New Error with subsetting in Seurat V5

[aLostMedStudent]

I was previously capable of manipulating this Seurat object with the subset function no without issue. Yesterday I updated my R from 4.1 to 4.3.2 and updated to Seurat V5. The weird thing is that this error is specific to this seurat object (i.e. when I loaded in a different object and used the subset function there was no such error). I would prefer not to have to re-create this object as it is rather larger. Thank you in advance!

The error is listed below:

fibroblasts <- subset(fibroblasts, idents = c("0", "1", "4"))
Error in getClassDef(x@superClass, package = packageSlot(x))@virtual :
no applicable method for @ applied to an object of class "NULL"

sessionInfo()
R version 4.3.2 (2023-10-31)
Platform: x86_64-apple-darwin20 (64-bit)
Running under: macOS Sonoma 14.1

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-x86_64/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: America/New_York
tzcode source: internal

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

other attached packages:
[1] Seurat_5.0.0 SeuratObject_5.0.0 sp_2.1-1

loaded via a namespace (and not attached):
[1] RColorBrewer_1.1-3 rstudioapi_0.15.0 jsonlite_1.8.7 magrittr_2.0.3 spatstat.utils_3.0-4 farver_2.1.1
[7] fs_1.6.3 vctrs_0.6.4 ROCR_1.0-11 memoise_2.0.1 spatstat.explore_3.2-5 htmltools_0.5.7
[13] usethis_2.2.2 sctransform_0.4.1 parallelly_1.36.0 KernSmooth_2.23-22 htmlwidgets_1.6.2 ica_1.0-3
[19] plyr_1.8.9 plotly_4.10.3 zoo_1.8-12 cachem_1.0.8 igraph_1.5.1 mime_0.12
[25] lifecycle_1.0.4 pkgconfig_2.0.3 Matrix_1.6-2 R6_2.5.1 fastmap_1.1.1 fitdistrplus_1.1-11
[31] future_1.33.0 shiny_1.7.5.1 digest_0.6.33 colorspace_2.1-0 patchwork_1.1.3 ps_1.7.5
[37] tensor_1.5 RSpectra_0.16-1 irlba_2.3.5.1 pkgload_1.3.3 labeling_0.4.3 progressr_0.14.0
[43] fansi_1.0.5 spatstat.sparse_3.0-3 httr_1.4.7 polyclip_1.10-6 abind_1.4-5 compiler_4.3.2
[49] remotes_2.4.2.1 withr_2.5.2 fastDummies_1.7.3 pkgbuild_1.4.2 MASS_7.3-60 sessioninfo_1.2.2
[55] tools_4.3.2 lmtest_0.9-40 httpuv_1.6.12 future.apply_1.11.0 goftest_1.2-3 glue_1.6.2
[61] callr_3.7.3 nlme_3.1-163 promises_1.2.1 grid_4.3.2 Rtsne_0.16 cluster_2.1.4
[67] reshape2_1.4.4 generics_0.1.3 gtable_0.3.4 spatstat.data_3.0-3 tidyr_1.3.0 data.table_1.14.8
[73] utf8_1.2.4 spatstat.geom_3.2-7 RcppAnnoy_0.0.21 ggrepel_0.9.4 RANN_2.6.1 pillar_1.9.0
[79] stringr_1.5.0 spam_2.10-0 RcppHNSW_0.5.0 later_1.3.1 splines_4.3.2 dplyr_1.1.3
[85] lattice_0.22-5 survival_3.5-7 deldir_1.0-9 tidyselect_1.2.0 miniUI_0.1.1.1 pbapply_1.7-2
[91] gridExtra_2.3 scattermore_1.2 devtools_2.4.5 matrixStats_1.1.0 stringi_1.7.12 lazyeval_0.2.2
[97] codetools_0.2-19 tibble_3.2.1 cli_3.6.1 uwot_0.1.16 xtable_1.8-4 reticulate_1.34.0
[103] munsell_0.5.0 processx_3.8.2 Rcpp_1.0.11 globals_0.16.2 spatstat.random_3.2-1 png_0.1-8
[109] parallel_4.3.2 ellipsis_0.3.2 ggplot2_3.4.4 prettyunits_1.2.0 dotCall64_1.1-0 profvis_0.3.8
[115] urlchecker_1.0.1 listenv_0.9.0 viridisLite_0.4.2 scales_1.2.1 ggridges_0.5.4 leiden_0.4.3
[121] purrr_1.0.2 crayon_1.5.2 rlang_1.1.2 cowplot_1.1.1

[RijndertAriese]

Hi, I have the same error unfortunately. It appeared that this was a result from the graphs embedded. If I removed the nn/snn graphs it will subset just fine for me, but I cannot use the subset for subsequent analysis. What worked for me was downgrading to a v4.x Seurat package.

