scran

library(Seurat)
library(scran)

pbmc.0 <- readRDS("pmbc_1683691139445.rds")
obj <- pbmc.0


mat <- obj@assays$RNA@counts |> as.matrix()
sce <- SingleCellExperiment(assays=list(counts=mat))
clusters <- quickCluster(sce)
sce <- computeSumFactors(sce, clusters=clusters)
summary(sizeFactors(sce))
sce <- logNormCounts(sce)

dec <- modelGeneVar(sce)
plot(dec$mean, dec$total, xlab="Mean log-expression", ylab="Variance")
curve(metadata(dec)$trend(x), col="blue", add=TRUE)

top.hvgs <- getTopHVGs(dec, n=2000)
sce <- fixedPCA(sce, subset.row=top.hvgs)
reducedDimNames(sce)

sced <- denoisePCA(sce, dec, subset.row=top.hvgs)
ncol(reducedDim(sced, "PCA"))
reducedDim(sce, "PCAsub") <-reducedDim(sced, "PCA")


# output <- getClusteredPCs(reducedDim(sce))
# npcs <- metadata(output)$chosen
# reducedDim(sce, "PCAsub") <- reducedDim(sce, "PCA")[,1:npcs,drop=FALSE]
# npcs

g <- buildSNNGraph(sce, use.dimred="PCAsub")
cluster <- igraph::cluster_walktrap(g)$membership

colLabels(sce) <- factor(cluster)
table(colLabels(sce))

library(scater)
sce <- runTSNE(sce, dimred="PCAsub")
plotTSNE(sce, colour_by="label", text_by="label")



marker_gene_list <- list(
  "Naive CD4+ T" = c("IL7R", "CCR7"),
  "CD14+ Mono" = c("CD14", "LYZ"),
  "Memory CD4+" = c("IL7R", "S100A4"),
  "B" = c("MS4A1"),
  "CD8+ T" = c("CD8A"),
  "FCGR3A+ Mono" = c("FCGR3A","MS4A7"),
  "NK" = c("GNLY","NKG7"),
  "DC" = c("FCER1A","CST3"),
  "Platelet" = c("PPBP")
)


marker_gene_mat <- marker_list_to_mat(marker_gene_list)
s <- sizeFactors(sce)

fit  <- cellassign(sce[rownames(marker_gene_mat),],
                   marker_gene_info = marker_gene_mat,
                   s =s,
                   learning_rate = 1e-2,
                   shrinkage = TRUE,
                   verbose = FALSE)
pheatmap::pheatmap(cellprobs(fit))

sce@colData$cellassign <-celltypes(fit)
pt1 <- plotTSNE(sce, colour_by="label", text_by="label")
pt2 <- plotTSNE(sce, colour_by="cellassign", text_by="cellassign")
pt1+pt2

https://bioconductor.org/packages/release/bioc/vignettes/scran/inst/doc/scran.html