Add samples IDs to Seurat object when integrating different samples to do differential expression analysis

Question

[Sara]

I have scRNAseq data and I am trying to do differential comparing 2 conditions (for each condition I have 3 samples). to do so I am using Seurat R package and to integrate the samples I used the following code. the part1 has code only for 1 sample (for other 5 samples I did the same) but the part2 is complete

part1:

med1 <- Read10X("AM-g001-raw-feature-bc-matrix")
med1 <- CreateSeuratObject(counts = med1, project = "med", min.cells = 3, min.features = 200)
med1 <- PercentageFeatureSet(med1, pattern = "^MT-", col.name = "percent.mt") 
med1 <- subset((med1, subset = nFeature_RNA < 4000 & nCount_RNA < 20000 & pecent.mt < 10 )

part2:

med_isa.list <- list(med1 = med1, med2 = med2, med3 = med3, iSA1 = iSA1, iSA2 = iSA2, iSA3 = iSA3)

med_isa.list <- lapply(x = med_isa.list, FUN = function(x) {
    x <- NormalizeData(x)
    x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 2000)
}) 
features <- SelectIntegrationFeatures(object.list = med_isa.list)
med_isa.anchores <- FindIntegrationAnchors(object.list = med_isa.list, anchor.features = features)
med_isa.combined <- IntegrateData(anchorset = med_isa.anchores)
DefaultAssay(med_isa.combined) <- "integrated"
med_isa.combined <- ScaleData(med_isa.combined, verbose = FALSE)
med_isa.combined <- RunPCA(med_isa.combined, npcs = 50, verbose = FALSE)
med_isa.combined <- RunUMAP(med_isa.combined, reduction = "pca", dims = 1:30)
med_isa.combined <- FindNeighbors(med_isa.combined, reduction = "pca", dims = 1:30)
med_isa.combined <- FindClusters(med_isa.combined, resolution = 0.6)
saveRDS(med_isa.combined, file="med_isa.combined.rds")

when I look at med_isa.combined I would expect to see samples IDs as well but it does not exist. I only see the following items:

1. orig.ident: which is either med or isa
2. seurat_clusters
3. nCount_RNA
4. nFeature_RNA
5. percent.mt

How can I add another item to med_isa.combined which shows the sample IDs?

Answer 1

[fracarb8]

When I integrate multiple samples together, I make sure to have, in each object :

• a column of the metadata table ([e-mail]$SampleID) with the sampleID
• the sampleID at the beginning of each barcode (sample1_AAATGGTAGAGAT-1)

The first point allow you to easily group/split your integrated object, and the second point avoid any confusion/duplication of barcodes between samples.

In your case you don't see it because your project is "med" (project name is saved into orig.ident) for each sample. I would either

sample_name <- "med1"
CreateSeuratObject(counts = med1, project = sample_name,..

or simply [e-mail]$SampleID <- "med1"