Imported from swaruplab/operon (protocols/gene-regulatory-networks-multiomics-grn/SKILL.md). Install upstream with npx skills add swaruplab/operon --skill gene-regulatory-networks-multiomics-grn. Copyright stays with the author.

Version Compatibility

Reference examples tested with: SCENIC+ (current Snakemake workflow), pycisTopic 2.0+, pycistarget 1.0+, scanpy 1.10+, MACS3 3.0+; FigR/Signac/ArchR (R).

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

SCENIC+ has undergone major API churn: the manual-object API (cisTopicObject, separate pycistarget, SCENICPLUS objects) is superseded by a Snakemake workflow (scenicplus init_snakemake). Pre-2024 tutorials are stale; verify against scenicplus.readthedocs.io before coding.

Multiomics GRN Inference

"Build an enhancer-driven gene regulatory network from my multiome data" -> Integrate scRNA-seq and scATAC-seq to identify eRegulons: transcription-factor -> enhancer-region -> target-gene triplets that link TF motif occupancy in accessible chromatin to gene expression.

• Python: SCENIC+ Snakemake pipeline (pycisTopic -> pycistarget -> eRegulons)
• Python: CellOracle base GRN (motif scan in Cicero co-accessible regions); R: Pando / FigR

The Single Most Important Modern Insight -- Accessibility Defines the Candidate Enhancers, but Every Inference Arrow Leaks

The advance of multiomic GRN inference over expression-only methods is where the candidate regulatory regions come from: expression-only tools can only search promoter-proximal motifs, while scATAC nominates the actual distal enhancers active in these cells -- and most cell-type-specific regulatory information is distal. So an eGRN edge is a triplet (TF -> region -> gene), with the region as the mechanistic anchor available for validation (ChIP/CUT&Tag, CRISPRi, reporter). But the reasoning chain leaks at every step: motif-present does not mean the TF binds (motifs are short, degenerate, and shared across a family -- the model cannot tell GATA1 from GATA2); accessible does not mean this TF holds the peak open; and peak-gene correlation does not mean the peak controls the gene. Treat an eGRN as a prioritized hypothesis list, not a wiring diagram.

The hardest and least-appreciated step is peak-to-gene linking, which is confounded by cell-type composition: across a heterogeneous dataset, any peak open in a cell type correlates with any gene expressed in that same type, whether or not the peak regulates it -- cell identity is a massive shared latent factor. Genome-wide peak-gene correlation therefore mostly recovers co-marker pairs. Mitigations (distance window, within-cell-type or GC/accessibility-matched null, requiring a motif, requiring the TF to be co-expressed) reduce but never eliminate it. Metacell aggregation, needed to beat scATAC sparsity, then introduces pseudo-replication: metacell-derived p-values are not calibrated significance and should be treated as ranking scores.

eGRN Method Taxonomy

Decision Tree by Scenario

SCENIC+ Pipeline (current Snakemake workflow)

Goal: Assemble eRegulons (TF -> enhancer -> gene) from paired or separate scRNA + scATAC.

Approach: Topic-model the ATAC with pycisTopic, call consensus peaks from per-cell-type pseudobulk (so cell-type labels are needed before peak calling), run pycistarget motif enrichment, then region-to-gene and TF-to-gene GBM regression to build triplets; orchestrate with Snakemake.

# Initialize and run the Snakemake workflow (the supported modern entry point).
# init_snakemake scaffolds a config.yaml pointing at the scATAC fragments, the scRNA
# AnnData, the motif/cisTarget databases, and the cell-type annotation used for pseudobulk.
# scenicplus init_snakemake --out_dir scenicplus_run
# (edit scenicplus_run/Snakemake/config/config.yaml, then run from inside that dir:)
# cd scenicplus_run/Snakemake && snakemake --cores 16

# Region-to-gene search space defaults to min 1kb / max 150kb from the gene, capped at the
# nearest neighboring gene's promoter -- narrower than the +/-500kb used by ArchR/Signac.

# Inspect the resulting eRegulons (TF -> region -> gene triplets). The output filename and
# directory are set in config.yaml (output_data); the direct (high-confidence) and extended
# (motif-similarity-inferred) tables are written there. The exact spelling has varied across
# versions, so resolve it by glob rather than hard-coding.
import glob, pandas as pd
ereg_file = glob.glob('scenicplus_run/**/eRegulon*direct*.tsv', recursive=True)[0]
eregulons = pd.read_csv(ereg_file, sep='\t')
summary = (eregulons.groupby('TF')
           .agg(n_regions=('Region', 'nunique'), n_genes=('Gene', 'nunique'))
           .sort_values('n_genes', ascending=False))
# Direct vs extended is a motif-to-TF annotation CONFIDENCE distinction, not topology:
# direct = curated/orthology; _extended adds motif-similarity-inferred (larger, noisier).

CellOracle Base GRN (works without paired multiome)

Goal: Build a base GRN -- the candidate TF -> gene scaffold -- from accessibility, as a prior for context-specific modeling.

