Issues with RunPCA on Seurat object with BPCells matrix directory

[fingeram]

Hi everyone,

I am having problems with running dimensionality reduction on my seurat object created from a .h5ad file using BPCells. I checked the count matrix and everything looks completely fine (no NAs, values >=0). However, once I reach the step of RunPCA in my pipeline and then check the result using an Elbow plot, something looks very wrong and I simply cannot figure out what is the cause of it. Like why is the standard deviation so large and why does it drop after the 1st PC? Also, when trying to run clustering after RunPCA, it returns an error (see below).

I have the same problem if I subset my seurat object to reduce its size and transform the BPCells count matrix into a dgCMatrix...PCA embeddings and ElbowPlot still make no sense.

#import raw data from h5ad file
raw <- open_matrix_anndata_hdf5(path="/novo/projects/departments/compbio/sysbio/Projects/mouse_liver_models/single_cell_and_nuclei/concatenated.dir/concatenated.h5ad")

#write matrix directory for on-disk storage
write_matrix_dir(mat = raw, dir = "/novo/projects/shared_projects/liver_biology_colab/people/aqnf/mouse_sc_sn_AQNF_June24/BPcells/mouse_counts")

#load data from matrix directory to generate seurat object
raw.mat <- open_matrix_dir(dir = "/novo/projects/shared_projects/liver_biology_colab/people/aqnf/mouse_sc_sn_AQNF_June24/BPcells/mouse_counts")

#merge meta data
meta <- merge(x= metadata_BSCK, y= metadata_CPDM, by.x = "LibraryID", by.y = "library_id", all.y=T)
rownames(meta) <- meta$barcode

# generate Seurat object from on-disk matrix
sobj <- CreateSeuratObject(counts = raw.mat, meta.data = meta)

vals <- as(sobj[["RNA"]]$counts[1:500,1:500], "dgCMatrix")
anyNA(vals)
all(vals >= 0)
any(vals == 0)

# process seurat object
sobj <- NormalizeData(sobj)
sobj <- FindVariableFeatures(sobj)
sobj <- ScaleData(sobj)
sobj <- RunPCA(sobj)
ElbowPlot(sobj.sketch, ndims = 50, reduction = 'pca')

sobj <- FindNeighbors(sobj, dims = 1:50)
Computing nearest neighbor graph
Computing SNN
Error: std::bad_alloc