ExpressPath

RNA-seq time course analysis pipeline — DESeq2 differential expression → temporal & cross-cell-line classification → GSEA pathway enrichment → TF enrichment → Pathview KEGG maps → interactive HTML report.

Metadata-driven. Single Snakemake command from raw counts to browsable results.

Quick Start

1. Open pipeline/setup_design.html in your browser
2. Define cell lines, time points, treatment, replicates
3. Browse for your TSV file → map columns to samples
4. Download design.yaml → save to data/
5. Put your TSV file in data/ (same name as shown in the GUI)
6. Run ./run.sh (add -j8 for more cores, -n for dry-run)
7. Open the report URL printed when the pipeline finishes

Or skip the GUI and edit data/design.example.yaml by hand:

cp data/design.example.yaml data/design.yaml
# tweak cell lines, time points, and column_map, then:
./run.sh

For a detailed explanation of every output file, QC plots, analysis decisions, and troubleshooting, see pipeline/README_explanation.md.

Requirements

• conda (or mamba)
• snakemake (conda install -c bioconda -c conda-forge snakemake)

Input

Tab-separated file with gene expression counts. Required columns:

Column	Header	Example
Gene ID	(unnamed — first column)	ENSG00000000003
Gene name	gene_name	TSPAN6
Count data	any names	A549_mock_1_count, Sample_A, …

Your count columns can have any names — you map them to samples in the setup GUI. Optional annotation columns (GO, KEGG, COG, etc.) are carried through to outputs if present.

See data/design.example.yaml for the experiment configuration format.

Output

All in results/<timestamp>/:

File	Description
tables/combined_results.tsv	All genes — LRT p-values + all pairwise log2FC and padj
tables/signif_lrt.tsv	Genes with LRT padj < 0.05
tables/counts_matrix.tsv	Filtered count matrix (DESeq2 input)
tables/vst_normalized_counts.tsv	VST-transformed counts
cross_temporal/persistence_classes.tsv	Per-cell-line temporal persistence categories
cross_temporal/gene_activity.tsv	Per-gene log2FC and significance at each timepoint
cross_temporal/venn_genelists.tsv	DEG sets per cell line × timepoint (for Venn/UpSet)
cross_temporal/cross_cellline_shared.tsv	Genes DE in both cell lines at each timepoint (concordance + magnitude divergence)
cross_temporal/cross_cellline_specific.tsv	Genes DE in only one cell line at each timepoint
cross_cellline/cross_temporal_persistence.tsv	Cross-cell-line temporal divergence categories
cross_cellline/cross_temporal_gene_activity.tsv	Per-gene between-cell-line log2FC at each timepoint
cross_cellline/cross_temporal_shared.tsv	Between-cell-line DEGs shared across timepoints
cross_cellline/cross_temporal_specific.tsv	Between-cell-line DEGs specific to one timepoint
pathway/gsea_kegg_signif.tsv	Enriched KEGG pathways (GSEA)
pathway/gsea_go_signif.tsv	Enriched GO terms (GSEA)
pathway/gsva_scores.tsv	Per-sample pathway activity scores
pathway/pathview_output/	KEGG pathway maps with log2FC overlay
pathway/interactive_report.html	Self-contained browsable report
tf/tf_enrichment_results.tsv	TF target enrichment (enrichR) — all contrasts
tf/tf_enrichment_heatmap.pdf	Heatmap of TF enrichment significance per analysis dimension
tf/tf_regulatory_network_*.html	Per-cell-line / shared / divergence TF–target regulatory networks

Directory naming note: cross_temporal/ holds treatment-response analyses (within-cell-line and cross-cell-line at each timepoint), while cross_cellline/ holds between-cell-line temporal divergence analyses. Files within each directory carry the opposite prefix (e.g., cross_cellline/cross_temporal_persistence.tsv) — this is intentional: it reflects the analysis dimension applied within that context.

pathview_output/ PNGs and tf_regulatory_network_*.html files are generated as side outputs by the R scripts and are not tracked by Snakemake rules; newer pipeline runs overwrite them in place.

