How do I subset a Xenium SeuratObject based on gene list

[An17aV0]

Discussed in https://github.com/satijalab/seurat/discussions/8459

^{Originally posted by **An17aV0** February 12, 2024} Hello everyone! I would like to subset my data based on the genes in the 10X gene panel and my custom gene panel and analyze them separately. I am using the subset function like this xenium.obj.subset <- subset(xenium.obj, features = keep.genes) The number of features (genes) is reduced after subsetting but it appears the expression data and coordinates are still present in the new object. Below is a reproducible example where I am removing 4 genes from the "mouse-brain-10x-genomics-xenium-in-situ " and then plot these 4 genes as molecules in ImageDimPlot before and after subsetting. The plots look basically the same although after subsetting these 4 features should not be present anymore. I also get a couple of warnings while subsetting such as : 1: Not validating FOV objects 2: Not validating Centroids objects ... 9: Not validating Seurat objects 1.) How do I accurately subset the SeuratObject and resolve those warnings? 2.) On a side note, how do I deal with the signal detected outside the tissue (as visible in the Images below)? Thank you, any help is very much appreciated. Data and most code from here: https://satijalab.org/seurat/articles/seurat5_spatial_vignette_2#mouse-brain-10x-genomics-xenium-in-situ wget https://cf.10xgenomics.com/samples/xenium/1.0.2/Xenium_V1_FF_Mouse_Brain_Coronal_Subset_CTX_HP/Xenium_V1_FF_Mouse_Brain_Coronal_Subset_CTX_HP_outs.zip unzip Xenium_V1_FF_Mouse_Brain_Coronal_Subset_CTX_HP_outs.zip ### Load the Xenium data xenium.obj <- LoadXenium("Xenium_V1_FF_Mouse_Brain_Coronal_Subset_CTX_HP_outs", fov = "fov") Output: 10X data contains more than one type and is being returned as a list containing matrices of each type. Output: Warning: Feature names cannot have underscores ('_'), replacing with dashes ('-') ### Remove cells with 0 counts xenium.obj <- subset(xenium.obj, subset = nCount_Xenium > 0) Output: Warning messages: #1: Not validating FOV objects #2: Not validating Centroids objects [...] #9: Not validating Seurat objects dim(xenium.obj) # [1] 248 36553 ### Remove 4 genes molecules.to.remove = c("Gad1", "Sst", "Pvalb", "Gfap") all.features <-Features(xenium.obj) # grab all genes from the Seurat object length(all.features) # [1] 248 keep.genes <- all.features[!all.features %in% molecules.to.remove] length(keep.genes) # [1] 244 ### Subset Seurat object xenium.obj.subset <- subset(xenium.obj, features = keep.genes) Output: Warning messages: #1: Not validating FOV objects #2: Not validating Centroids objects [...] #9: Not validating Seurat objects dim(xenium.obj.subset) # [1] 244 36553 ### Plot data before and after subsetting plot.xenium.obj <- ImageDimPlot(xenium.obj, fov = "fov", molecules = c("Gad1", "Sst", "Pvalb", "Gfap"), nmols = 20000)+ggtitle("Before") plot.xenium.obj.subset <- ImageDimPlot(xenium.obj.subset, fov = "fov", molecules = c("Gad1", "Sst", "Pvalb", "Gfap"), nmols = 20000) +ggtitle("After subsetting") ggarrange(plot.xenium.obj, plot.xenium.obj.subset)

[alikhuseynov]

Hi, this can help I hope, though it's a temporary solution. Also check this discussion #7462 this would be the run: xenium.obj.subset <- subset_opt(xenium.obj, features = keep.genes)