Cheers

[frac2738]

Same here.

options(Seurat.object.assay.version = "v3")
> Idents(exp_Integrated) <- "ScType"
> exp_Integrated <- subset(exp_Integrated, idents = c("Platelet","HSPC","Eryth","CD4 Proliferating"), invert = TRUE) 
Error in getClassDef(x@superClass, package = packageSlot(x))@virtual :    
no applicable method for `@` applied to an object of class "NULL"

> class(exp_Integrated)
 [1] "Seurat"
 attr(,"package")
 [1] "SeuratObject"


> sessionInfo()
R version 4.3.1 (2023-06-16)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.6 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/libf77blas.so.3.10.3 
LAPACK: /usr/lib/x86_64-linux-gnu/atlas/liblapack.so.3.10.3;  LAPACK version 3.9.0

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=fr_FR.UTF-8        LC_COLLATE=en_US.UTF-8         LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=en_US.UTF-8    LC_PAPER=fr_FR.UTF-8      
 [8] LC_NAME=C                  LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       

time zone: Europe/Paris
tzcode source: system (glibc)

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] dplyr_1.1.3                 HGNChelper_0.8.1            scR_0.9.4                   ggpubr_0.6.0                doParallel_1.0.17               iterators_1.0.14            foreach_1.5.2              
 [8] pROC_1.18.5                 Boruta_8.0.0                ranger_0.15.1               caretEnsemble_2.0.3         caret_6.0-94                lattice_0.22-5              corrplot_0.92              
[15] kableExtra_1.3.4            SCpubr_2.0.2                DESeq2_1.40.2               RColorBrewer_1.1-3          RUVSeq_1.34.0               edgeR_3.42.4                limma_3.56.2               
[22] EDASeq_2.34.0               ShortRead_1.58.0            GenomicAlignments_1.36.0    SummarizedExperiment_1.30.2 MatrixGenerics_1.12.3       matrixStats_1.1.0           Rsamtools_2.16.0           
[29] GenomicRanges_1.52.1        Biostrings_2.68.1           GenomeInfoDb_1.36.4         XVector_0.40.0              IRanges_2.34.1              S4Vectors_0.38.2            BiocParallel_1.34.2        
[36] Biobase_2.60.0              BiocGenerics_0.46.0         factoextra_1.0.7            Seurat_5.0.0                SeuratObject_5.0.0          sp_2.1-1                    ggrepel_0.9.4              
[43] data.table_1.14.8           ggplot2_3.4.4               viridis_0.6.4               viridisLite_0.4.2           pheatmap_1.0.12            

