Run scATOMIC-26-09-2025-All_samples_merged-Looped

Load libraries————————————

1. Load SS seurat object

# Load full Seurat object
All_samples <- readRDS("../../0-Seurat_RDS_OBJECT_FINAL/Seurat_object_Final_changes/All_samples_Merged_with_STCAT_Annotation_final-5-09-2025.rds")

DefaultAssay(All_samples) <- "RNA"

Idents(All_samples) <- "orig.ident"

2. Running scATOMIC and Loop through each cell group

# Define groups exactly as in metadata
cell_groups <- c("L1","L2","L3","L4","L5","L6","L7","CD4T_lab","CD4T_10x")

# Initialize empty list
predictions_all <- list()

for (grp in cell_groups){
  
  message("Processing group: ", grp)
  
  # Subset Seurat object by orig.ident only
  subset_obj <- subset(All_samples, idents = grp)
  
  # Check that the subset has cells
  if(ncol(subset_obj) == 0){
    warning("No cells found for group: ", grp)
    next
  }
  
  # Get counts and filter zero-count genes
  sparse_matrix <- GetAssayData(subset_obj, assay="RNA", slot="counts")
  sparse_matrix_filtered <- sparse_matrix[rowSums(sparse_matrix) > 0, ]
  
  # Run scATOMIC
  cell_predictions <- run_scATOMIC(sparse_matrix_filtered)
  
  # Create summary matrix
  summary_matrix <- create_summary_matrix(
    prediction_list = cell_predictions,
    use_CNVs = FALSE,
    modify_results = TRUE,
    mc.cores = 1,
    raw_counts = sparse_matrix,
    min_prop = 0.5
  )
  
  # Interactive tree (optional)
  tree_results_Interactive <- scATOMICTree(
    predictions_list = cell_predictions,
    summary_matrix = summary_matrix,
    interactive_mode = TRUE,
    collapsed = TRUE,
    save_results = FALSE,
    height = 700,
    width = 1000
  )
  print(tree_results_Interactive)
  
  # Non-interactive tree (optional)
  tree_results_Non_Interactive <- scATOMICTree(
    predictions_list = cell_predictions,
    summary_matrix = summary_matrix,
    interactive_mode = FALSE,
    collapsed = TRUE,
    save_results = FALSE
  )
  print(tree_results_Non_Interactive)
  
  # Store summary matrix in list
  predictions_all[[grp]] <- summary_matrix
  
  # Clean up memory properly
  rm(
    subset_obj,
    sparse_matrix,
    sparse_matrix_filtered,
    cell_predictions,
    summary_matrix,
    tree_results_Interactive,
    tree_results_Non_Interactive
  )
  gc()
}

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"
[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"
[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"
[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"
[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 4 Soft Tissue Neuro"
[1] "Done Layer 4 Soft Tissue Neuro"
[1] "Starting Layer 2 Blood"
[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5825 x 36601) matrix will require approximately 1.59GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 100
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 71
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 34
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.15"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"
[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"
[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"
[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"
[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 2 Blood"
[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 4 CD8 NK"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD8 NK"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5935 x 36601) matrix will require approximately 1.62GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5155 x 36601) matrix will require approximately 1.41GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (3587 x 36601) matrix will require approximately 0.98GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 37
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.33"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 4 Soft Tissue Neuro"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Soft Tissue Neuro"
[1] "Starting Layer 2 Blood"
[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 3 Myeloid"
[1] "Done Layer 3 Myeloid"
[1] "Starting Layer 4 Macrophage Monocyte"
[1] "nothing to score in this layer"
[1] "Done Layer 4 Macrophage Monocyte"
[1] "Starting Layer 5 Macrophage"
[1] "nothing to score in this layer"
[1] "Done Layer 5 Macrophage"
[1] "Starting Layer 3 B Cell"
[1] "Done Layer 3 B Cell"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (6428 x 36601) matrix will require approximately 1.75GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.92"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 2 Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 3 B Cell"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 41
  warnings.warn(

