# Get started with CraftGRN

CraftGRN is an R package for constructing and comparing condition-specific gene regulatory networks from matched ATAC-seq and RNA-seq data. The workflow follows three modules:

1. Predict TF binding sites from footprint or peak signal.
2. Connect TF binding sites to candidate target genes.
3. Learn regulatory topics and visualize differential GRNs.

The full pipeline is designed around a project YAML file that records paths, sample metadata, threshold values, genome settings, and output directories. For day-to-day analysis, the recommended workflow is wrapper-first: run one wrapper per module, then use the step functions when you want to inspect or customize a specific stage.

    remotes::install_github("oncologylab/craftgrn")
    library(craftgrn)

## Demo data

CraftGRN keeps demo datasets outside the source package so installation remains small and CRAN-friendly. The package helper reports any configured external demo bundles:

    craftgrn_demo_data_info()

No external demo bundle is currently configured. To run your own project, point CraftGRN at a project-level YAML file and use the normal Module 1 preparation function:

    config <- "project.yaml"
    module1_dir <- file.path(tempdir(), "predict_tf_binding_sites")

    omics <- load_prep_multiomic_data(
      config = config,
      label_col = "strict_match_rna",
      do_preprocess = FALSE,
      verbose = TRUE
    )

To run Module 1:

    module1 <- predict_tfbs(
      omics_data = omics,
      out_dir = module1_dir,
      output_format = "auto",
      write_outputs = TRUE,
      write_stats = FALSE,
      verbose = TRUE
    )

For project-level reproducibility, keep the run code in a short script beside the project YAML. The GSE192390 demo script follows this pattern: wrapper modes run full modules with one call, while step modes expose intermediate objects.

    Sys.setenv(CRAFTGRN_DEMO_RUN_MODE = "full_wrapper")
    source("11_pipeline_gse192390_tcell_fibroblast_demo.R")

    Sys.setenv(CRAFTGRN_DEMO_RUN_MODE = "module3_steps")
    source("11_pipeline_gse192390_tcell_fibroblast_demo.R")

Supported project-script modes should stay simple and explicit:

- `module1_wrapper`, `module2_wrapper`, and `module3_wrapper` run one module.
- `module1_steps`, `module2_steps`, and `module3_steps` expose intermediate objects for inspection.
- `full_wrapper` runs Modules 1, 2, and 3 through wrapper calls.
- `full_steps` runs Modules 1, 2, and 3 through step calls.

Troubleshooting demo data:

- If `craftgrn_demo_data_info()` returns zero rows, no public demo bundle is currently advertised by this package version.
- If file paths fail after moving the project, pass the `project.yaml` path explicitly. A portable config should use `base_dir: "."` so paths are relative to the config location.
- If the computer has limited memory, first run only `load_prep_multiomic_data()` and Module 1. Module 2 is larger because it computes TF-target correlations before restricting FP-target candidates to predicted TFBS.

## Module 1: Predict TF binding sites

Module 1 combines normalized ATAC signal, RNA expression, footprint summaries, and sample metadata into a multiomic object. It aligns footprint sites, builds condition-specific binding flags, normalizes footprint scores, and predicts TF binding sites with a single user-facing workflow. Internally, CraftGRN first correlates each aligned footprint with the TFs represented by its canonical motifs, applies configured correlation cutoffs, and labels footprints with at least one canonical-bound TF. By default, only these canonical-bound footprints are used for the all-expressed-TF prediction step.

### Inputs

Module 1 expects:

- A merged ATAC peak master table with normalized signal and overlap flags.
- A normalized RNA-seq count or expression matrix.
- A directory of per-condition motif footprint score and binding overview files generated by TOBIAS or a TOBIAS-compatible workflow.
- A metadata table mapping samples to conditions.
- A YAML configuration file with file paths, thresholds, and genome settings. The TFBS workflow uses `threshold_fp_tf_corr_r`, and can also use optional `threshold_fp_tf_corr_p` and `threshold_fp_tf_corr_fdr` fields when a project should require p-value or adjusted p-value support.

Supported built-in motif resources include `hg38` and `mm10`.

For large projects, keep `write_stats = FALSE` and use `output_format = "auto"`. Module 1 keeps full correlation tables as audit outputs, but the primary handoff to Module 2 is `predicted_tfbs`: a table with only aligned canonical-bound FPs and the TF names predicted to bind each FP.

