1. Load Libraries
2. load seurat object
#Load Seurat Object L7
load("../0-robj/5-Harmony_Integrated_All_samples_Merged_CD4Tcells_final_Resolution_Selected_0.8_ADT_Normalized_cleaned_mt.robj")
All_samples_Merged
An object of class Seurat
62900 features across 49305 samples within 6 assays
Active assay: SCT (26176 features, 3000 variable features)
3 layers present: counts, data, scale.data
5 other assays present: RNA, ADT, prediction.score.celltype.l1, prediction.score.celltype.l2, prediction.score.celltype.l3
5 dimensional reductions calculated: integrated_dr, ref.umap, pca, umap, harmony
3. Set Up Identifiers for Clustering
# Assign cluster identities to the Seurat object
Idents(All_samples_Merged) <- "seurat_clusters"
DimPlot(All_samples_Merged, reduction = "umap", group.by = "cell_line",label = T, label.box = T)

DimPlot(All_samples_Merged, reduction = "umap", group.by = "seurat_clusters",label = T, label.box = T)

4. Differential Expression Analysis
4.1 Using MAST with SCT and Latent Variables (cell_line)
# Load necessary libraries
library(Seurat)
# Set the identities to cell line vs. control
Idents(All_samples_Merged) <- "cell_line"
# Define the control group and the list of cell lines
control_group <- c("PBMC", "PBMC_10x")
cell_lines <- c("L1", "L2", "L3", "L4", "L5", "L6", "L7")
# Create an empty list to store DE results
de_results <- list()
# Run MAST for each cell line vs. control and save results to CSV
for (cell_line in cell_lines) {
# Run MAST with "orig.ident" as a covariate (e.g., batch or patient)
de_results[[cell_line]] <- FindMarkers(
object = All_samples_Merged,
ident.1 = cell_line,
ident.2 = control_group,
test.use = "MAST",
logfc.threshold = 0,
min.pct = 0.01,
latent.vars = c("cell_line") # Adjust for batch/patient effect
)
# Save the results to CSV
output_file <- paste0("DE_Results_", cell_line, "_vs_Control.csv")
write.csv(de_results[[cell_line]], output_file, row.names = TRUE)
}
Avis : sparse->dense coercion: allocating vector of size 1.4 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Avis : sparse->dense coercion: allocating vector of size 1.6 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Avis : sparse->dense coercion: allocating vector of size 1.6 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Avis : sparse->dense coercion: allocating vector of size 1.5 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Avis : sparse->dense coercion: allocating vector of size 1.4 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Avis : sparse->dense coercion: allocating vector of size 1.5 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.
This likely indicates a bug in an upstream package.Avis : Coefficients cell_linePBMC_10x are never estimible and will be dropped.
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Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
# Check one of the results
head(de_results[["L1"]])
NA
5. Compute Mean Expression for Groups
# Load necessary libraries
library(Seurat)
library(dplyr)
library(tibble)
# Define the control group and the list of cell lines
control_group <- c("PBMC", "PBMC_10x")
cell_lines <- c("L1", "L2", "L3", "L4", "L5", "L6", "L7")
# Extract normalized expression values for SCT assay
expression_data_SCT <- GetAssayData(All_samples_Merged, assay = "SCT", slot = "data") # SCT-normalized counts
Avis : The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
Please use the `layer` argument instead.
# Function to calculate mean expression for each group
calculate_mean_expression <- function(markers, group1_cells, group2_cells, expression_data) {
# Calculate mean expression for each group
group1_mean <- rowMeans(expression_data[, group1_cells, drop = FALSE], na.rm = TRUE)
group2_mean <- rowMeans(expression_data[, group2_cells, drop = FALSE], na.rm = TRUE)
# Append mean expression to markers dataframe
markers <- markers %>%
rownames_to_column("gene") %>%
mutate(mean_expr_group1 = group1_mean[gene],
mean_expr_group2 = group2_mean[gene])
return(markers)
}
# Loop through each cell line to compute and save mean expression
for (cell_line in cell_lines) {
# Load the existing DE results for this cell line
existing_results <- de_results[[cell_line]]
# Define cell groups for mean expression calculation
group1_cells <- WhichCells(All_samples_Merged, idents = cell_line) # Malignant CD4+ T cells
