I use SingleR to get cell type annotation for each cell, then try to annotate each cluster, but the clustering result seems not to be consistent with output of SingleR, where is wrong?
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6 months ago
FantasticAI ▴ 60

Hi

I just finished my first task on scRNAseq analysis, please feel free to make any comments. Very appreciate your help. Thank you!

I use SingleR to get cell type annotation. And my general workflow for 4 control samples as follows:


GL1101_data <- Read10X(data.dir = "~/GL1101/", strip.suffix = TRUE)
GL1111_data <- Read10X(data.dir = "~/GL1111/", strip.suffix = TRUE)
GL1131_data <- Read10X(data.dir = "~/GL1131/", strip.suffix = TRUE)
GL1141_data <- Read10X(data.dir = "~/GL1141/", strip.suffix = TRUE)

data1101<- CreateSeuratObject(counts = GL1101_data, project = "GL1101", min.cells = 3, min.features = 200)
data1111<- CreateSeuratObject(counts = GL1111_data, project = "GL1111", min.cells = 3, min.features = 200)
data1131<- CreateSeuratObject(counts = GL1131_data, project = "GL1131", min.cells = 3, min.features = 200)
data1141<- CreateSeuratObject(counts = GL1141_data, project = "GL1141", min.cells = 3, min.features = 200)

data1101 <- subset(data1101, subset = nFeature_RNA > 200 & nFeature_RNA < 4000)
data1111 <- subset(data1111, subset = nFeature_RNA > 400 & nFeature_RNA < 3600)
data1131 <- subset(data1131, subset = nFeature_RNA > 500 & nFeature_RNA < 4000)
data1141 <- subset(data1141, subset = nFeature_RNA > 300 & nFeature_RNA < 4000)

data1101<-NormalizeData(data1101, normalization.method = "LogNormalize", scale.factor = 10000)
data1111<-NormalizeData(data1111, normalization.method = "LogNormalize", scale.factor = 10000)
data1131<-NormalizeData(data1131, normalization.method = "LogNormalize", scale.factor = 10000)
data1141<-NormalizeData(data1141, normalization.method = "LogNormalize", scale.factor = 10000)

data1101$sampleID<-"GL1101"
data1111$sampleID<-"GL1111"
data1131$sampleID<-"GL1131"
data1141$sampleID<-"GL1141"

obj.list<-list(GL1101 = data1101, GL1111 = data1111, GL1131 = data1131, GL1141 = data1141)
# identify variable features for each dataset independently
obj.list <- lapply(X = obj.list, FUN = function(x) {
  x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 2000)
})

features <- SelectIntegrationFeatures(object.list = obj.list)
data.anchors <- FindIntegrationAnchors(object.list = obj.list, normalization.method = "LogNormalize",
                                       anchor.features = features)
data.combined <- IntegrateData(anchorset = data.anchors, normalization.method = "LogNormalize")

data.combined <- ScaleData(data.combined, verbose = FALSE)
data.combined <- RunPCA(data.combined, verbose = FALSE)
data.combined <- RunUMAP(data.combined, reduction = "pca", dims = 1:40)
data.combined<-FindNeighbors(data.combined, reduction = "pca", dim = 1:40)
data.combined<-FindClusters(data.combined, resolution = 0.5)

#load reference dataset, which has cell type annotation information.  A SingleCellExpreiment Object
sce.ref<-readRDS("~/sce_ref.rds")

DefaultAssay(data.combined)<- "RNA"
sce.data.combined<-as.SingleCellExperiment(data.combined)

library(SingleR)
pred.data.combined <- SingleR(test=sce.data.combined,
                         ref=sce.ref, labels=sce.ref$celllabel, de.method="wilcox", de.n = 50)

#Calculating the Rand Index
DefaultAssay(data.combined) <- "integrated"
library(bluster)
pairwiseRand(data.combined$integrated_snn_res.0.5, pred.data.combined$labels, mode="index")

#plot heat map
tab <- table(cluster=data.combined$integrated_snn_res.0.5, label=pred.data.combined$labels) 
pheatmap::pheatmap(log10(tab+10))

The rand Index ARI between unsupervised cluster and assigned cell type label is below 0.5, only 0.461. What's worse is that the cluster and cell type is not quite one-to-one map, maybe it has some nested structure, but I barely have any biological and medical background, it's hard for me to tell. heatmap

Also I use the following command to plot the clustering after I have assigned the cell type name to each cell.

data.combined$cell.type.label<-pred.data.combined$labels
DimPlot(data.combined, reduction = "umap", group.by = "cell.type.label", label = TRUE, pt.size = 3, label.size = 5)

At the left corner, there are two clusters which does not be labeled, I guess it is because those cells share the same label to the other clusters which actually has already been labeled, it might be just a "label location" issue. But I am wondering whether is it safe to say the cell annotation results are reasonable and solid? clustering

SingleR scRNAseq • 419 views
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