第一步 下载自带的参考数据库(仅需一次,已经运行过了,跳过)
library(celldex)
ref_HPCD <- HumanPrimaryCellAtlasData()
ref_BFD <- BlueprintEncodeData()
ref_MRD <- MouseRNAseqData()
ref_IGD <- ImmGenData()
ref_DICED <- DatabaseImmuneCellExpressionData()
ref_NHD <- NovershternHematopoieticData()
ref_MID <- MonacoImmuneData()
save(list = c('ref_HPCD', 'ref_BFD', 'ref_MRD', 'ref_IGD', 'ref_DICED', 'ref_NHD', 'ref_MID'), file = '~/upload/zl_liu/celldexData/celldex.rdata')
- BlueprintEncodeData(人)
Blueprint (Martens and Stunnenberg 2013) and Encode (The ENCODE Project Consortium 2012) - DatabaseImmuneCellExpressionData (人)
The Database for Immune Cell Expression(/eQTLs/Epigenomics)(Schmiedel et al. 2018) - HumanPrimaryCellAtlasData(人)
The Human Primary Cell Atlas (Mabbott et al. 2013) - MonacoImmuneData (人)
Monaco Immune Cell Data - GSE107011 (Monaco et al. 2019) - NovershternHematopoieticData (人)
Novershtern Hematopoietic Cell Data - GSE24759 - ImmGenData(鼠)
The murine ImmGen (Heng et al. 2008) - MouseRNAseqData(鼠)
A collection of mouse data sets downloaded from GEO (Benayoun et al. 2019).
第二步 进行自动注释
- 加载参考数据集
library(celldex)
load(file = '~/upload/zl_liu/celldexData/celldex.rdata')
- 读入待注释对象
library(SingleR)
library(Seurat)
sce <- readRDS('Myeloid.rds')
[email protected] <- readRDS('myeloid_metadata.rds')
DimPlot(sce, reduction = 'umap', label = T, repel = T)
- 进行分群
require(dplyr)
f_FindNeighbors_integrated_s <- function(scRNA){
DefaultAssay(scRNA) <- 'integrated'
scRNA <- ScaleData(scRNA)
scRNA <- RunPCA(scRNA)
scRNA <- RunUMAP(scRNA, dims = 1:30)
}
f_FindNeighbors_integrated <- function(scRNA, resolution = 0.5, reduction='pca'){
scRNA <- scRNA %>% FindNeighbors(reduction = reduction) %>% FindClusters(resolution = resolution)
scRNA[[paste0('h_resolution_', resolution)]] <- Idents(scRNA)
scRNA
}
sce <- f_FindNeighbors_integrated_s(sce)
sce <- f_FindNeighbors_integrated(sce, 1.5)
DimPlot(sce, reduction = 'umap', label = T, repel = T)
- 进行预测
sce_for_SingleR <- GetAssayData(sce, slot="data")
clusters <- sce[['h_resolution_1.5']][[1]]
pred_MID <- SingleR(test = sce_for_SingleR, ref = ref_MID, labels = ref_MID$label.main,
method = "cluster", clusters = clusters,
assay.type.test = "logcounts", assay.type.ref = "logcounts")
pred_NHD <- SingleR(test = sce_for_SingleR, ref = ref_NHD, labels = ref_NHD$label.main,
method = "cluster", clusters = clusters,
assay.type.test = "logcounts", assay.type.ref = "logcounts")
cellType=data.frame(ClusterID=levels([email protected]$seurat_clusters),
pred_MID=pred_MID$labels,
pred_NHD=pred_NHD$labels )
head(cellType)
[email protected]$pred_MID <- cellType[match(clusters,cellType$ClusterID), 'pred_MID']
[email protected]$pred_NHD <- cellType[match(clusters,cellType$ClusterID), 'pred_NHD']
第三步 查看注释质量
- 肉眼观察
DimPlot(sce, reduction = 'umap', group.by = 'pred_MID', label = T, repel = T)
DimPlot(sce, reduction = 'umap', group.by = 'pred_NHD', label = T, repel = T)
DimPlot(sce, reduction = 'umap', group.by = 'majority_voting', label = T, repel = T)
DimPlot(sce, reduction = 'umap', group.by = 'immune_annotion', label = T, repel = T)
- plotScoreHeatmap(pred_MID, show.labels = T, show.pruned = T, show_colnames = T)
- plotDeltaDistribution(pred_MID)
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