group2_cells <- WhichCells(All_samples_Merged, idents = control_group) # Normal CD4+ T cells
# Calculate mean expression for SCT assay
updated_results <- calculate_mean_expression(existing_results, group1_cells, group2_cells, expression_data_SCT)
# Save the updated results to CSV
output_file <- paste0("Updated_DE_Results_", cell_line, "_with_MeanExpr.csv")
write.csv(updated_results, output_file, row.names = TRUE)
}
# Check one of the updated results
head(de_results[["L1"]])
NA
6. Summarize Results
6.1 Summary Function
summarize_markers <- function(markers) {
num_pval0 <- sum(markers$p_val_adj == 0)
num_pval1 <- sum(markers$p_val_adj == 1)
num_significant <- sum(markers$p_val_adj < 0.05)
num_upregulated <- sum(markers$avg_log2FC > 1)
num_downregulated <- sum(markers$avg_log2FC < -1)
cat("Number of genes with p_val_adj = 0:", num_pval0, "\n")
cat("Number of genes with p_val_adj = 1:", num_pval1, "\n")
cat("Number of significant genes (p_val_adj < 0.05):", num_significant, "\n")
cat("Number of upregulated genes (avg_log2FC > 1):", num_upregulated, "\n")
cat("Number of downregulated genes (avg_log2FC < -1):", num_downregulated, "\n")
}
Summarize Markers (L1)
cat("Markers1 Summary (L1):\n")
Markers1 Summary (L1):
summarize_markers(de_results$L1)
Number of genes with p_val_adj = 0: 1843
Number of genes with p_val_adj = 1: 2080
Number of significant genes (p_val_adj < 0.05): 10996
Number of upregulated genes (avg_log2FC > 1): 7752
Number of downregulated genes (avg_log2FC < -1): 1190
Summarize Markers (L2)
cat("Markers1 Summary (L2):\n")
Markers1 Summary (L2):
summarize_markers(de_results$L2)
Number of genes with p_val_adj = 0: 3000
Number of genes with p_val_adj = 1: 1647
Number of significant genes (p_val_adj < 0.05): 12057
Number of upregulated genes (avg_log2FC > 1): 9648
Number of downregulated genes (avg_log2FC < -1): 1160
Summarize Markers (L3)
cat("Markers1 Summary (L3):\n")
Markers1 Summary (L3):
summarize_markers(de_results$L3)
Number of genes with p_val_adj = 0: 2162
Number of genes with p_val_adj = 1: 2327
Number of significant genes (p_val_adj < 0.05): 11284
Number of upregulated genes (avg_log2FC > 1): 7293
Number of downregulated genes (avg_log2FC < -1): 1780
Summarize Markers (L4)
cat("Markers1 Summary (L4):\n")
Markers1 Summary (L4):
summarize_markers(de_results$L4)
Number of genes with p_val_adj = 0: 3253
Number of genes with p_val_adj = 1: 1598
Number of significant genes (p_val_adj < 0.05): 12909
Number of upregulated genes (avg_log2FC > 1): 10493
Number of downregulated genes (avg_log2FC < -1): 1088
Summarize Markers (L5)
cat("Markers1 Summary (L5):\n")
Markers1 Summary (L5):
summarize_markers(de_results$L5)
Number of genes with p_val_adj = 0: 2682
Number of genes with p_val_adj = 1: 1970
Number of significant genes (p_val_adj < 0.05): 11812
Number of upregulated genes (avg_log2FC > 1): 8553
Number of downregulated genes (avg_log2FC < -1): 1477
Summarize Markers (L6)
cat("Markers1 Summary (L6):\n")
Markers1 Summary (L6):
summarize_markers(de_results$L6)
Number of genes with p_val_adj = 0: 1999
Number of genes with p_val_adj = 1: 2230
Number of significant genes (p_val_adj < 0.05): 11408
Number of upregulated genes (avg_log2FC > 1): 8119
Number of downregulated genes (avg_log2FC < -1): 1269
Summarize Markers (L7)
cat("Markers1 Summary (L7):\n")
Markers1 Summary (L7):
summarize_markers(de_results$L7)
Number of genes with p_val_adj = 0: 2321
Number of genes with p_val_adj = 1: 1819
Number of significant genes (p_val_adj < 0.05): 11788
Number of upregulated genes (avg_log2FC > 1): 8406
Number of downregulated genes (avg_log2FC < -1): 1356
7. Visualization
7.1 Volcano Plot for Cell lines with Normal Control
# Load necessary libraries
library(EnhancedVolcano)
library(ggplot2)
# Define a function to create and display volcano plots for each cell line
create_volcano_plot <- function(de_results, cell_line) {
# Create volcano plot
volcano <- EnhancedVolcano(de_results[[cell_line]],
lab = rownames(de_results[[cell_line]]),
x = "avg_log2FC",
y = "p_val_adj",
title = paste0(cell_line, " vs Normal CD4+ T Cells"),
pCutoff = 0.0001,
FCcutoff = 1.0)
# Display the plot in the notebook
print(volcano)
}
# Loop through each cell line and generate volcano plots
for (cell_line in cell_lines) {
create_volcano_plot(de_results, cell_line)
}
Avis : One or more p-values is 0. Converting to 10^-1 * current lowest non-zero p-value...






