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Commit 19c00ea7 authored by John Lafin's avatar John Lafin
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Complete annotation script

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scripts/04_annotation.R
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# Annotation and stress score filtering
# Author: John T Lafin
# Updated: 2024-10-15
# Updated: 2024-10-16
print("Script begin at: ")
Sys.time()
......@@ -70,12 +70,12 @@ dir.create(annotdir, showWarnings=FALSE)
## Create list of labels
annot.list <- list()
annot.list$Endothelia <- c("Venous endothelia",
"Venous endothelia",
"Capillary endothelia",
"Capillary endothelia",
"Arterial endothelia",
"Lymphatic endothelia")
annot.list$Endothelia <- c("venous endothelia",
"venous endothelia",
"capillary endothelia",
"capillary endothelia",
"arterial endothelia",
"lymphatic endothelia")
annot.list$Epithelia <- c("transitional basal epithelia of urethra",
"basal epithelia of prostate",
"transitional basal epithelia of urethra",
......@@ -92,7 +92,7 @@ annot.list$Epithelia <- c("transitional basal epithelia of urethra",
"squamous intermediate epithelia of urethra",
"squamous intermediate epithelia of urethra",
"basal epithelia of prostate",
"transitional basal epithelia of urethra",
"squamous basal epithelia of urethra",
"basal epithelia of prostate",
"proliferative cells",
"squamous luminal epithelia of urethra",
......@@ -100,15 +100,15 @@ annot.list$Epithelia <- c("transitional basal epithelia of urethra",
"transitional basal epithelia of urethra",
"basal epithelia of prostate")
annot.list$Leukocyte <- c("T cell",
"Macrophage",
"Mast cell",
"macrophage",
"mast cell",
"T cell",
"T cell",
"NK cell",
"Proliferating leukocyte",
"proliferating leukocyte",
"B cell",
"Dendritic cell",
"Plasma cell")
"dendritic cell",
"plasma cell")
annot.list$Stroma <- c("pericyte",
"major interstitial fibroblast",
"smooth muscle",
......@@ -150,7 +150,7 @@ VlnPlot(lin.list$Epithelia, "NE.score", group.by = "orig.ident") +
ggsave(paste0(annotdir, "NE_score_cutoff.png"))
lin.list$Epithelia@meta.data <- lin.list$Epithelia@meta.data %>%
mutate(cellannotation = if_else(NE.score > 0.3, "Neuroendocrine cell", cellannotation))
mutate(cellannotation = if_else(NE.score > 0.3, "neuroendocrine cell", cellannotation))
## Add ionocytes
VlnPlot(lin.list$Epithelia, "FOXI1", group.by = "orig.ident") +
......@@ -159,10 +159,7 @@ VlnPlot(lin.list$Epithelia, "FOXI1", group.by = "orig.ident") +
ggsave(paste0(annotdir, "FOXI1_score_cutoff.png"))
ionocyte.barcodes <- WhichCells(lin.list$Epithelia, expression = FOXI1 > 0.3)
lin.list$Epithelia@meta.data[ionocyte.barcodes, "cellannotation"] <- "Ionocyte"
## Save updates to object
lin.list$Epithelia <- StashIdent(lin.list$Epithelia, "cellannotation")
lin.list$Epithelia@meta.data[ionocyte.barcodes, "cellannotation"] <- "ionocyte"
## Split periepithelial fibroblasts by sex
fib_rename <- WhichCells(lin.list$Stroma,
......@@ -170,7 +167,10 @@ fib_rename <- WhichCells(lin.list$Stroma,
expression = Sex == "male")
levels(lin.list$Stroma$cellannotation) <- c(levels(lin.list$Stroma$cellannotation), "periepithelial fibroblast of prostate")
lin.list$Stroma@meta.data[fib_rename, "cellannotation"] <- "periepithelial fibroblast of prostate"
Idents(stroma) <- "cellannotation"
## Reset idents
Idents(lin.list$Epithelia) <- "cellannotation"
Idents(lin.list$Stroma) <- "cellannotation"
## Save labeled UMAP plots
for (lin in lins) {
......@@ -192,9 +192,7 @@ Idents(lin.list$Epithelia) <- "RNA_snn_res.1.5"
for (lin in names(lin.list)) {
VlnPlot(lin.list[[lin]], "Stress.score", pt.size=0) +
NoLegend()
ggsave(paste0(stressdir, lin, "_pre-filter.png"),
width=11,
height=8.5)
ggsave(paste0(stressdir, lin, "_pre-filter.png"))
}
## Define stressed clusters based on violin plots
......@@ -209,24 +207,59 @@ for (lin in names(lin.list)) {
lin.list[[lin]] <- subset(lin.list[[lin]],
idents = stressed.cells[[lin]],
invert = TRUE)
lin.list[[lin]] <- preprocess(lin.list[[lin]], default.res="cellannotation")
}
lin.list <- lapply(lin.list, preprocess)
## Save violin plots after first round of filtering
for (lin in names(lin.list)) {
VlnPlot(lin.list[[lin]], "Stress.score", pt.size=0, group.by="RNA_snn_res.1") +
NoLegend()
ggsave(paste0(stressdir, lin, "_first_filter.png"))
}
### TO-DO
# Second round of stress filtering on stroma
# Revisit epithelia to relabel
# Make sure that ionocytes and NE cells are present in epi labels
## Second round of stressed cell removal in stroma
Idents(lin.list$Stroma) <- "RNA_snn_res.1"
lin.list$Stroma <- subset(lin.list$Stroma,
idents = 8,
invert = TRUE) %>%
preprocess(default.res="cellannotation")
## Save violin plot
VlnPlot(lin.list$Stroma, "Stress.score", pt.size=0, group.by="RNA_snn_res.1") +
NoLegend()
ggsave(paste0(stressdir, lin, "_second_filter.png"))
## Reannotate endothelia
lin.list$Endothelia@meta.data <- lin.list$Endothelia@meta.data %>%
mutate(cellannotation = if_else(RNA_snn_res.1 == 7, "venous endothelia", cellannotation))
Idents(lin.list$Endothelia) <- "cellannotation"
## Save labeled UMAP plots
for (lin in lins) {
DimPlot(lin.list[[lin]])
ggsave(paste0(annotdir, lin,"_umap-2.png"))
}
# Merge -------------------------------------------------------------------
# Produce post-filter stress plots
for (lin in names(lin.list)) {
VlnPlot(lin.list[[lin]], "Stress.score", pt.size=0) +
NoLegend()
ggsave(paste0(stressdir, lin, "_first_filter.png"),
width=11,
height=8.5)
seu <- merge(lin.list[[1]], lin.list[2:length(lin.list)])
plan(sequential)
seu <- preprocess(seu, default.res="cellannotation")
## Save UMAP plot
DimPlot(seu, label=TRUE, repel=TRUE, label.size=3) +
NoLegend()
ggsave(paste0(annotdir, "global_umap.png"))
# Save outputs ------------------------------------------------------------
objdir <- paste0(outdir, "objects/")
dir.create(objdir, showWarnings=FALSE)
saveRDS(seu, paste0(objdir, "global.rds"))
for (lin in names(lin.list)){
saveRDS(lin.list[[lin]], paste0(objdir, lin, ".rds"))
}
## Save session info
capture.output(sessionInfo(), file = paste0(outdir, "session_info.txt"))
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