[1] "Done Layer 3 B Cell"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (6006 x 36601) matrix will require approximately 1.64GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5457 x 36601) matrix will require approximately 1.49GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (4144 x 36601) matrix will require approximately 1.13GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.84"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 4 Soft Tissue Neuro"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Soft Tissue Neuro"
[1] "Starting Layer 2 Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (6022 x 36601) matrix will require approximately 1.64GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5190 x 36601) matrix will require approximately 1.42GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.42"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 4 Soft Tissue Neuro"
[1] "Done Layer 4 Soft Tissue Neuro"
[1] "Starting Layer 2 Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 4 CD8 NK"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD8 NK"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"
[1] "Starting Layer 3 Myeloid"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 24
  warnings.warn(

[1] "Done Layer 3 Myeloid"
[1] "Starting Layer 4 Dendritic"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 24
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 4 Dendritic"
[1] "Starting Layer 5 cDC"
[1] "nothing to score in this layer"
[1] "Done Layer 5 cDC"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5148 x 36601) matrix will require approximately 1.40GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (4215 x 36601) matrix will require approximately 1.15GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (3038 x 36601) matrix will require approximately 0.83GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.07"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Non Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Non Blood"
[1] "Starting Layer 3 Non Stromal"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 3 Non Stromal"
[1] "Starting Layer 4 Non GI"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Non GI"
[1] "Starting Layer 5 Breast Lung Prostate"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 5 Breast Lung Prostate"
[1] "Starting Layer 4 Soft Tissue Neuro"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 4 Soft Tissue Neuro"
[1] "Starting Layer 2 Blood"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(

[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"

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/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (5331 x 36601) matrix will require approximately 1.45GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (4657 x 36601) matrix will require approximately 1.27GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:455: UserWarning: Returning imputed values for all genes on a (3518 x 36601) matrix will require approximately 0.96GB of memory. Suppress this warning with `genes='all_genes'`
  warnings.warn(

[1] "Added MAGIC output to MAGIC_RNA. To use it, pass assay='MAGIC_RNA' to downstream methods or set seurat_object@active.assay <- 'MAGIC_RNA'."
[1] "Sample classification confidence = 0.02"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Blood"
[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 4 CD8 NK"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD8 NK"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"

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[1] "Sample classification confidence = 1.00"

[1] "Starting Layer 1"
[1] "Done Layer 1"
[1] "Starting Layer 2 Blood"
[1] "Done Layer 2 Blood"
[1] "Starting Layer 3 TNK"
[1] "Done Layer 3 TNK"
[1] "Starting Layer 4 CD4 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD4 CD8"
[1] "Starting Layer 4 CD8 NK"
[1] "nothing to score in this layer"
[1] "Done Layer 4 CD8 NK"
[1] "Starting Layer 5 CD4"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD4"
[1] "Starting Layer 5 CD8"
[1] "nothing to score in this layer"
[1] "Done Layer 5 CD8"
[1] "Starting Layer 3 Myeloid"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 22
  warnings.warn(

[1] "Done Layer 3 Myeloid"
[1] "Starting Layer 4 Macrophage Monocyte"

/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/magic/magic.py:425: UserWarning: Input matrix contains unexpressed genes. Please remove them prior to running MAGIC.
  warnings.warn(
/home/bioinfo/.virtualenvs/r-reticulate/lib/python3.12/site-packages/graphtools/base.py:165: RuntimeWarning: Cannot perform PCA to 100 dimensions on data with min(n_samples, n_features) = 22
  warnings.warn(

[1] "nothing to score in this layer"
[1] "Done Layer 4 Macrophage Monocyte"
[1] "Starting Layer 5 Monocyte"
[1] "nothing to score in this layer"
[1] "Done Layer 5 Monocyte"

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[1] "Sample classification confidence = 1.00"

gc()

             used    (Mb) gc trigger    (Mb)   max used    (Mb)
Ncells    6027482   322.0   10776783   575.6   10776783   575.6
Vcells 1517319639 11576.3 3905287830 29795.0 3905287199 29795.0

