Issue with SeuratObject version leads to different numbers of observation from FindAllMarkers

[eunksung]

Since I updated Seurat from v4 to v5, I kept getting this error message when I was loading Seurat: library(Seurat)

Loading required package: SeuratObject Loading required package: sp ‘SeuratObject’ was built under R 4.3.1 but the current version is 4.3.3; it is recomended that you reinstall ‘SeuratObject’ as the ABI for R may have changed ‘SeuratObject’ was built with package ‘Matrix’ 1.6.3 but the current version is 1.6.5; it is recomended that you reinstall ‘SeuratObject’ as the ABI for ‘Matrix’ may have changed

When I ran FindAllMarkers after PrepSCTFindMarkers, the number of observation was changed from 7,141 to 46,367. This is the code that I used: ALL.MARKERS.WNN <- FindAllMarkers(seg_status, assay = "SCT", recorrect_umi = FALSE)

My previous version of Seurat and SeuratObject was 4.4.0 and 4.1.4, respectively. The Matrix version was 1.6-5.

I deleted SeuratObject and Matrix package and re-install, and I restarted R whenever I deleted or re-installed them. I also deleted Seurat and Signac and re-installed them, but it kept giving me the same error. I even uninstalled R and RStudio and re-downloaded them. After that, I installed Seurat and Signac, non other packages. However, I am still getting same error.

I tried to update my Seurat object. The version of my Seurat object (CF_MultiModal_WNN) was 4.1.4: seg_status@version

‘4.1.4’

I used this code to update the object: seg_status <- UpdateSeuratObject(seg_status)

Validating object structure Updating object slots Ensuring keys are in the proper structure Updating matrix keys for DimReduc ‘pca’ Updating matrix keys for DimReduc ‘umap’ Updating matrix keys for DimReduc ‘lsi’ Updating matrix keys for DimReduc ‘umap.atac’ Updating matrix keys for DimReduc ‘harmony’ Updating matrix keys for DimReduc ‘wnn.umap’ Ensuring keys are in the proper structure Ensuring feature names don't have underscores or pipes Updating slots in RNA Updating slots in ATAC Updating slots in SCT Updating slots in integrated Updating slots in integrated_nn Setting default assay of integrated_nn to integrated Updating slots in integrated_snn Setting default assay of integrated_snn to integrated Updating slots in ATAC_nn Setting default assay of ATAC_nn to ATAC Updating slots in ATAC_snn Setting default assay of ATAC_snn to ATAC Updating slots in wknn Cannot find wknn in the object, setting default assay of wknn to ATAC Updating slots in wsnn Cannot find wsnn in the object, setting default assay of wsnn to ATAC Updating slots in pca Updating slots in umap Setting umap DimReduc to global Updating slots in lsi Updating slots in umap.atac Setting umap.atac DimReduc to global Updating slots in harmony Updating slots in wnn.umap Setting wnn.umap DimReduc to global No assay information could be found for FindIntegrationAnchors No assay information could be found for withCallingHandlers Setting assay used for ScaleData.integrated to integrated Setting assay used for RunPCA.integrated to integrated Setting assay used for FindNeighbors.integrated.pca to integrated Setting assay used for RunUMAP.integrated.pca to integrated Setting assay used for RunUMAP.ATAC.lsi to ATAC Setting assay used for RunUMAP.ATAC.harmony to ATAC Setting assay used for FindNeighbors.ATAC.harmony to ATAC No assay information could be found for FindModalityWeights No assay information could be found for FindMultiModalNeighbors Setting assay used for RunUMAP.ATAC.pca to integrated No assay information could be found for FindClusters Validating object structure for Assay ‘RNA’ Validating object structure for ChromatinAssay ‘ATAC’ Validating object structure for SCTAssay ‘SCT’ Validating object structure for Assay ‘integrated’ Validating object structure for Graph ‘integrated_nn’ Validating object structure for Graph ‘integrated_snn’ Validating object structure for Graph ‘ATAC_nn’ Validating object structure for Graph ‘ATAC_snn’ Validating object structure for Graph ‘wknn’ Validating object structure for Graph ‘wsnn’ Validating object structure for DimReduc ‘pca’ Validating object structure for DimReduc ‘umap’ Validating object structure for DimReduc ‘lsi’ Validating object structure for DimReduc ‘umap.atac’ Validating object structure for DimReduc ‘harmony’ Validating object structure for DimReduc ‘wnn.umap’ Object representation is consistent with the most current Seurat version Warning messages: 1: Adding a command log without an assay associated with it 2: Adding a command log without an assay associated with it 3: Adding a command log without an assay associated with it 4: Adding a command log without an assay associated with it 5: Adding a command log without an assay associated with it

The version of the object changed to 5.0.1, but this didn't help me to solve the issue.

