IntegrateLayers: Error in UseMethod(generic = "Assays", object = object) : no applicable method for 'Assays' applied to an object of class "NULL"

[changve]

Hello, I am working with a log normalized data set from: http://gerg.gsc.riken.jp/SC2018/. I'm trying to go through the integration introduction vignette. I have gone through the steps in this section but proceeding to
IntegrateLayers(object = stripped.seu, method = CCAIntegration, orig.reduction = "pca", new.reduction = "integrated.cca", verbose = FALSE) on my Seurat object, I'm getting the following error:

  |                                                  | 0 % ~calculating  Error in UseMethod(generic = "Assays", object = object) : 
  no applicable method for 'Assays' applied to an object of class "NULL"

I only have one assay which is "RNA". I have already tried running both [email protected] = "RNA" and DefaultAssay(stripped.seu) <- "RNA" before integration.

This is my session info:

R version 4.4.0 (2024-04-24)
Platform: aarch64-apple-darwin20
Running under: macOS Sonoma 14.2

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

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/Toronto
tzcode source: internal

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

other attached packages:
 [1] ggpubr_0.6.0                forcats_1.0.0               reshape2_1.4.4              EnsDb.Hsapiens.v86_2.99.0   ensembldb_2.28.0            AnnotationFilter_1.28.0     GenomicFeatures_1.56.0     
 [8] AnnotationDbi_1.66.0        msigdbr_7.5.1               clusterProfiler_4.12.0      dbplyr_2.5.0                SingleR_2.6.0               clustree_0.5.1              ggraph_2.2.1               
[15] Matrix_1.7-0                pheatmap_1.0.12             viridis_0.6.5               viridisLite_0.4.2           Seurat_5.1.0                SeuratObject_5.0.2          sp_2.1-4                   
[22] SingleCellExperiment_1.26.0 SummarizedExperiment_1.34.0 GenomicRanges_1.56.0        GenomeInfoDb_1.40.1         IRanges_2.38.0              S4Vectors_0.42.0            MatrixGenerics_1.16.0      
[29] matrixStats_1.3.0           kableExtra_1.4.0            RColorBrewer_1.1-3          gprofiler2_0.2.3            ggplot2_3.5.1               dplyr_1.1.4                 plyr_1.8.9                 
[36] tinytex_0.51                knitr_1.47                  Biobase_2.64.0              BiocGenerics_0.50.0         biomaRt_2.60.0              edgeR_4.2.0                 limma_3.60.2               

