This repository contains the complete bioinformatics workflow used in:
Pandey RS, Kotredes KP, Sasner M, Howell GR, Carter GW. Differential splicing of neuronal genes in a Trem2*R47H mouse model mimics alterations associated with Alzheimer's disease. BMC Genomics. 2023;24:172. https://doi.org/10.1186/s12864-023-09280-x
We performed differential gene expression and differential splicing analyses on whole-brain transcriptomes from aging mouse models carrying humanized APOE4 and/or the Trem2*R47H variant on a C57BL/6J background. This workflow documents every computational step from raw FASTQ processing through functional annotation, enabling full reproduction of published results.
Key findings:
- Differentially expressed genes in Trem2*R47H mice were enriched in immune and metabolic pathways
- Differentially spliced genes were enriched in neuronal functions (GABAergic and glutamatergic synapse)
- Significant overlap was observed between spliced genes in Trem2*R47H mice and human AD subjects
- These effects were absent in APOE4 mice and suppressed in APOE4·Trem2*R47H double mutant mice
The full step-by-step workflow is documented in WORKFLOW.md.
| Step | Tool | Version | Purpose |
|---|---|---|---|
| 1 | FastQC | v0.11.3 | Read quality assessment |
| 2 | Trimmomatic | v0.33 | Adapter and quality trimming |
| 3 | STAR | v2.5.3 | Alignment to reference genome |
| 4 | HTSeq | v0.8.0 | Gene-level read counting |
| 5 | RSEM | v1.3.3 | Isoform-level quantification |
| Step | Tool | Version | Purpose |
|---|---|---|---|
| 6 | DESeq2 | v1.16.1 | Differential gene expression |
| 7 | DEXSeq | v1.40.0 | Differential exon usage |
| 8 | IsoformSwitchAnalyzeR | v1.20.0 | Isoform switch analysis |
| 9 | clusterProfiler | — | KEGG and GO enrichment |
| 10 | — | — | Cell type enrichment (Fisher's exact test) |
| 11 | RBPmap | — | RNA-binding protein site prediction |
| 12 | — | — | Overlap with human AD splicing studies |
Raw sequencing data and processed count matrices are available via the AD Knowledge Portal:
https://adknowledgeportal.synapse.org/Explore/Studies/DetailsPage/StudyDetails?Study=syn66318364
Trem2-RNAseq-splicing-workflow/
│
├── README.md # This file
├── WORKFLOW.md # Complete 12-step documented workflow
├── LICENSE # MIT license
│
└── scripts/
├── DEG_function.R # DESeq2 wrapper function
├── ENRICHGO_function.R # GO enrichment function
└── cell_type_enrichment.R # Fisher's exact test for cell types
| Tool | Version | Installation |
|---|---|---|
| FastQC | v0.11.3 | bioinformatics.babraham.ac.uk |
| Trimmomatic | v0.33 | usadellab.org |
| STAR | v2.5.3 | github.com/alexdobin/STAR |
| Picard | v1.95 | broadinstitute.github.io/picard |
| SAMtools | v1.10 | samtools.sourceforge.net |
| HTSeq | v0.8.0 | htseq.readthedocs.io |
| RSEM | v1.3.3 | github.com/deweylab/RSEM |
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install(c(
"DESeq2",
"DEXSeq",
"IsoformSwitchAnalyzeR",
"clusterProfiler",
"org.Mm.eg.db",
"AnnotationDbi",
"BiocParallel",
"BSgenome.Mmusculus.UCSC.mm10"
))
install.packages(c("tidyverse", "xlsx"))All analyses used the mouse reference genome GRCm38/mm10 with Ensembl annotation (release 97).
If you use this workflow, please cite:
Pandey RS, Kotredes KP, Sasner M, Howell GR, Carter GW. Differential splicing of neuronal genes in a Trem2*R47H mouse model mimics alterations associated with Alzheimer's disease. BMC Genomics. 2023;24:172. https://doi.org/10.1186/s12864-023-09280-x
MIT — see LICENSE for details.
Ravi S. Pandey GitHub: @pandeyravi15