loaded via a namespace (and not attached):
  [1] R.methodsS3_1.8.2       progress_1.2.2          nnet_7.3-19             goftest_1.2-3           vctrs_0.6.4             spatstat.random_3.2-1   digest_0.6.33           png_0.1-8              
  [9] shape_1.4.6             deldir_1.0-9            parallelly_1.36.0       MASS_7.3-60             reshape2_1.4.4          httpuv_1.6.12           withr_2.5.2             xfun_0.41              
 [17] ellipsis_0.3.2          survival_3.5-7          memoise_2.0.1           systemfonts_1.0.5       ragg_1.2.6              zoo_1.8-12              GlobalOptions_0.1.2     pbapply_1.7-2          
 [25] R.oo_1.25.0             prettyunits_1.2.0       KEGGREST_1.40.1         promises_1.2.1          httr_1.4.7              rstatix_0.7.2           restfulr_0.0.15         globals_0.16.2         
 [33] fitdistrplus_1.1-11     ps_1.7.5                rstudioapi_0.15.0       miniUI_0.1.1.1          generics_0.1.3          processx_3.8.2          curl_5.1.0              zlibbioc_1.46.0        
 [41] polyclip_1.10-6         GenomeInfoDbData_1.2.10 desc_1.4.2              xtable_1.8-4            stringr_1.5.0           evaluate_0.23           S4Arrays_1.0.6          BiocFileCache_2.8.0    
 [49] hms_1.1.3               irlba_2.3.5.1           colorspace_2.1-0        filelock_1.0.2          ROCR_1.0-11             reticulate_1.34.0       spatstat.data_3.0-3     magrittr_2.0.3         
 [57] lmtest_0.9-40           later_1.3.1             spatstat.geom_3.2-7     future.apply_1.11.0     scattermore_1.2             XML_3.99-0.15           cowplot_1.1.1           RcppAnnoy_0.0.21       
 [65] class_7.3-22            pillar_1.9.0            nlme_3.1-163            compiler_4.3.1          RSpectra_0.16-1         stringi_1.7.12              gower_1.0.1             tensor_1.5             
 [73] lubridate_1.9.3         plyr_1.8.9              crayon_1.5.2            abind_1.4-5             BiocIO_1.10.0           gridGraphics_0.5-1      locfit_1.5-9.8          bit_4.0.5              
 [81] codetools_0.2-19        textshaping_0.3.7       recipes_1.0.8           plotly_4.10.3           mime_0.12               splines_4.3.1           circlize_0.4.15         Rcpp_1.0.11            
 [89] fastDummies_1.7.3       dbplyr_2.4.0            interp_1.1-4            knitr_1.45              blob_1.2.4              utf8_1.2.4              fs_1.6.3                listenv_0.9.0          
 [97] pkgbuild_1.4.2          openxlsx_4.2.5.2        ggsignif_0.6.4          ggplotify_0.1.2         tibble_3.2.1            Matrix_1.6-2            callr_3.7.3             svglite_2.1.2          
[105] pkgconfig_2.0.3         tools_4.3.1             cachem_1.0.8            RSQLite_2.3.3           rvest_1.0.3             DBI_1.1.3               fastmap_1.1.1           rmarkdown_2.25         
[113] scales_1.2.1            grid_4.3.1              ica_1.0-3               broom_1.0.5             patchwork_1.1.3         dotCall64_1.1-0         carData_3.0-5           RANN_2.6.1             
[121] rpart_4.1.21            farver_2.1.1            yaml_2.3.7              latticeExtra_0.6-30     rtracklayer_1.60.1      cli_3.6.1               purrr_1.0.2             webshot_0.5.5          
[129] leiden_0.4.3            lifecycle_1.0.4         uwot_0.1.16             lava_1.7.3              backports_1.4.1         timechange_0.2.0        gtable_0.3.4            rjson_0.2.21           
[137] ggridges_0.5.4          progressr_0.14.0        jsonlite_1.8.7          RcppHNSW_0.5.0          bitops_1.0-7            bit64_4.0.5             assertthat_0.2.1        Rtsne_0.16             
[145] yulab.utils_0.1.0       spatstat.utils_3.0-4    zip_2.3.0               R.utils_2.12.2          timeDate_4022.108       lazyeval_0.2.2          shiny_1.7.5.1           htmltools_0.5.7        
[153] sctransform_0.4.1       rappdirs_0.3.3          glue_1.6.2              spam_2.10-0             RCurl_1.98-1.13         rprojroot_2.0.4         jpeg_0.1-10             gridExtra_2.3          
[161] igraph_1.5.1            R6_2.5.1                tidyr_1.3.0             forcats_1.0.0           labeling_0.4.3          GenomicFeatures_1.52.2  cluster_2.1.4           ipred_0.9-14           
[169] DelayedArray_0.26.7     tidyselect_1.2.0        xml2_1.3.5              car_3.1-2               AnnotationDbi_1.62.2    future_1.33.0           ModelMetrics_1.2.2.2    munsell_0.5.0          
[177] KernSmooth_2.23-22      htmlwidgets_1.6.2       aroma.light_3.30.0      hwriter_1.3.2.1         biomaRt_2.56.1          rlang_1.1.2             spatstat.sparse_3.0-3   spatstat.explore_3.2-5 
[185] remotes_2.4.2.1         fansi_1.0.5             hardhat_1.3.0           prodlim_2023.08.28 

I also get the same error when I try to split.by the feature plot in SCpubr.

[mhkowalski]

Hi, thanks for reporting this issue. Could you please try running UpdateSeuratObject on your object and then subsetting?

