Differential Expression Analysis using Harmony Integrated Clusters

1. Load Libraries

2. Load Seurat Object

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 = "seurat_clusters",label = T, label.box = T, repel = T) + 
  ggtitle("Harmony Integration - By Clusters")

4. Differential Expression Analysis

4.1 Using MAST with Latent Variables (Batch Correction)

markers_mast_batch <- FindMarkers(
  All_samples_Merged,
  ident.1 = c("1", "5", "9", "2", "6", "8", "0", "4", "7", "11", "12", "13"), # Cell lines-Malignant
  ident.2 = c("3", "10"), # Normal CD4 T cells
  assay = "SCT",
  test.use = "MAST",
  latent.vars = c("cell_line"), # Adjust for batch effects
  min.pct = 0,
  logfc.threshold = 0
)

Avis : sparse->dense coercion: allocating vector of size 9.6 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.  
                    This likely indicates a bug in an upstream package.
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Done!
Combining coefficients and standard errors
Calculating log-fold changes

Processed 217471 groups out of 471168. 46% done. Time elapsed: 3s. ETA: 3s.
Processed 288106 groups out of 471168. 61% done. Time elapsed: 4s. ETA: 2s.
Processed 350864 groups out of 471168. 74% done. Time elapsed: 5s. ETA: 1s.
Processed 415611 groups out of 471168. 88% done. Time elapsed: 6s. ETA: 0s.
Processed 471168 groups out of 471168. 100% done. Time elapsed: 6s. ETA: 0s.

Calculating likelihood ratio tests
Refitting on reduced model...

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Done!

write.csv(markers_mast_batch, file = "0-robj/1-MAST_with_batch_as_Covariate.csv", row.names = TRUE)

4.2 Using Wilcoxon Test (Without Batch Correction)

markers_wilcox <- FindMarkers(
  All_samples_Merged,
 ident.1 = c("1", "5", "9", "2", "6", "8", "0", "4", "7", "11", "12", "13"), # Cell lines-Malignant
  ident.2 = c("3", "10"), # Normal CD4 T cells
  assay = "SCT",
  test.use = "wilcox",
  min.pct = 0,
  logfc.threshold = 0
)
write.csv(markers_wilcox, file = "0-robj/2-Wilcox_min.pct_logfcT-0.csv", row.names = TRUE)

4.3 Using MAST Without Latent Variables (No Batch Correction)

markers_mast_no_batch <- FindMarkers(
  All_samples_Merged,
 ident.1 = c("1", "5", "9", "2", "6", "8", "0", "4", "7", "11", "12", "13"), # Cell lines-Malignant
  ident.2 = c("3", "10"), # Normal CD4 T cells
  assay = "SCT",
  test.use = "MAST",
  min.pct = 0,
  logfc.threshold = 0
)

Avis : sparse->dense coercion: allocating vector of size 9.6 GiBAvis : Dropping illegal slot(s) norm.method for class BayesGLMlike.  
                    This likely indicates a bug in an upstream package.
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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!

View(markers_wilcox)
write.csv(markers_mast_no_batch, file = "0-robj/3-MAST_without_batch_as_Covariate.csv", row.names = TRUE)

5. Compute Mean Expression for Groups

# Define cell groups
group1_cells <- WhichCells(All_samples_Merged, idents = c("1", "5", "9", "2", "6", "8", "0", "4", "7", "11", "12", "13"))
group2_cells <- WhichCells(All_samples_Merged, idents = c("3", "10"))

# Extract normalized expression values
expression_data <- GetAssayData(All_samples_Merged, slot = "data")  # Log-normalized values

Avis : The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
Please use the `layer` argument instead.

# Calculate mean expression for each group
calculate_mean_expression <- function(markers, group1_cells, group2_cells, expression_data) {
  group1_mean <- rowMeans(expression_data[, group1_cells])
  group2_mean <- rowMeans(expression_data[, group2_cells])
  
  markers <- markers %>%
    rownames_to_column("gene") %>%
    mutate(mean_expr_group1 = group1_mean[gene],
           mean_expr_group2 = group2_mean[gene])
  
  return(markers)
}

markers_mast_batch <- calculate_mean_expression(markers_mast_batch, group1_cells, group2_cells, expression_data)
markers_wilcox <- calculate_mean_expression(markers_wilcox, group1_cells, group2_cells, expression_data)
markers_mast_no_batch <- calculate_mean_expression(markers_mast_no_batch, group1_cells, group2_cells, expression_data)

# Save updated results with mean expression
write.csv(markers_mast_batch, file = "0-robj/1-MAST_with_batch_as_Covariate_with_meanExpression.csv", row.names = TRUE)
write.csv(markers_wilcox, file = "0-robj/2-Wilcox_min.pct_logfcT-0_with_meanExpression.csv", row.names = TRUE)
write.csv(markers_mast_no_batch, file = "0-robj/3-MAST_without_batch_as_Covariate_with_meanExpression.csv", row.names = TRUE)

