression",
col = viridis(10),
cluster_rows = FALSE,
cluster_columns = FALSE,
row_names_side = "left",
column_title = "cluster_id",
column_title_side = "bottom",
column_names_side = "bottom",
column_names_rot = 0,
column_names_centered = TRUE,
rect_gp = gpar(col = "white"),
left_annotation = row_anno,
top_annotation = col_anno)
draw(hm, annotation_legend_list = list(col_lgd))
Save cluster markers to RDS
saveRDS(scran_markers, file.path("output", "TDP-06_scran_markers.rds"))
saveRDS(so, file.path("output", "so_TDP-06-cluster-analysis.rds"))
sessionInfo()R version 4.0.5 (2021-03-31)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 18.04.5 LTS
Matrix products: default
BLAS: /usr/local/R/R-4.0.5/lib/libRblas.so
LAPACK: /usr/local/R/R-4.0.5/lib/libRlapack.so
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
attached base packages:
[1] parallel stats4 grid stats graphics grDevices utils
[8] datasets methods base
other attached packages:
[1] BiocParallel_1.22.0 RCurl_1.98-1.3
[3] stringr_1.4.0 SeuratObject_4.0.1
[5] Seurat_4.0.1 scran_1.16.0
[7] SingleCellExperiment_1.10.1 SummarizedExperiment_1.18.1
[9] DelayedArray_0.14.0 matrixStats_0.56.0
[11] Biobase_2.48.0 GenomicRanges_1.40.0
[13] GenomeInfoDb_1.24.2 IRanges_2.22.2
[15] S4Vectors_0.26.1 BiocGenerics_0.34.0
[17] viridis_0.5.1 viridisLite_0.3.0
[19] RColorBrewer_1.1-2 purrr_0.3.4
[21] muscat_1.2.1 dplyr_1.0.2
[23] ggplot2_3.3.2 cowplot_1.0.0
[25] ComplexHeatmap_2.4.2 workflowr_1.6.2
loaded via a namespace (and not attached):
[1] reticulate_1.16 tidyselect_1.1.0
[3] lme4_1.1-23 RSQLite_2.2.0
[5] AnnotationDbi_1.50.1 htmlwidgets_1.5.1
[7] Rtsne_0.15 munsell_0.5.0
[9] codetools_0.2-16 ica_1.0-2
[11] statmod_1.4.34 future_1.17.0
[13] miniUI_0.1.1.1 withr_2.4.1
[15] colorspace_1.4-1 knitr_1.29
[17] ROCR_1.0-11 tensor_1.5
[19] listenv_0.8.0 labeling_0.3
[21] git2r_0.27.1 GenomeInfoDbData_1.2.3
[23] polyclip_1.10-0 farver_2.0.3
[25] bit64_0.9-7 glmmTMB_1.0.2.1
[27] rprojroot_1.3-2 vctrs_0.3.4
[29] generics_0.0.2 xfun_0.15
[31] R6_2.4.1 doParallel_1.0.15
[33] ggbeeswarm_0.6.0 clue_0.3-57
[35] rsvd_1.0.3 locfit_1.5-9.4
[37] spatstat.utils_2.1-0 bitops_1.0-6
[39] cachem_1.0.4 promises_1.1.1
[41] scales_1.1.1 beeswarm_0.2.3
[43] gtable_0.3.0 globals_0.12.5
[45] goftest_1.2-2 rlang_0.4.10
[47] genefilter_1.70.0 GlobalOptions_0.1.2
[49] splines_4.0.5 TMB_1.7.16
[51] lazyeval_0.2.2 spatstat.geom_2.1-0
[53] abind_1.4-5 yaml_2.2.1
[55] reshape2_1.4.4 backports_1.1.9
[57] httpuv_1.5.4 tools_4.0.5
[59] spatstat.core_2.1-2 ellipsis_0.3.1
[61] gplots_3.0.4 ggridges_0.5.2
[63] Rcpp_1.0.5 plyr_1.8.6
[65] progress_1.2.2 zlibbioc_1.34.0
[67] prettyunits_1.1.1 rpart_4.1-15
[69] deldir_0.2-10 pbapply_1.4-2
[71] GetoptLong_1.0.1 zoo_1.8-8
[73] ggrepel_0.8.2 cluster_2.1.0
[75] colorRamps_2.3 fs_1.5.0
[77] variancePartition_1.18.2 magrittr_1.5
[79] data.table_1.12.8 scattermore_0.7
[81] lmerTest_3.1-2 circlize_0.4.10
[83] lmtest_0.9-37 RANN_2.6.1
[85] whisker_0.4 fitdistrplus_1.1-1
[87] hms_0.5.3 patchwork_1.0.1
[89] mime_0.9 evaluate_0.14
[91] xtable_1.8-4 pbkrtest_0.4-8.6
[93] XML_3.99-0.4 gridExtra_2.3
[95] shape_1.4.4 compiler_4.0.5
[97] scater_1.16.2 tibble_3.0.3
[99] KernSmooth_2.23-17 crayon_1.3.4
[101] minqa_1.2.4 htmltools_0.5.0
[103] mgcv_1.8-31 later_1.1.0.1
[105] tidyr_1.1.0 geneplotter_1.66.0
[107] DBI_1.1.0 MASS_7.3-51.6
[109] rappdirs_0.3.1 boot_1.3-25
[111] Matrix_1.3-3 gdata_2.18.0
[113] igraph_1.2.5 pkgconfig_2.0.3
[115] numDeriv_2016.8-1.1 spatstat.sparse_2.0-0
[117] plotly_4.9.2.1 foreach_1.5.0
[119] annotate_1.66.0 vipor_0.4.5
[121] dqrng_0.2.1 blme_1.0-4
[123] XVector_0.28.0 digest_0.6.25
[125] sctransform_0.3.2 RcppAnnoy_0.0.18
[127] spatstat.data_2.1-0 rmarkdown_2.3
[129] leiden_0.3.3 uwot_0.1.10
[131] edgeR_3.30.3 DelayedMatrixStats_1.10.1
[133] shiny_1.5.0 gtools_3.8.2
[135] rjson_0.2.20 nloptr_1.2.2.2
[137] lifecycle_1.0.0 nlme_3.1-148
[139] jsonlite_1.7.2 BiocNeighbors_1.6.0
[141] limma_3.44.3 pillar_1.4.6
[143] lattice_0.20-41 fastmap_1.0.1
[145] httr_1.4.2 survival_3.2-3
[147] glue_1.4.2 png_0.1-7
[149] iterators_1.0.12 bit_1.1-15.2
[151] stringi_1.4.6 blob_1.2.1
[153] DESeq2_1.28.1 BiocSingular_1.4.0
[155] caTools_1.18.0 memoise_2.0.0
[157] irlba_2.3.3 future.apply_1.6.0