iSCanGuide’s native formats are .h5ad and 10x MTX, so Seurat .rds objects need to be converted first. We recommend anndataR, the scverse-maintained R package, which writes .h5ad directly from a Seurat object without requiring a Python environment — SeuratDisk is no longer actively maintained and fails on Seurat v5 assays. Please make sure raw counts are included in the converted file, since iSCanGuide runs its own normalization.