Hello, everyone.
I'm conducting Seurat analysis for GSE-deposited scRNA seq data. I performed integration of multiple data set (by "IntegrateData") and proceeded to gene expression profiling of each Seurat cluster for cell type annotation, but the following error message was appeared...
Tcell_markers<-c("CD4","CD8A","FOXP3") AverageExpression(My_integrated_data, group.by = "seurat_clusters", features=Tcell_markers ,verbose=FALSE)$RNA Warning: None of the features specified were found in the RNA assay. Warning: The following 3 features were not found in the integrated assay: CD4, CD8A, FOXP3 Warning: None of the features specified were found in the integrated assay. NULL
Curiously, when I run the same script last month, I could get proper results....
Scripts I used for data integration was following; Merged_data<-merge(P1,y=c(P10,P11,P12,P13,P14,P15,P16,P17,P18,P19,P2,P20,P21,P22,P23,P24,P25,P26,P27,P28,P29,P3,P30,P31,P32,P33,P34,P35,P36,P37,P38,P39,P4,P40,P41,P42,P5,P6,P7,P8,P9),add.cell.ids = ls()[1:42],project = "Merged_data")
(※ P1,P2,,,: Seurat objects)
Merged_data<-subset(Merged_data ,subset = nFeature_RNA>200 & nFeature_RNA<5000 & nCount_RNA<30000 & percent.mt<30
Merged_data.list<-SplitObject(Merged_data, split.by = "orig.ident")
Merged_data.list <- lapply(X = Merged_data.list, FUN = function(x) { x <- NormalizeData(x) x <- FindVariableFeatures(x, verbose=FALSE) })
features <- SelectIntegrationFeatures(object.list = Merged_data.list)
Merged_data.list <- lapply(X = Merged_data.list, FUN = function(x) { x <- ScaleData(x,features=features,verbose=FALSE) x <- RunPCA(x,features=features,verbose=FALSE) })
Merged_data.anchors <- FindIntegrationAnchors(object.list = Merged_data.list,reduction="rpca",dims=1:50)
My_integrated_data <- IntegrateData(anchorset = Merged_data.anchors,dims=1:50)
My_integrated_data <- ScaleData(My_integrated_data verbose = FALSE) My_integrated_data <- RunPCA(My_integrated_data, verbose = FALSE) My_integrated_data<- FindNeighbors(My_integrated_data,reduction="pca",dims=1:30) My_integrated_data<- FindClusters(My_integrated_data,resolution=0.3) My_integrated_data<- RunUMAP(My_integrated_data, reduction="pca",dims = 1:30)
If anyone knows of a solution, I would appreciate it if you could let us know.
Thank you.