3. Adding Results to Seurat object

# Create empty vector for predictions
All_samples$scATOMIC_pred <- NA

# Loop through groups and assign predictions per cell
for(grp in names(predictions_all)){
  preds <- predictions_all[[grp]]$scATOMIC_pred
  cells <- rownames(predictions_all[[grp]])
  
  # Assign predictions to Seurat object
  All_samples$scATOMIC_pred[cells] <- preds
}

4. plot our results

DimPlot(All_samples, group.by="scATOMIC_pred", reduction = "umap") + 
  ggtitle("scATOMIC Annotations for All Cell Lines and Controls") + 
  labs(fill="scATOMIC")

NA
NA

Table of predictions

# ========================
# 1. Heatmap: Clusters × Predictions
# ========================

library(pheatmap)
library(ggplot2)

# Cluster-level contingency table
table_clusters <- table(All_samples$seurat_clusters, All_samples$scATOMIC_pred)
prop_clusters  <- prop.table(as.matrix(table_clusters), margin = 1)

# Heatmap
pheatmap(
  prop_clusters,
  cluster_rows = TRUE,
  cluster_cols = TRUE,
  color = colorRampPalette(c("white", "red"))(100),
  main = "scATOMIC Predictions per Cluster"
)

# ========================
# 2. Barplot: Clusters × Predictions
# ========================

df_clusters <- as.data.frame(table(All_samples$seurat_clusters, All_samples$scATOMIC_pred))
colnames(df_clusters) <- c("Cluster", "Prediction", "Count")

ggplot(df_clusters, aes(x = Cluster, y = Count, fill = Prediction)) +
  geom_bar(stat = "identity", position = "fill") +
  scale_y_continuous(labels = scales::percent) +
  ggtitle("scATOMIC Predictions by Cluster") +
  theme_minimal()

# ========================
# 3. Heatmap: Cell lines × Predictions
# ========================

# Cell line-level contingency table
table_cellline <- table(All_samples$orig.ident, All_samples$scATOMIC_pred)
prop_cellline  <- prop.table(as.matrix(table_cellline), margin = 1)

# Heatmap
pheatmap(
  prop_cellline,
  cluster_rows = TRUE,
  cluster_cols = TRUE,
  color = colorRampPalette(c("white", "red"))(100),
  main = "scATOMIC Predictions per Cell Line"
)

# ========================
# 4. Barplot: Cell lines × Predictions
# ========================

df_cellline <- as.data.frame(table(All_samples$orig.ident, All_samples$scATOMIC_pred))
colnames(df_cellline) <- c("CellLine", "Prediction", "Count")

ggplot(df_cellline, aes(x = CellLine, y = Count, fill = Prediction)) +
  geom_bar(stat = "identity", position = "fill") +
  scale_y_continuous(labels = scales::percent) +
  ggtitle("scATOMIC Predictions by Cell Line") +
  theme_minimal()

5. Save updated Seurat object

saveRDS(All_samples, file="All_samples_Merged_scATOMIC_26-09-2025.rds")

sessionInfo()

sessionInfo()

R version 4.5.1 (2025-06-13)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 24.04.3 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.12.0 
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0  LAPACK version 3.12.0

locale:
 [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C               LC_TIME=fr_FR.UTF-8        LC_COLLATE=en_GB.UTF-8    
 [5] LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=en_GB.UTF-8    LC_PAPER=fr_FR.UTF-8       LC_NAME=C                 
 [9] 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  stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] future_1.67.0         cutoff.scATOMIC_0.1.0 agrmt_1.42.12         scATOMIC_2.0.3       
 [5] copykat_1.1.0         Rmagic_2.0.3          reticulate_1.43.0     caret_7.0-1          
 [9] lattice_0.22-7        randomForest_4.7-1.2  plyr_1.8.9            data.table_1.17.8    
[13] Matrix_1.7-4          pheatmap_1.0.13       ggplot2_4.0.0         patchwork_1.3.2      
[17] dplyr_1.1.4           Seurat_5.3.0          SeuratObject_5.2.0    sp_2.2-0             