How It Works

data/design.yaml + data/your_data.tsv
│
[extract_counts] Python — reads column_map from design
│
[deseq2_analysis] R/DESeq2 — LRT + pairwise Wald contrasts,
│ Mfuzz clustering, temporal persistence,
│ cross-cell-line comparisons
│
[pathway_analysis] R/clusterProfiler — GSEA + Pathview + GSVA
│
[tf_enrichment] R/enrichR — TF target enrichment + regulatory networks
│
[interactive_report] R/htmltools — self-contained HTML report

Contrasts are auto-generated from your experiment design — no hardcoded cell line or time point names. Add more time points or rename cell lines in data/design.yaml and everything adapts.

Analysis Categories

The pipeline classifies DEGs into temporal activity categories at three levels:

Per-Cell-Line Temporal Persistence

For each cell line, genes are classified by which treatment timepoints they are DE at (vs mock). Output in cross_temporal/persistence_classes.tsv.

Category	Meaning
Transient	DE only at the first treatment timepoint
Transient_Mid	DE at a single intermediate timepoint
Secondary_Deferred	DE only at the last treatment timepoint
Sustained	DE at the first AND last treatment timepoints, with contiguous significance across all intermediate timepoints
Partially_Sustained	DE contiguously from the first through an intermediate timepoint, but NOT at the last
Intermittent	DE at the first AND last treatment timepoints, but with gaps (non-contiguous)
Complex	Any other multi-timepoint pattern not fitting the above

Cross-Cell-Line at Each Timepoint

Compares DEG sets between two cell lines at each treatment timepoint. Output in cross_temporal/cross_cellline_shared.tsv and cross_temporal/cross_cellline_specific.tsv.

Category	Meaning
Concordant_Up	Both cell lines upregulated (same direction)
Concordant_Down	Both cell lines downregulated (same direction)
Discordant	One up, one down (labeled as {CL}_Up_{CL}_Down)
Magnitude Divergent	Shared gene where |log2FC ratio| between cell lines > 2
Cell-line-specific	DE in one cell line only (absent from the other)

Cross-Cell-Line Temporal Divergence (Part G)

Classifies how the between-cell-line difference evolves over time. Uses the cell-line-vs-cell-line contrasts at each timepoint (e.g. E6 vs A549 at mock, 1h, 3h). Output in cross_cellline/cross_temporal_persistence.tsv.

Category	Meaning
Constitutive	Between-cell-line difference significant at ALL timepoints
Baseline_Only	Difference only at the reference timepoint (pre-existing, disappears after treatment)
Emergent_Early	Difference appears only at the first treatment timepoint
Emergent_Mid	Difference appears at a single mid-treatment timepoint
Emergent_Late	Difference appears only at the last treatment timepoint
Emergent_Sustained	Difference at ALL treatment timepoints but NOT at baseline — treatment-induced and persistent
Emergent_Complex	Difference at multiple (but not all) treatment timepoints, not at baseline
Convergent	Difference present at baseline but ABSENT by the last timepoint (cell lines become more similar)
Complex	Any other multi-timepoint pattern

DESeq2 Contrasts

The statistical model ~ batch + cell_line + time + cell_line:time produces four types of pairwise Wald contrasts, each gated by flags in data/design.yaml:

Comparison type	Flag	Example	What it tests
Within cell line	within_cell_line	A549_3h_vs_mock	Treatment effect per cell line
Progression	progression	A549_3h_vs_1h	Response evolution between consecutive timepoints
Between cell lines	between_cell_lines	E6_vs_A549_1h	Cell line difference at each timepoint
Interaction	interactions	interaction_3h	Does the time effect differ between cell lines?

An LRT (omnibus) test on ~ batch + cell_line + time + cell_line:time vs. ~ batch + cell_line identifies genes that change in any way across the experiment.

Configuration

Edit pipeline/config.yaml for pipeline parameters (filters, thresholds). Edit data/design.yaml for your experiment (cell lines, time points, column mapping, batch labels). Both can be set with setup_design.html.

License

MIT — see LICENSE

Authors

Dr. Bruno Pavletić — bruno.pavletic@irb.hr

Ruđer Bošković Institute, Zagreb, Croatia