### Run Module 1

    data_object <- load_prep_multiomic_data(
      config = "craftgrn_grn.yaml",
      genome = "hg38",
      gene_symbol_col = "HGNC"
    )
    module1_dir <- file.path(tempdir(), "predict_tf_binding_sites")

    module1 <- predict_tfbs(
      data_object,
      out_dir = module1_dir,
      write_outputs = TRUE,
      output_format = "auto"
    )

    module1_qc <- build_module1_qc_report(
      module1,
      output_dir = file.path(module1_dir, "reports"),
      scan_predicted_tfbs = TRUE
    )

### Outputs

Module 1 creates:

- A CraftGRN multiomic object with normalized signal matrices, binary flags, and feature annotations.
- `01_fp_scores_qn_<db>.csv`, the quantile-normalized footprint score matrix written for direct inspection and downstream external tools.
- Compact aligned footprint site, score, binding, and motif-support tables.
- Canonical-bound footprint summaries, including all canonical motifs retained for each aligned footprint.
- `predicted_tfbs`, the FP-to-TF handoff used by Module 2.
- Optional full TFBS correlation statistics for audit and debugging.
- `module1_qc_report.html`: an HTML QC report summarizing run parameters, input gates, motif-supported canonical support, correlation diagnostics, chunked predicted TFBS integrity, top TFs/FPs, condition support, motif complexity, warning checks, and related artifacts. The report uses static processing funnels, density curves, scatter summaries, heatmaps, lollipop rank plots, and cumulative curves.
- QC summaries reporting how many FPs, motif-supported pairs, canonical-bound FPs, prediction pairs, and predicted TFBS rows pass each step.

## Module 2: Connect TFs to target genes

Module 2 treats TF-target prediction as a relational problem. It joins the Module 1 `predicted_tfbs` relation with TF-target expression correlations, restricted FP-target correlations, TSS-window evidence, and optional generic regulatory-prior evidence. GeneHancer, HiC, promoter-capture, CRISPR enhancer screens, or user tables are all represented as regulatory priors rather than separate modes.

### Inputs

Module 2 uses:

- The CraftGRN multiomic object generated by Module 1.
- `predicted_tfbs`, with one row per predicted FP-TF binding event.
- A target gene TSS annotation table.
- Optional generic regulatory-prior FP-target links.
- YAML thresholds for TF-target and FP-target correlation filtering.

### Process

Module 2 first computes TF expression to target expression correlations and filters TF-target pairs. Only after that filter does it build FP-target candidates from FPs bound by the surviving TFs. FP score to target expression correlations are computed only for those restricted FP-target candidates within the TSS window or supported by regulatory prior evidence.

    module2 <- predict_tf_targets(
      multiomic_data = module1$omics_data,
      predicted_tfbs = module1$predicted_tfbs,
      gene_tss = gene_tss,
      regulatory_prior = regulatory_prior,
      project_config = "project.yaml",
      output_dir = file.path(tempdir(), "connect_tf_target_genes")
    )

    hnf4a_links <- query_predicted_links(module2, tf = "HNF4A")

    module2_qc <- build_module2_qc_report(
      module2,
      multiomic_data = module1$omics_data,
      output_dir = file.path(tempdir(), "connect_tf_target_genes", "reports"),
      scan_large_tables = TRUE,
      validate_integrity = TRUE
    )

### Outputs

Module 2 produces normalized tables:

- `module2_tf_target_corr`: one row per TF-target correlation.
- `module2_fp_target_candidates`: one row per FP-target candidate with distance-to-TSS and prior evidence.
- `module2_fp_target_corr`: one row per FP-target correlation.
- `module2_links`: final TF-FP-target links as keys and pass flags.
- `module2_condition_activity`: long condition-level activity flags.
- `module2_qc_summary`: counts before and after each filter.
- `module2_qc_report.html`: an HTML QC report summarizing handoff checks, TF-target and FP-target correlation filters, FP-target candidate source and distance-to-TSS evidence, final-link integrity checks, condition activity, warning checks, top TF/target/FP summaries, output manifests, and related HTML browser reports. The report uses relational flow diagrams, density and cumulative distance plots, scatter summaries, heatmaps, and lollipop rank plots.

## Module 3: Learn regulatory topics and visualize differential GRNs

Module 3 compares regulatory links between conditions, builds joint RNA and footprint document-term matrices, trains topic models, assigns links to topics, and summarizes topic-specific pathway and master TF programs.

### Inputs

Module 3 expects:

- Per-condition TF->TFBS->target link matrices from Module 2.
- A comparison metadata table defining pairwise contrasts.
- TF motif cluster assignments from Module 1.

### Run regulatory topics

The topic modeling workflow builds documents such as `comparison x TF-cluster x direction`. Terms are genes and TFBS features, and weights are derived from RNA log2 fold changes and footprint or peak signal deltas. The model balances RNA and footprint contributions before converting weights into pseudocounts for topic modeling. By default, WarpLDA uses CraftGRN’s native `warp_omp` sampler, which keeps doc/word Metropolis-Hastings semantics while using OpenMP threads inside each model fit. For fixed-seed validation runs, set `warplda_sampler = "warp_ref"`; this native sequential reference sampler is much slower than `warp_omp`.

