how to integrate multiple HD Visium data

[JuanXie19]

Dear Seurat team,

I am using Seurat v5.1.0 to analyze HD Visium data, which consists of two biological replicates from a spinal cord sample. We hope to combine/integrate the two datasets and then to run clustering. I read the vignette (https://satijalab.org/seurat/articles/visiumhd_analysis_vignette), which seems to focus on single dataset analysis. I also searched and found one related issue (issue #9007), but unfortunately didn't get much useful information. In addition, I read issue #7094 and tried to follow but failed.

Here is what I tried so far:

data1 <- Load10X_Spatial(data.dir = dir1, bin.size = c(8,16))
DefaultAssay(data1) <- 'Spatial.016um'

data2 <- Load10X_Spatial(data.dir = dir2, bin.size = c(8,16))
DefaultAssay(data2) <- "Spatial.016um"

data1 <- NormalizeData(data1)
data2 <- NormalizeData(data2)

data <- merge(x = data1, y = data2, add.cell.ids = c('rep1','rep2'))

I got an error when running the merge function:

I checked data1, and here is the basic info:

I also checked the spatial info, which is as follows:

Could you please help and see what might be the problem? I am new to HD Visium data and please pardon me if the question is too naive.

Thanks!

[JuanXie19]

Hello!

Just a follow-up for my attempts.

I recreated the Seurat object by loading only the 16um resolution for each data set, run normalization and scaling, and then merging the two data and RunPCA on the merged data:

data1 <- Load10X_Spatial(data.dir = dir1, bin.size = c(16))

data2 <- Load10X_Spatial(data.dir = dir2, bin.size = c(16))


data1 <- NormalizeData(data1)
data1 <- FindVariableFeatures(data1)
data1 <- ScaleData(data1)

data2 <- NormalizeData(data2)
data2 <- FindVariableFeatures(data2)
data2 <- ScaleData(data2)

data <- merge(x = data1, y = data2, add.cell.ids = c('rep1','rep2'))
data <- NormalizedData(data)
data <- FindVariableFeatures(data)
data <- ScaleData(data)

data <- RunPCA(data)
data <- FindNeighbors(DATA, dims = 1:50, reduction = 'pca')
data <- FindClusters(DATA, resolution = 0.5)

I didn't get error message during the process, but I am not sure if this is the appropriate process.

In addition, I saw HD Visium vignette uses sketched cells, is it possible to combine multiple datasets using sketched cells? Furthermore, is it possible to do integration using sketched cells + SCTransformation?