Deseq2 Analysis

DESeq2 Pseudobulk Analysis Skill

When to Use

• Differential expression between wound conditions
• Pseudobulk aggregation per sample/condition
• MAST single-cell DE as alternative

Pseudobulk DESeq2 Workflow (R)

library(DESeq2)
library(Seurat)

# Aggregate to pseudobulk
pseudo <- AggregateExpression(sobj, group.by = c("condition", "sample"),
                              return.seurat = TRUE)

# Run DESeq2
dds <- DESeqDataSetFromMatrix(
  countData = GetAssayData(pseudo, slot = "counts"),
  colData = [email protected],
  design = ~ condition
)
dds <- DESeq(dds)
res <- results(dds, contrast = c("condition", "wound_7d", "control"),
               lfcThreshold = 0, alpha = 0.05)
res <- lfcShrink(dds, coef = "condition_wound_7d_vs_control", type = "ashr")

Thresholds