loaded via a namespace (and not attached):
  [1] ProtGenerics_1.36.0       fs_1.6.4                  spatstat.sparse_3.0-3     bitops_1.0-7              enrichplot_1.24.0         HDO.db_0.99.1             httr_1.4.7               
  [8] backports_1.5.0           tools_4.4.0               sctransform_0.4.1         utf8_1.2.4                R6_2.5.1                  lazyeval_0.2.2            uwot_0.2.2               
 [15] withr_3.0.0               prettyunits_1.2.0         gridExtra_2.3             progressr_0.14.0          cli_3.6.2                 spatstat.explore_3.2-7    fastDummies_1.7.3        
 [22] scatterpie_0.2.3          spatstat.data_3.0-4       ggridges_0.5.6            pbapply_1.7-2             yulab.utils_0.1.4         Rsamtools_2.20.0          systemfonts_1.1.0        
 [29] gson_0.1.0                DOSE_3.30.1               svglite_2.1.3             parallelly_1.37.1         rstudioapi_0.16.0         RSQLite_2.3.7             gridGraphics_0.5-1       
 [36] generics_0.1.3            BiocIO_1.14.0             ica_1.0-3                 spatstat.random_3.2-3     car_3.1-2                 GO.db_3.19.1              fansi_1.0.6              
 [43] abind_1.4-5               lifecycle_1.0.4           yaml_2.3.8                carData_3.0-5             qvalue_2.36.0             SparseArray_1.4.8         BiocFileCache_2.12.0     
 [50] Rtsne_0.17                grid_4.4.0                blob_1.2.4                promises_1.3.0            crayon_1.5.2              miniUI_0.1.1.1            lattice_0.22-6           
 [57] beachmat_2.20.0           cowplot_1.1.3             KEGGREST_1.44.0           pillar_1.9.0              fgsea_1.30.0              rjson_0.2.21              future.apply_1.11.2      
 [64] codetools_0.2-20          fastmatch_1.1-4           leiden_0.4.3.1            glue_1.7.0                ggfun_0.1.5               data.table_1.15.4         remotes_2.5.0            
 [71] treeio_1.28.0             vctrs_0.6.5               png_0.1-8                 spam_2.10-0               gtable_0.3.5              cachem_1.1.0              xfun_0.44                
 [78] S4Arrays_1.4.1            mime_0.12                 tidygraph_1.3.1           tidyverse_2.0.0           survival_3.5-8            statmod_1.5.0             fitdistrplus_1.1-11      
 [85] ROCR_1.0-11               nlme_3.1-164              ggtree_3.12.0             bit64_4.0.5               progress_1.2.3            filelock_1.0.3            RcppAnnoy_0.0.22         
 [92] irlba_2.3.5.1             KernSmooth_2.23-22        colorspace_2.1-0          DBI_1.2.3                 DESeq2_1.44.0             tidyselect_1.2.1          bit_4.0.5                
 [99] compiler_4.4.0            curl_5.2.1                httr2_1.0.1               xml2_1.3.6                DelayedArray_0.30.1       plotly_4.10.4             shadowtext_0.1.3         
[106] rtracklayer_1.64.0        scales_1.3.0              lmtest_0.9-40             rappdirs_0.3.3            stringr_1.5.1             digest_0.6.35             goftest_1.2-3            
[113] spatstat.utils_3.0-4      rmarkdown_2.27            XVector_0.44.0            htmltools_0.5.8.1         pkgconfig_2.0.3           sparseMatrixStats_1.16.0  fastmap_1.2.0            
[120] rlang_1.1.4               htmlwidgets_1.6.4         UCSC.utils_1.0.0          DelayedMatrixStats_1.26.0 shiny_1.8.1.1             farver_2.1.2              zoo_1.8-12               
[127] jsonlite_1.8.8            BiocParallel_1.38.0       GOSemSim_2.30.0           BiocSingular_1.20.0       RCurl_1.98-1.14           magrittr_2.0.3            ggplotify_0.1.2          
[134] GenomeInfoDbData_1.2.12   dotCall64_1.1-1           patchwork_1.2.0           munsell_0.5.1             Rcpp_1.0.12               babelgene_22.9            ape_5.8                  
[141] reticulate_1.37.0         stringi_1.8.4             zlibbioc_1.50.0           MASS_7.3-60.2             parallel_4.4.0            listenv_0.9.1             ggrepel_0.9.5            
[148] deldir_2.0-4              Biostrings_2.72.1         graphlayouts_1.1.1        splines_4.4.0             tensor_1.5                hms_1.1.3                 locfit_1.5-9.9           
[155] igraph_2.0.3              spatstat.geom_3.2-9       ggsignif_0.6.4            RcppHNSW_0.6.0            ScaledMatrix_1.12.0       XML_3.99-0.16.1           evaluate_0.24.0          
[162] BiocManager_1.30.23       tweenr_2.0.3              httpuv_1.6.15             RANN_2.6.1                tidyr_1.3.1               purrr_1.0.2               polyclip_1.10-6          
[169] future_1.33.2             scattermore_1.2           ggforce_0.4.2             rsvd_1.0.5                broom_1.0.6               xtable_1.8-4              restfulr_0.0.15          
[176] tidytree_0.4.6            RSpectra_0.16-1           rstatix_0.7.2             later_1.3.2               tibble_3.2.1              aplot_0.2.2               memoise_2.0.1            
[183] GenomicAlignments_1.40.0  cluster_2.1.6             globals_0.16.3          

[Alexis-Varin]

Hello, you likely haven't split your layers before running IntegrateLayers.

This line, above the section in the tutorial you have linked, is absolutely crucial :
ifnb[["RNA"]] <- split(ifnb[["RNA"]], f = ifnb$stim)

This replaces the SplitObject function in Seurat v4 integration pipeline. Here the f parameter needs to be one of the metadata column, it could be orig.ident, condition, treatment etc