This is my sessionInfo:

R version 4.3.3 (2024-02-29) Platform: aarch64-apple-darwin20 (64-bit) Running under: macOS Sonoma 14.4

Matrix products: default BLAS: /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.11.0

locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: US/Pacific tzcode source: internal

attached base packages: [1] stats graphics grDevices utils datasets methods base

other attached packages: [1] Seurat_5.0.3 SeuratObject_5.0.1 sp_2.1-3

loaded via a namespace (and not attached): [1] deldir_2.0-4 pbapply_1.7-2 gridExtra_2.3
[4] rlang_1.1.3 magrittr_2.0.3 RcppAnnoy_0.0.22
[7] matrixStats_1.2.0 ggridges_0.5.6 compiler_4.3.3
[10] spatstat.geom_3.2-9 png_0.1-8 vctrs_0.6.5
[13] reshape2_1.4.4 stringr_1.5.1 pkgconfig_2.0.3
[16] fastmap_1.1.1 utf8_1.2.4 promises_1.2.1
[19] purrr_1.0.2 jsonlite_1.8.8 goftest_1.2-3
[22] later_1.3.2 spatstat.utils_3.0-4 irlba_2.3.5.1
[25] parallel_4.3.3 cluster_2.1.6 R6_2.5.1
[28] ica_1.0-3 stringi_1.8.3 RColorBrewer_1.1-3
[31] spatstat.data_3.0-4 reticulate_1.35.0 parallelly_1.37.1
[34] lmtest_0.9-40 scattermore_1.2 Rcpp_1.0.12
[37] tensor_1.5 future.apply_1.11.2 zoo_1.8-12
[40] sctransform_0.4.1 httpuv_1.6.15 Matrix_1.6-5
[43] splines_4.3.3 igraph_2.0.3 tidyselect_1.2.1
[46] rstudioapi_0.16.0 abind_1.4-5 spatstat.random_3.2-3 [49] codetools_0.2-19 miniUI_0.1.1.1 spatstat.explore_3.2-7 [52] listenv_0.9.1 lattice_0.22-5 tibble_3.2.1
[55] plyr_1.8.9 shiny_1.8.1 ROCR_1.0-11
[58] Rtsne_0.17 future_1.33.2 fastDummies_1.7.3
[61] survival_3.5-8 polyclip_1.10-6 fitdistrplus_1.1-11
[64] pillar_1.9.0 KernSmooth_2.23-22 plotly_4.10.4
[67] generics_0.1.3 RcppHNSW_0.6.0 ggplot2_3.5.0
[70] munsell_0.5.0 scales_1.3.0 globals_0.16.3
[73] xtable_1.8-4 glue_1.7.0 pheatmap_1.0.12
[76] lazyeval_0.2.2 tools_4.3.3 data.table_1.15.2
[79] RSpectra_0.16-1 RANN_2.6.1 leiden_0.4.3.1
[82] dotCall64_1.1-1 cowplot_1.1.3 grid_4.3.3
[85] tidyr_1.3.1 colorspace_2.1-0 nlme_3.1-164
[88] patchwork_1.2.0 cli_3.6.2 spatstat.sparse_3.0-3 [91] spam_2.10-0 fansi_1.0.6 viridisLite_0.4.2
[94] dplyr_1.1.4 uwot_0.1.16 gtable_0.3.4
[97] digest_0.6.35 progressr_0.14.0 ggrepel_0.9.5
[100] htmlwidgets_1.6.4 htmltools_0.5.8 lifecycle_1.0.4
[103] httr_1.4.7 mime_0.12 MASS_7.3-60.0.1