20230703 -- scRNAseq from gastric cancer

文章标题：《Single-cell atlas of lineage states, tumor microenvironment and subtypespecific expression programs in gastric cancer》
DOI: 10.1158/2159-8290.CD-21-0683

数据集组织形式快照：

• step1
  利用Seurat包整合数据

#! conda env R4

library(Seurat)
library(tidyverse)
library(cowplot)

# output dir
workdir <- ""
# where dataset 
projectdir <- ""

file.list <- list.files(projectdir,pattern="csv$")

# create  SeuratObject
scelist <-lapply(file.list,function(x){CreateSeuratObject(counts = read.csv(paste(projectdir,x,sep="/"),header=T,row.names=1,sep=","), min.cells = 3,min.features  = 200)})

# 把数据集中包含细胞数少于500的去掉
numcol <- lapply(scelist,function(x){ncol(x)})
numcol <- unlist(numcol
scelist <- scelist[numcol > 500]


#log-normalization and identify variable features
sceList1 <- lapply(scelist, function(obj){
		obj[["percent.mt"]] <- PercentageFeatureSet(object = obj, pattern = "^MT", assay = 'RNA')
		obj <- subset(obj, subset = nFeature_RNA > 200 & nFeature_RNA < 5000 & percent.mt < 20)
        obj <- NormalizeData(obj, normalization.method = "LogNormalize")
        obj <- FindVariableFeatures(obj, selection.method = "vst", nfeatures = 2000, verbose = T)
})

features <- SelectIntegrationFeatures(object.list = sceList1)

sceList1 <- lapply(X = sceList1, FUN = function(x) {
    x <- ScaleData(x, features = features, verbose = FALSE)
    x <- RunPCA(x, features = features,npcs = 30, verbose = FALSE)
})


# then identify anchors using the FindIntegrationAnchors() function, which takes a list of Seurat objects as input
immune.anchors <- FindIntegrationAnchors(object.list = sceList1,reduction = "rpca")
# and use these anchors to integrate the two datasets together with IntegrateData()
immune.combined <- IntegrateData(anchorset = immune.anchors)

immune.combined <- ScaleData(immune.combined, verbose = FALSE)
immune.combined <- RunPCA(immune.combined, npcs = 30, verbose = FALSE)
immune.combined <- RunUMAP(immune.combined, reduction = "pca", dims = 1:30)
immune.combined <- FindNeighbors(immune.combined, reduction = "pca", dims = 1:30)
immune.combined <- FindClusters(immune.combined, resolution = 0.5)

cat("move to SingleR\n")
# singleR 注释
library(celldex)
library(SingleR)

hpca.se <- HumanPrimaryCellAtlasData()
counts <- GetAssayData(immune.combined[["RNA"]], slot="counts")
pred.sce <- SingleR(test = counts, ref = hpca.se, labels = hpca.se$label.main)

immune.combined@meta.data$singleR <- pred.sce$labels
 
saveRDS(immune.combined,file = paste(workdir,"/Annotation/immune.combined.rds",sep=""))