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")
}

6.2 Summarize Markers1 (MAST with Batch Correction)

cat("Markers1 Summary (MAST with Batch Correction):\n")

Markers1 Summary (MAST with Batch Correction):

summarize_markers(markers_mast_batch)

Number of genes with p_val_adj = 0: 74 
Number of genes with p_val_adj = 1: 19337 
Number of significant genes (p_val_adj < 0.05): 5615 
Number of upregulated genes (avg_log2FC > 1): 15048 
Number of downregulated genes (avg_log2FC < -1): 2281 

6.3 Summary for Markers2 (Wilcoxon Test)

cat("Markers2 Summary (Wilcoxon Test):\n")

Markers2 Summary (Wilcoxon Test):

summarize_markers(markers_wilcox)

Number of genes with p_val_adj = 0: 4208 
Number of genes with p_val_adj = 1: 10428 
Number of significant genes (p_val_adj < 0.05): 15043 
Number of upregulated genes (avg_log2FC > 1): 15048 
Number of downregulated genes (avg_log2FC < -1): 2281 

6.4 Summary for Markers3 (MAST without Batch Correction)

cat("Markers3 Summary (MAST without Batch Correction):\n")

Markers3 Summary (MAST without Batch Correction):

summarize_markers(markers_mast_no_batch)

Number of genes with p_val_adj = 0: 4679 
Number of genes with p_val_adj = 1: 9855 
Number of significant genes (p_val_adj < 0.05): 15579 
Number of upregulated genes (avg_log2FC > 1): 15048 
Number of downregulated genes (avg_log2FC < -1): 2281 

7. Visualization

7.1 Volcano Plot for MAST with Batch Correction

EnhancedVolcano(markers_mast_batch,
                lab = markers_mast_batch$gene,
                x = "avg_log2FC",
                y = "p_val_adj",
                title = "MAST with Batch Correction",
                pCutoff = 0.05,
                FCcutoff = 1.0)

Avis : One or more p-values is 0. Converting to 10^-1 * current lowest non-zero p-value...

7.2 Volcano Plot for Wilcoxon Test

EnhancedVolcano(markers_wilcox,
                lab = markers_wilcox$gene,
                x = "avg_log2FC",
                y = "p_val_adj",
                title = "MAST with Batch Correction",
                pCutoff = 0.05,
                FCcutoff = 1.0)

Avis : One or more p-values is 0. Converting to 10^-1 * current lowest non-zero p-value...

7.3 Volcano Plot for MAST without Batch Correction

EnhancedVolcano(markers_mast_no_batch,
                lab = markers_mast_no_batch$gene,
                x = "avg_log2FC",
                y = "p_val_adj",
                title = "MAST with Batch Correction",
                pCutoff = 0.05,
                FCcutoff = 1.0)

Avis : One or more p-values is 0. Converting to 10^-1 * current lowest non-zero p-value...

7.4 Heatmap of Top Differentially Expressed Genes

# Load required package
library(pheatmap)

# Define cell groups
group1_cells <- WhichCells(All_samples_Merged, idents = c("1", "5", "9", "2", "6", "8", "0", "4", "7", "11", "12", "13"))
group2_cells <- WhichCells(All_samples_Merged, idents = c("3", "10"))

# Define thresholds for adjusted p-value and avg_log2fc
p_val_adj_threshold <- 0.05
avg_logFC_threshold <- 1

# Ensure avg_log2fc column is numeric
markers_mast_batch$avg_log2fc <- as.numeric(as.character(markers_mast_batch$avg_log2FC))

# Identify and remove rows where avg_log2fc is NA after conversion
markers_mast_batch <- markers_mast_batch[!is.na(markers_mast_batch$avg_log2fc), ]

# Filter genes based on both adjusted p-value and avg_log2fc
filtered_genes <- markers_mast_batch[markers_mast_batch$p_val_adj < p_val_adj_threshold & 
                                     markers_mast_batch$avg_log2fc > avg_logFC_threshold, ]

# Define top genes based on adjusted p-value
top_genes <- head(filtered_genes$gene[order(filtered_genes$p_val_adj)], 20)

# Extract the corresponding expression data
expression_data <- GetAssayData(All_samples_Merged, slot = "data")  # Log-normalized values
heatmap_data <- expression_data[top_genes, c(group1_cells, group2_cells)]

# Create annotation data
annotation_data <- data.frame(
  CellLine = factor(All_samples_Merged$cell_line[c(group1_cells, group2_cells)]),
  Cluster = factor(All_samples_Merged$seurat_clusters[c(group1_cells, group2_cells)])
)

# Set row names for annotation data to match column names of heatmap_data
rownames(annotation_data) <- c(group1_cells, group2_cells)

# Generate the heatmap
pheatmap(heatmap_data, 
         cluster_rows = TRUE, 
         cluster_cols = TRUE, 
         show_rownames = TRUE,
         show_colnames = FALSE,  # Hide cell labels below the heatmap
         annotation_col = annotation_data,
         fontsize = 10)  # Adjust font size for readability