Expected Outputs

Upon successful completion, CARLISLE generates a comprehensive directory structure under WORKDIR/results. Each subdirectory contains outputs corresponding to specific stages of the workflow — from raw alignment statistics to annotated peak results and quality control summaries.

Directory Overview

  • alignment_stats/ – Contains detailed alignment reports for each sample, including mapping efficiency, read depth, and spike-in alignment metrics.

  • bam/ – Stores sorted and indexed BAM files for all samples. This directory also includes per-sample and spike-in alignment statistics useful for downstream normalization and QC.

  • bedgraph/ – Includes BEDGRAPH coverage tracks summarizing read density across the genome. These files serve as intermediates for visualization and peak-calling validation.

  • bigwig/ – Contains BigWig files generated from normalized coverage data, suitable for visualization in genome browsers such as UCSC Genome Browser or IGV.

  • fragments/ – Stores fragment length distributions and deduplicated fragment data (particularly important for CUT&RUN and CUT&Tag experiments). Useful for assessing fragment size enrichment and MNase digestion efficiency.

  • peaks/ – The core results directory containing called peaks, differential comparisons, and annotations.

  • Subdirectories are organized by quality thresholds (e.g., 0.05, 0.01), representing the significance cutoffs applied during peak calling.

  • Each quality threshold directory includes:

    • contrasts/ – Contains results of differential binding analyses defined in the contrast manifest, including:
    • Differential enrichment results from DESeq2 (AUC-based and fragment-based)
    • 3-column BED files for up-regulated peaks in each group (up_group1.bed, up_group2.bed)
    • homer_deg/ – HOMER motif enrichment analysis for differentially enriched peaks
    • ame_deg/ – AME (Analysis of Motif Enrichment) results for differentially enriched peaks using HOCOMOCO motifs

    • <peak_caller>/ – Subdirectories for each peak caller (e.g., macs2, seacr, gopeaks). Each includes raw peak calls and annotated results.

    • peak_output/ – Raw peak calls organized by control mode:

      • individual/ – Peaks called using individual replicate controls (present for all analyses)
      • pooled/ – Peaks called using merged high-depth controls (present when pool_controls: true)

    • annotation/ – Contains enriched feature and pathway analyses, organized by control mode:

      • go_enrichment/ – Results from ChIP-Enrich gene set enrichment, generated when run_go_enrichment: true is enabled.
      • homer/ – Output from HOMER motif discovery and annotation.
      • rose/ – Output from ROSE super-enhancer analysis, generated when run_rose: true is specified.

  • qc/ – Centralized quality control directory containing comprehensive MultiQC summaries, FastQC metrics, and spike-in normalization reports (when applicable).

Example Directory Layout

Below is an example of the CARLISLE output structure for a typical CUT&RUN experiment:

results/
├── alignment_stats/
├── bam/
│   └── pooled_controls/           # Merged control BAMs (when pool_controls: true)
├── bedgraph/
│   └── pooled_controls/           # Pooled control bedgraphs (when pool_controls: true)
├── bigwig/
├── fragments/
│   └── pooled_controls/           # Pooled control fragments (when pool_controls: true)
├── peaks/
│   ├── 0.05/
│   │   ├── contrasts/
│   │   │   ├── homer_deg/         # DEG motif enrichment (HOMER)
│   │   │   ├── ame_deg/           # DEG motif enrichment (AME/HOCOMOCO)
│   │   │   ├── up_group1.bed      # 3-column BED for group1 up-regulated peaks
│   │   │   └── up_group2.bed      # 3-column BED for group2 up-regulated peaks
│   │   ├── gopeaks/
│   │   │   ├── peak_output/
│   │   │   │   ├── individual/    # Peaks with individual controls
│   │   │   │   └── pooled/        # Peaks with pooled controls
│   │   │   └── annotation/
│   │   │       ├── individual/
│   │   │       │   ├── go_enrichment/
│   │   │       │   ├── homer/
│   │   │       │   └── rose/
│   │   │       └── pooled/
│   │   │           ├── go_enrichment/
│   │   │           ├── homer/
│   │   │           └── rose/
│   │   ├── macs2/
│   │   │   ├── peak_output/
│   │   │   │   ├── individual/
│   │   │   │   └── pooled/
│   │   │   └── annotation/
│   │   │       ├── individual/
│   │   │       │   ├── go_enrichment/
│   │   │       │   ├── homer/
│   │   │       │   └── rose/
│   │   │       └── pooled/
│   │   │           ├── go_enrichment/
│   │   │           ├── homer/
│   │   │           └── rose/
│   │   └── seacr/
│   │       ├── peak_output/
│   │       │   ├── individual/
│   │       │   └── pooled/
│   │       └── annotation/
│   │           ├── individual/
│   │           │   ├── go_enrichment/
│   │           │   ├── homer/
│   │           │   └── rose/
│   │           └── pooled/
│   │               ├── go_enrichment/
│   │               ├── homer/
│   │               └── rose/
│   └── 0.01/
│       └── ...
└── qc/
    ├── fastqc_raw/
    └── fqscreen_raw/

🧭 Tip: The structure is intentionally hierarchical, enabling automated report generation and simplifying downstream integration with visualization tools and statistical frameworks.