For regular analysis, keep one selected document construction method in the project YAML config. A standard run writes a flat single-method layout: `topic_documents/`, `topic_models/`, `topic_extraction/`, `review/`, and `reports/`. Benchmark-only method grids can be stored separately in the config and left disabled for normal package runs.

    topic_method: comparison_aggr_multivi
    topic_k: 10
    warplda_iterations: 2000
    topic_link_output: pass
    topic_vae_device: auto
    topic_vae_batch_size: 512
    pathway_backend: enrichly

    topic_benchmark_enabled: false
    topic_benchmark_methods: []
    topic_benchmark_k_grid: []

Use `pathway_backend: enrichly` for local cached pathway enrichment when the optional `enrichly` package is installed. Use `pathway_backend: enrichr` when you intentionally want the Enrichr web API backend.

    run_topic_modeling(
      filtered_dir = "differential_links",
      comparisons = "module3_comparisons.csv",
      output_dir = file.path(tempdir(), "regulatory_topics"),
      project_config = "project.yaml",
      run_training = TRUE,
      run_extraction = TRUE,
      run_reports = TRUE
    )

The lower-level calls remain available when you want to inspect inputs, separate model training from topic extraction, or rerun only one stage.

    module3_prepare_differential_links(
      module2 = "connect_tf_target_genes",
      multiomic_data = module3_multiomic,
      compar = "module3_comparisons.csv",
      project_config = "project.yaml",
      output_dir = file.path(tempdir(), "regulatory_topics", "differential_links")
    )

    module3_construct_docs(
      filtered_dir = file.path(tempdir(), "regulatory_topics", "differential_links"),
      output_dir = file.path(tempdir(), "regulatory_topics", "topic_documents"),
      tf_cluster_map = NULL,
      doc_mode = "tf",
      doc_design = "comparison",
      fp_term_mode = "aggregate",
      gene_term_mode = "unique",
      count_method = "log",
      include_tf_terms = TRUE
    )

    module3_train_topic_models(
      k_grid = 10,
      filtered_dir = file.path(tempdir(), "regulatory_topics", "differential_links"),
      output_dir = file.path(tempdir(), "regulatory_topics", "topic_models"),
      flat_output = TRUE,
      tf_cluster_map = NULL,
      doc_mode = "tf",
      doc_design = "comparison",
      fp_term_mode = "aggregate",
      gene_term_mode = "unique",
      count_method = "log",
      include_tf_terms = TRUE,
      backend = "vae",
      vae_variant = "multivi_encoder",
      vae_device = "auto"
    )

    module3_extract_topics(
      k = 10,
      model_dir = file.path(tempdir(), "regulatory_topics", "topic_models"),
      output_dir = file.path(tempdir(), "regulatory_topics", "topic_extraction"),
      topic_input_dir = file.path(tempdir(), "regulatory_topics", "topic_models"),
      backend = "vae",
      vae_variant = "multivi_encoder",
      doc_mode = "tf",
      weight_label = "peak_log2fc_fp_gene_fc_expr",
      flatten_single_output = TRUE,
      topic_report_args = list(link_topic_output = "pass")
    )

When `k_grid` contains more than one value, the standard Module 3 output keeps the K-review tables and topic-model diagnostics for the selected method. Broad multi-method benchmarks are developer workflows and are not part of the normal package tutorial.

### Review topic modeling and differential GRNs

    build_module3_qc_report(
      topic_dir = file.path(tempdir(), "regulatory_topics"),
      differential_links_dir = "differential_links"
    )

    visualize_topic_modeling_results(
      topic_dir = file.path(tempdir(), "regulatory_topics"),
      output_dir = file.path(tempdir(), "regulatory_topics", "reports")
    )

    visualize_differential_grns(
      differential_links_dir = "differential_links",
      output_dir = file.path(tempdir(), "regulatory_topics", "reports"),
      top_tf_n = 10,
      top_link_n = 300
    )

### Outputs

Module 3 creates:

- Differential link tables, comparison-level plots, and an interactive differential GRN network browser with comparison, direction, Top TF, and Top link controls.
- Model selection metrics across topic-number choices.
- Topic-term and topic-link probability tables.
- Topic assignments for regulatory links.
- Standard topic review tables and HTML reports under `review/`.
- Optional benchmark review tables and HTML reports under a separate benchmark output directory when `topic_benchmark_enabled: true`.
- `module3_qc_report.html`: a QC report summarizing topic inputs, method plan, theta separation scores, compact topic-link pass counts, and output files.
- Pathway enrichment summaries and plots.
- Master TF connectivity summaries and plots.
- Topic-stratified differential GRN visualizations.

## Next steps

After running the three modules, inspect the model selection metrics, choose a topic number, review topic-specific pathway enrichments, and use master TF summaries to prioritize condition-specific regulatory programs for follow-up.