matrisome-analysis

A Snakemake workflow for differential expression and extracellular matrix (matrisome) analysis of bulk RNA-seq, comparing tumour samples against normal brain reference samples.

Workflow summary

1. Differential expression. DESeq2 on tumour versus normal brain counts. Normal-brain technical replicates are averaged per donor first, and genes are reconciled on symbol.
2. Functional enrichment. g:Profiler results rendered as GOplot chord diagrams for molecular function, biological process and cellular component.
3. Principal component analysis. PCA across all genes and across housekeeping genes, per sample and per averaged brain region, as a check that the tumour and normal cohorts separate on biology rather than on library composition.
4. Matrisome annotation. Differentially expressed genes annotated with MatrisomeAnalyzeR, split into core matrisome and matrisome-associated.
5. Figures. Barplots of core matrisome differential expression, and dotplots of effect size and significance across matrisome categories.

Usage

snakemake -s workflow/Snakefile --cores 4

Or in the pinned container, from the repository root:

docker build -t matrisome-analysis -f env/Dockerfile .
docker run --rm -v "$PWD":/project -w /project matrisome-analysis \
  snakemake -s workflow/Snakefile -c1

rule all declares the full target set, so any single output can also be requested by name.

Inputs

Not included in this repository. data/ is gitignored.

Input	Format
Tumour RNA-seq counts	TSV, genes by samples, from a controlled-access cohort
Normal brain RNA-seq counts	TSV, compound ENSG|SYMBOL gene identifiers, multiple columns per donor
Sample metadata	XLSX with Patient_ID and Type columns
g:Profiler enrichment exports	CSV, one per ontology

Paths are declared per rule in the Snakefile.

Outputs

Written to results/ and gitignored.

Output	Contents
DESEQ2_results.csv, DESEQ2_results_sig.csv	All and significant DESeq2 results
Matrisome_DESEQ_results_annotated*.csv	Results annotated by matrisome division
CoreMatrisome_DESEQ_results_sig.csv	Significant core matrisome genes
enrichment/GOplot_chord_{MF,BP,CC}.pdf	Enrichment chord diagrams
CoreMatrisome_DEG_barplot.pdf, dotplot_*.pdf, pvalue_dotplot_core_matrisome.pdf	Figures
pca_*.pdf, housekeeping_boxplots/	PCA and housekeeping-gene diagnostics

Parameters

Set at the top of scripts/differential_expression.R.

Parameter	Default	Meaning
thr	10	A gene is kept if it exceeds this count in more than half the samples
lfc_cutoff	2	Absolute log2 fold change required for significance, alongside adjusted p ≤ 0.05

Environment

R 4.4.1 with Bioconductor 3.19 and Snakemake 9.9.0. All 148 R package versions are pinned in renv.lock, and env/Dockerfile builds the exact environment from that lockfile, so a clean rebuild restores the same versions rather than whatever is current.

Licence

MIT. See LICENSE.

Author

Philipp Graber. ORCID 0000-0002-3157-1434