BUG: PseudobulkExpression ignores user-specified scale.factor argument

[anncfs]

Issue Description

PseudobulkExpression (and AggregateExpression) do not pass user-specified scale.factor to NormalizeData

Reproducing Code Example

library(Seurat)

# create two pseudobulk expression Seurat objects using "groups" metadata annotation of pbmc_small
pb_1e6 <- PseudobulkExpression(pbmc_small, return.seurat = T, method = "aggregate", group.by = "groups", normalization.method = "RC", scale.factor = 1e6)
pb_default <- PseudobulkExpression(pbmc_small, return.seurat = T, method = "aggregate", group.by = "groups", normalization.method = "RC")

# default scale.factor is used for RC normalisation regardless of whether this is specified above
colSums(pb_1e6[["RNA"]]$data)
#   g1    g2 
# 10000 10000 
colSums(pb_default[["RNA"]]$data)
#   g1    g2 
# 10000 10000 

# you need to rerun Normalize Data directly to use a different scale.factor
colSums(NormalizeData(pb_1e6, normalization.method = "RC", scale.factor = 1e6))
#   g1    g2 
# 1e+06 1e+06 

Error Message

Additional Comments

I believe this is due to the scale.factor argument specified when using either AggregateExpression or PseudobulkExpression not being passed to NormalizeData in lines 1546-1550 of seurat/R/utilities.R

Session Info

R version 4.4.2 (2024-10-31)
Platform: aarch64-apple-darwin20.0.0
Running under: macOS Ventura 13.6.7

Matrix products: default
BLAS/LAPACK: /Users/ab1511/miniconda3/envs/singlecell/lib/libopenblas.0.dylib;  LAPACK version 3.12.0

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

time zone: Europe/London
tzcode source: system (macOS)

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

other attached packages:
[1] Seurat_5.2.1       SeuratObject_5.0.2 sp_2.2-0          

loaded via a namespace (and not attached):
  [1] deldir_2.0-4           pbapply_1.7-2          gridExtra_2.3         
  [4] rlang_1.1.5            magrittr_2.0.3         RcppAnnoy_0.0.22      
  [7] matrixStats_1.5.0      ggridges_0.5.6         compiler_4.4.2        
 [10] spatstat.geom_3.3-5    png_0.1-8              vctrs_0.6.5           
 [13] reshape2_1.4.4         stringr_1.5.1          pkgconfig_2.0.3       
 [16] fastmap_1.2.0          promises_1.3.2         purrr_1.0.4           
 [19] jsonlite_1.9.0         goftest_1.2-3          later_1.4.1           
 [22] spatstat.utils_3.1-2   irlba_2.3.5.1          parallel_4.4.2        
 [25] cluster_2.1.8          R6_2.6.1               ica_1.0-3             
 [28] stringi_1.8.4          RColorBrewer_1.1-3     spatstat.data_3.1-4   
 [31] reticulate_1.40.0      parallelly_1.42.0      spatstat.univar_3.1-1 
 [34] lmtest_0.9-40          scattermore_1.2        Rcpp_1.0.14           
 [37] tensor_1.5             future.apply_1.11.3    zoo_1.8-12            
 [40] sctransform_0.4.1      httpuv_1.6.15          Matrix_1.7-2          
 [43] splines_4.4.2          igraph_2.1.4           tidyselect_1.2.1      
 [46] abind_1.4-8            codetools_0.2-20       spatstat.random_3.3-2 
 [49] miniUI_0.1.1.1         spatstat.explore_3.3-4 listenv_0.9.1         
 [52] lattice_0.22-6         tibble_3.2.1           plyr_1.8.9            
 [55] shiny_1.10.0           ROCR_1.0-11            Rtsne_0.17            
 [58] future_1.34.0          fastDummies_1.7.5      survival_3.8-3        
 [61] polyclip_1.10-7        fitdistrplus_1.2-2     pillar_1.10.1         
 [64] KernSmooth_2.23-26     plotly_4.10.4          generics_0.1.3        
 [67] RcppHNSW_0.6.0         ggplot2_3.5.1          munsell_0.5.1         
 [70] scales_1.3.0           globals_0.16.3         xtable_1.8-4          
 [73] glue_1.8.0             lazyeval_0.2.2         tools_4.4.2           
 [76] data.table_1.16.4      RSpectra_0.16-2        RANN_2.6.2            
 [79] dotCall64_1.2          cowplot_1.1.3          grid_4.4.2            
 [82] tidyr_1.3.1            colorspace_2.1-1       nlme_3.1-167          
 [85] patchwork_1.3.0        cli_3.6.4              spatstat.sparse_3.1-0 
 [88] spam_2.11-1            viridisLite_0.4.2      dplyr_1.1.4           
 [91] uwot_0.2.2             gtable_0.3.6           digest_0.6.37         
 [94] progressr_0.15.1       ggrepel_0.9.6          htmlwidgets_1.6.4     
 [97] farver_2.1.2           htmltools_0.5.8.1      lifecycle_1.0.4       
[100] httr_1.4.7             mime_0.12              MASS_7.3-64

[mhkowalski]

Can confirm that this a bug, thank you for reporting this! We will have a fix out as soon as we can.