8  Comparison of Differential Expression Analysis Methods

Venn diagram analysis of DEGs across analytical approaches

8.1 Overview

We compared three analytical approaches for identifying differentially expressed genes (DEGs) in liver tissue in response to Cre-mediated ApoE4→ApoE2 conversion:

1. Method 1: Combined batches without batch adjustment (~ Sex + group)
   • Cross-batch replicates collapsed by averaging
   • Significance: FDR < 0.01, |log₂FC| > 1
2. Method 2: Combined batches with batch adjustment (~ batch + Sex + group)
   • Cross-batch replicates collapsed by averaging
   • Significance: FDR < 0.01, |log₂FC| > 1
3. Method 3: Batch 2 only (~ Sex + group)
   • No cross-batch replicates; complete independence
   • Significance: FDR < 0.01, |log₂FC| > 1

8.2 Liver

8.2.1 Summary of Methods

Method	Design Formula	Samples Used	DEGs in Liver
Method 1: Combined, no batch adj.	~ Sex + group	Both batches (collapsed replicated)	63
Method 2: Combined, with batch adj.	~ batch + Sex + group	Both batches (collapsed replicated)	26
Method 3: Batch 2 only	~ Sex + group	Batch 2 only	20

8.2.2 Venn Diagram: All Three Methods

8.2.3 Core High-Confidence DEGs

Genes identified by all three methods represent the most robust findings, independent of analytical approach:

### 3 genes were identified by all three methods

Abca2, Ccdc148, Slc41a2

–

	Method 1		Method 2		Method 3
Gene	log₂FC	FDR	log₂FC	FDR	log₂FC	FDR
Ccdc148	1.146	0	1.295	0.000	1.055	0.009
Abca2	-1.121	0	-1.246	0.003	-1.050	0.008
Slc41a2	-1.400	0	-1.908	0.000	-1.912	0.000

8.3 Brain

8.3.1 Summary of Methods

Method	Design Formula	Samples Used	DEGs in Brain
Method 1: Combined, no batch adj.	~ Sex + group	Both batches (collapsed replicated)	69
Method 2: Combined, with batch adj.	~ batch + Sex + group	Both batches (collapsed replicated)	5
Method 3: Batch 2 only	~ Sex + group	Batch 2 only	0

8.3.2 Venn Diagram: Methods 1 and 2

Since Method 3 (Batch 2 only) yielded no significant DEGs in brain tissue, we compare only the two combined-batch approaches:

8.3.3 Core High-Confidence DEGs in Brain

Genes identified by both methods represent robust findings in brain tissue:

### 5 genes were identified by both methods

Aldoart1, Cdr1, Eno1b, Sap18b, Uba52rt

8.3.3.1 Details of Core Brain DEGs

	Method 1		Method 2
Gene	log₂FC	FDR	log₂FC	FDR
Uba52rt	-2.269	0	-1.350	0.000
Eno1b	-3.137	0	-2.116	0.000
Sap18b	-2.261	0	-1.365	0.007
Aldoart1	-3.473	0	-2.536	0.001
Cdr1	1.310	0	1.238	0.005