loaded via a namespace (and not attached):
  [1] RcppAnnoy_0.0.22       splines_4.5.1          later_1.4.4            tibble_3.3.0          
  [5] polyclip_1.10-7        hardhat_1.4.2          pROC_1.19.0.1          rpart_4.1.24          
  [9] fastDummies_1.7.5      lifecycle_1.0.4        rprojroot_2.1.1        globals_0.18.0        
 [13] MASS_7.3-65            tree_1.0-45            magrittr_2.0.4         plotly_4.11.0         
 [17] sass_0.4.10            rmarkdown_2.29         jquerylib_0.1.4        yaml_2.3.10           
 [21] httpuv_1.6.16          sctransform_0.4.2      spam_2.11-1            spatstat.sparse_3.1-0 
 [25] cowplot_1.2.0          pbapply_1.7-4          RColorBrewer_1.1-3     lubridate_1.9.4       
 [29] abind_1.4-8            Rtsne_0.17             purrr_1.1.0            nnet_7.3-20           
 [33] rappdirs_0.3.3         ipred_0.9-15           lava_1.8.1             data.tree_1.2.0       
 [37] ggrepel_0.9.6          irlba_2.3.5.1          listenv_0.9.1          spatstat.utils_3.2-0  
 [41] goftest_1.2-3          RSpectra_0.16-2        spatstat.random_3.4-2  fitdistrplus_1.2-4    
 [45] parallelly_1.45.1      codetools_0.2-20       tidyselect_1.2.1       farver_2.1.2          
 [49] matrixStats_1.5.0      stats4_4.5.1           spatstat.explore_3.5-3 jsonlite_2.0.0        
 [53] progressr_0.16.0       ggridges_0.5.7         survival_3.8-3         iterators_1.0.14      
 [57] bbmle_1.0.25.1         foreach_1.5.2          tools_4.5.1            ica_1.0-3             
 [61] Rcpp_1.1.0             glue_1.8.0             prodlim_2025.04.28     gridExtra_2.3         
 [65] dlm_1.1-6.1            xfun_0.53              here_1.0.2             amap_0.8-20           
 [69] withr_3.0.2            numDeriv_2016.8-1.1    fastmap_1.2.0          digest_0.6.37         
 [73] parallelDist_0.2.6     timechange_0.3.0       R6_2.6.1               mime_0.13             
 [77] scattermore_1.2        tensor_1.5.1           dichromat_2.0-0.1      spatstat.data_3.1-8   
 [81] DiagrammeR_1.0.11      tidyr_1.3.1            generics_0.1.4         recipes_1.3.1         
 [85] class_7.3-23           httr_1.4.7             htmlwidgets_1.6.4      uwot_0.2.3            
 [89] ModelMetrics_1.2.2.2   pkgconfig_2.0.3        gtable_0.3.6           timeDate_4041.110     
 [93] rsconnect_1.5.1        lmtest_0.9-40          S7_0.2.0               htmltools_0.5.8.1     
 [97] dotCall64_1.2          scales_1.4.0           png_0.1-8              gower_1.0.2           
[101] spatstat.univar_3.1-4  knitr_1.50             rstudioapi_0.17.1      reshape2_1.4.4        
[105] visNetwork_2.1.4       nlme_3.1-168           bdsmatrix_1.3-7        cachem_1.1.0          
[109] zoo_1.8-14             stringr_1.5.2          KernSmooth_2.23-26     miniUI_0.1.2          
[113] pillar_1.11.1          grid_4.5.1             vctrs_0.6.5            RANN_2.6.2            
[117] promises_1.3.3         xtable_1.8-4           cluster_2.1.8.1        evaluate_1.0.5        
[121] mvtnorm_1.3-3          cli_3.6.5              compiler_4.5.1         rlang_1.1.6           
[125] crayon_1.5.3           future.apply_1.20.0    labeling_0.4.3         stringi_1.8.7         
[129] viridisLite_0.4.2      deldir_2.0-4           lazyeval_0.2.2         spatstat.geom_3.6-0   
[133] RcppHNSW_0.6.0         shiny_1.11.1           ROCR_1.0-11            igraph_2.1.4          
[137] RcppParallel_5.1.11-1  bslib_0.9.0            collapsibleTree_0.1.8