[aLostMedStudent]

Hi, I have done this and it did not fix the error. Thank you for your suggestion though!

…

On Nov 10, 2023 at 3:58 PM -0500, mhkowalski ***@***.***>, wrote: Hi, thanks for reporting this issue. Could you please try running UpdateSeuratObject on your object and then subsetting? — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[longmanz]

Hi,
Thanks for reporting this. This has been reported multiple times (#7983, #7993, #7999), and is caused by the Matrix package version 1.6-2. Please down-grade your Matrix back to 1.6-1.1 to temporarily avoid this error: remotes::install_version("Matrix", version = "1.6-1.1").

[afletch00]

I downgraded Matrix and I still cannot subset...

[ABauer26]

Hello!
I've also updated to Seurat v5 under R/4.2.3. I'm reaching a similar subsetting error with the spatial transcriptomics 10X Genomics Visium analysis under the seurat5 spatial vignette. While using the provided mouse brain spatial data and brain object, I run into the error that an object of type 'S4' is not subsettable while trying to execute a GroupCorrelation command. I have tried the referenced solutions above, including the UpdateSeuratObject as well as the Matrix package down-grade to 1.6-1.1. Nothing seems to work, so I thought I would also chime in to see if solutions become available. Cheers ~

[frac2738]

Downgrading to Matrix 1.6-1 (which is the minimum required by seurat5 ) fixed it.

> sessionInfo()
R version 4.3.1 (2023-06-16)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.6 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/libf77blas.so.3.10.3 
LAPACK: /usr/lib/x86_64-linux-gnu/atlas/liblapack.so.3.10.3;  LAPACK version 3.9.0

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=fr_FR.UTF-8        LC_COLLATE=en_US.UTF-8     LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=en_US.UTF-8    LC_PAPER=fr_FR.UTF-8      
 [8] LC_NAME=C                  LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       

time zone: Europe/Paris
tzcode source: system (glibc)

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] Matrix_1.6-1                dplyr_1.1.3                 HGNChelper_0.8.1            scR_0.9.4                   ggpubr_0.6.0                doParallel_1.0.17          
 [7] iterators_1.0.14            foreach_1.5.2               pROC_1.18.5                 Boruta_8.0.0                ranger_0.15.1               caretEnsemble_2.0.3        
[13] caret_6.0-94                lattice_0.22-5              corrplot_0.92               kableExtra_1.3.4            SCpubr_2.0.2                DESeq2_1.40.2              
[19] RColorBrewer_1.1-3          wesanderson_0.3.7           paletteer_1.5.0             RUVSeq_1.34.0               edgeR_3.42.4                limma_3.56.2               
[25] EDASeq_2.34.0               ShortRead_1.58.0            GenomicAlignments_1.36.0    SummarizedExperiment_1.30.2 MatrixGenerics_1.12.3       matrixStats_1.1.0          
[31] Rsamtools_2.16.0            GenomicRanges_1.52.1        Biostrings_2.68.1           GenomeInfoDb_1.36.4         XVector_0.40.0              IRanges_2.34.1             
[37] S4Vectors_0.38.2            BiocParallel_1.34.2         Biobase_2.60.0              BiocGenerics_0.46.0         factoextra_1.0.7            Seurat_5.0.0               
[43] SeuratObject_5.0.0          sp_2.1-1                    ggrepel_0.9.4               data.table_1.14.8           ggplot2_3.4.4               viridis_0.6.4              
[49] viridisLite_0.4.2           pheatmap_1.0.12            