Approach: Define active regions by Cicero co-accessibility (in R), scan them for TF motifs, and format the result as the base GRN; or load a prebuilt base GRN and skip ATAC entirely.

import celloracle as co
import pandas as pd

# Custom base GRN: peaks already filtered to Cicero co-accessible, promoter-linked regions.
peaks = pd.read_parquet('processed_peak_file.parquet')   # columns: peak_id, gene_short_name
tfi = co.motif_analysis.TFinfo(peak_data_frame=peaks, ref_genome='hg38')
tfi.scan(fpr=0.02)                                       # motif FPR
tfi.filter_motifs_by_score(threshold=10)
tfi.make_TFinfo_dataframe_and_dictionary()
base_grn = tfi.to_dataframe()

# Or skip ATAC: prebuilt base GRN as a prior (CellOracle ships ~10 species + mouse atlas).
# base_grn = co.data.load_mouse_scATAC_atlas_base_GRN()

The base GRN constrains which TF -> gene edges are allowed; the context-specific weights are then learned per cluster in perturbation-simulation.

FigR (paired, DORC-based, R)

Goal: Identify domains of regulatory chromatin (DORCs) and the TFs that regulate them.

Approach: Correlate peaks to genes, call DORCs (genes with an unusually large number of significant peaks), then score TF-DORC associations from motif enrichment plus TF expression correlation.

library(FigR)
# Step 1: peak-gene correlations (smoothed over KNN metacells for sparsity)
cisCor <- runGenePeakcorr(ATAC.se = atac_se, RNAmat = rna_mat,
                          genome = 'hg38', nCores = 8, p.cut = 0.05)
# Step 2: DORC scores then TF-DORC regulation scores (signed: activator/repressor)
dorcMat <- getDORCScores(atac_se, cisCor, geneList = unique(cisCor$Gene), nCores = 8)
figR <- runFigRGRN(ATAC.se = atac_se, dorcTab = cisCor, dorcMat = dorcMat,
                   rnaMat = rna_mat, genome = 'hg38', nCores = 8)

Per-Method Failure Modes

Cell-composition confound in peak-gene linking

Trigger: genome-wide peak-gene correlation with no within-type control. Mechanism: any peak open in a cell type correlates with any gene expressed in it. Symptom: "links" that are just cell-type co-markers. Fix: restrict to the distance window, use a matched null (Signac) or within-type correlation, and require a motif.

Metacell p-value laundering

Trigger: reporting astronomically small p-values from KNN-smoothed metacells. Mechanism: metacells are not independent (overlapping cells). Symptom: millions of "significant" links. Fix: treat metacell p-values as ranking scores; apply FDR honest about pseudo-replication.

Motif-family overclaiming

Trigger: naming a specific TF (GATA1) from a family motif. Mechanism: paralogs share near-identical motifs. Symptom: confident single-TF claims where only a family is detectable. Fix: report the motif/family and require orthogonal evidence to single out a member.

Unstated/wrong pairing in unpaired data

Trigger: computing "joint" correlations on separately-measured scRNA and scATAC. Mechanism: cells were computationally matched, not co-measured. Symptom: no pairing method or accuracy reported. Fix: integrate with GLUE/anchors first; report pairing quality; treat links as upper-bounded by it.

Treating eGRN edges as ground truth

Trigger: a published wiring diagram with no orthogonal validation. Mechanism: accessibility != binding != regulation, and motif/proximity validation is circular. Symptom: no ChIP/CRISPRi/perturbation check; validation uses the same motif DB used to build the net. Fix: validate against independent ChIP-seq or perturbation data.

Quantitative Thresholds

Common Errors

References

• Bravo Gonzalez-Blas C, et al. 2023. SCENIC+: single-cell multiomic inference of enhancers and gene regulatory networks. Nat Methods 20(9):1355-1367.
• Kamimoto K, et al. 2023. Dissecting cell identity via network inference and in silico gene perturbation (CellOracle). Nature 614(7949):742-751.
• Fleck JS, et al. 2023. Inferring and perturbing cell fate regulomes in human brain organoids (Pando). Nature 621:365-372.
• Kartha VK, et al. 2022. Functional inference of gene regulation using single-cell multi-omics (FigR). Cell Genomics 2(9):100166.
• Zhang L, Zhang J, Nie Q. 2022. DIRECT-NET. Sci Adv 8(22):eabl7393.
• Jiang Y, et al. 2022. TRIPOD: nonparametric single-cell multiomic trio characterization. Cell Syst 13(9):737-751.e4.
• Li Z, et al. 2023. scMEGA. Bioinform Adv 3(1):vbad003.
• Cao ZJ, Gao G. 2022. Multi-omics integration and regulatory inference with graph-linked embedding (GLUE). Nat Biotechnol 40(9):1458-1466.
• Pliner HA, et al. 2018. Cicero: cis-regulatory DNA interactions from single-cell accessibility. Mol Cell 71(5):858-871.e8.

Related Skills

• scenic-regulons - RNA-only regulon inference (promoter-proximal motif pruning)
• perturbation-simulation - in silico TF perturbation built on a CellOracle base GRN
• coexpression-networks - undirected co-expression baseline
• single-cell/scatac-analysis - scATAC preprocessing with Signac/ArchR
• atac-seq/atac-peak-calling - peak calling for the region universe
• chip-seq/motif-analysis - motif enrichment and TF binding for validation