loaded via a namespace (and not attached):
  [1] spatstat.sparse_3.0-3   bitops_1.0-7            lubridate_1.9.3         httr_1.4.7              webshot_0.5.5           backports_1.4.1         tools_4.3.1             sctransform_0.4.1      
  [9] utf8_1.2.4              R6_2.5.1                lazyeval_0.2.2          uwot_0.1.16             withr_2.5.2             prettyunits_1.2.0       gridExtra_2.3           progressr_0.14.0       
 [17] cli_3.6.1               spatstat.explore_3.2-5  fastDummies_1.7.3       spatstat.data_3.0-3     ggridges_0.5.4          pbapply_1.7-2           systemfonts_1.0.5       svglite_2.1.2          
 [25] R.utils_2.12.2          parallelly_1.36.0       rstudioapi_0.15.0       RSQLite_2.3.3           shape_1.4.6             generics_0.1.3          BiocIO_1.10.0           hwriter_1.3.2.1        
 [33] ica_1.0-3               spatstat.random_3.2-1   car_3.1-2               zip_2.3.0               interp_1.1-4            fansi_1.0.5             abind_1.4-5             R.methodsS3_1.8.2      
 [41] lifecycle_1.0.4         yaml_2.3.7              carData_3.0-5           recipes_1.0.8           BiocFileCache_2.8.0     Rtsne_0.16              grid_4.3.1              blob_1.2.4             
 [49] promises_1.2.1          crayon_1.5.2            miniUI_0.1.1.1          cowplot_1.1.1           GenomicFeatures_1.52.2  KEGGREST_1.40.1         pillar_1.9.0            knitr_1.45             
 [57] rjson_0.2.21            future.apply_1.11.0     codetools_0.2-19        leiden_0.4.3            glue_1.6.2              remotes_2.4.2.1         vctrs_0.6.4             png_0.1-8              
 [65] spam_2.10-0             gtable_0.3.4            rematch2_2.1.2          cachem_1.0.8            gower_1.0.1             aroma.light_3.30.0      xfun_0.41               openxlsx_4.2.5.2       
 [73] prodlim_2023.08.28      S4Arrays_1.0.6          mime_0.12               survival_3.5-7          timeDate_4022.108       hardhat_1.3.0           lava_1.7.3              ellipsis_0.3.2         
 [81] fitdistrplus_1.1-11     ipred_0.9-14            ROCR_1.0-11             nlme_3.1-163            bit64_4.0.5             progress_1.2.2          filelock_1.0.2          RcppAnnoy_0.0.21       
 [89] irlba_2.3.5.1           rpart_4.1.21            KernSmooth_2.23-22      colorspace_2.1-0        DBI_1.1.3               nnet_7.3-19             tidyselect_1.2.0        processx_3.8.2         
 [97] bit_4.0.5               compiler_4.3.1          curl_5.1.0              rvest_1.0.3             xml2_1.3.5              DelayedArray_0.26.7     plotly_4.10.3           rtracklayer_1.60.1     
[105] scales_1.2.1            lmtest_0.9-40           callr_3.7.3             rappdirs_0.3.3          stringr_1.5.0           digest_0.6.33           goftest_1.2-3           spatstat.utils_3.0-4   
[113] rmarkdown_2.25          htmltools_0.5.7         pkgconfig_2.0.3         jpeg_0.1-10             dbplyr_2.4.0            fastmap_1.1.1           GlobalOptions_0.1.2     rlang_1.1.2            
[121] htmlwidgets_1.6.2       shiny_1.7.5.1           zoo_1.8-12              jsonlite_1.8.7          ModelMetrics_1.2.2.2    R.oo_1.25.0             RCurl_1.98-1.13         magrittr_2.0.3         
[129] GenomeInfoDbData_1.2.10 dotCall64_1.1-0         patchwork_1.1.3         munsell_0.5.0           Rcpp_1.0.11             reticulate_1.34.0       stringi_1.7.12          zlibbioc_1.46.0        
[137] MASS_7.3-60             plyr_1.8.9              pkgbuild_1.4.2          listenv_0.9.0           deldir_1.0-9            splines_4.3.1           tensor_1.5              circlize_0.4.15        
[145] hms_1.1.3               locfit_1.5-9.8          ps_1.7.5                igraph_1.5.1            spatstat.geom_3.2-7     ggsignif_0.6.4          RcppHNSW_0.5.0          reshape2_1.4.4         
[153] biomaRt_2.56.1          XML_3.99-0.15           evaluate_0.23           latticeExtra_0.6-30     httpuv_1.6.12           RANN_2.6.1              tidyr_1.3.0             purrr_1.0.2            
[161] polyclip_1.10-6         future_1.33.0           scattermore_1.2         broom_1.0.5             xtable_1.8-4            restfulr_0.0.15         RSpectra_0.16-1         rstatix_0.7.2          
[169] later_1.3.1             class_7.3-22            tibble_3.2.1            memoise_2.0.1           AnnotationDbi_1.62.2    cluster_2.1.4           timechange_0.2.0        globals_0.16.2