Output Reference

This page documents all outputs generated by the pipeline. Understanding these outputs helps with debugging, quality control, and downstream analysis.

How Outputs Are Organized¶

The pipeline organizes outputs into two main directories:

• results/images/ - All processed image files organized by batch, processing stage, arm, and plate

• results/workspace/ - Analysis outputs and QC reports

Within these, outputs follow a consistent naming pattern: {batch}/{subfolder}/{arm}/{plate}/{plate}-{well}-{site}/

Directory Structure Overview¶

Default and No-Stitch workflows¶

results/
├── images/
│   └── {batch}/
│       ├── illum/                          # Illumination functions
│       ├── images_corrected/               # Illumination-corrected images, intra-arm aligned, channel balanced if 4-color SBS chemistry
│       ├── images_aligned/                 # Aligned barcoding images
│       ├── images_segmentation/            # Segmentation check outputs
│       ├── images_corrected_stitched/      # Stitched images (default only)
│       ├── images_corrected_cropped/       # Cropped images (default only)
│       └── images_corrected_stitched_10X/  # 10X downsampled stitched images (default only)
├── workspace/
│   ├── analysis/                           # Combined analysis outputs
│   └── qc_reports/                         # Quality control reports
├── multiqc/                                # MultiQC summary reports
└── pipeline_info/                          # Nextflow execution reports and metadata

Stitch-Align-Crop workflow¶

results/
├── images/
│   └── {batch}/
│       ├── illum/                          # Illumination functions
│       ├── images_corrected/               # Illumination-corrected images
│       ├── images_corrected_stitched/      # Stitched images
│       └── images_corrected_stitched_10X/  # 10X downsampled stitched images
│       ├── images_aligned_intraarm/        # Cross-cycle aligned images
│       ├── images_aligned_combined/        # Cross-arm aligned images
│       ├── images_aligned_cropped/         # Cropped images
│       ├── images_preprocessed/            # Preprocessed barcoding images (STITCH_ALIGN_CROP only)
│       └── images_segmentation/            # Segmentation check outputs
├── workspace/
│   ├── analysis/                           # Combined analysis outputs
│   └── qc_reports/                         # Quality control reports - numbering
├── multiqc/                                # MultiQC summary reports
└── pipeline_info/                          # Nextflow execution reports and metadata

Cell Painting Outputs¶

Illumination Functions¶

Location: results/images/{batch}/illum/{plate}/

Files: {plate}_Illum{channel}.npy

Description: NumPy arrays containing illumination correction functions for each channel. Used to correct uneven illumination across the field of view.

Example:

results/images/Batch1/illum/Plate1/
  Plate1_IllumDAPI.npy
  Plate1_IllumGFP.npy
  Plate1_IllumRFP.npy

Corrected Images¶

Location: results/images/{batch}/images_corrected/painting/{plate}/{plate}-{well}-{site}/

Files: Plate_{plate}_Well_{well}_Site_{site}_Corr{channel}.tiff

Description: Multi-frame TIFF images with illumination correction applied. One file per channel per site.

Example:

results/images/Batch1/images_corrected/painting/Plate1/Plate1-A1-1/
  Plate_Plate1_Well_A1_Site_1_CorrDAPI.tiff
  Plate_Plate1_Well_A1_Site_1_CorrGFP.tiff
  Plate_Plate1_Well_A1_Site_1_CorrRFP.tiff

Segmentation Check¶

Location: results/images/{batch}/images_segmentation/painting/{plate}/{plate}-{well}/

Files: Segmentation preview images (*.png) and CSV summaries

Description: QC outputs from the segmentation check step.

Stitched Images¶

Location: results/images/{batch}/images_corrected_stitched/painting/{plate}/{plate}-{well}/

Files: *.tiff, TileConfiguration.txt

Description: Stitched full-resolution images for each well. Default entrypoint: generated only if qc_painting_passed == true, and includes the FIJI TileConfiguration file for reference. STITCH_ALIGN_CROP: always generated (not gated by qc_painting_passed), stitched with ashlar from the illumination-corrected images, one file per well, cycle and channel named Plate_{plate}_Well_{well}_Site_1_Stitched_Cycle{NN}_Corr{channel}.tiff (no TileConfiguration file).

Cropped Images¶

Location: results/images/{batch}/images_corrected_cropped/painting/{plate}/{plate}-{well}/ (default) or results/images/{batch}/images_aligned_cropped/painting/{plate}/{plate}-{well}/ (STITCH_ALIGN_CROP - by this point the images have gone through cross-cycle/cross-arm alignment, not just illumination correction)

Files: *.tiff

Description: Cropped images with overlap regions removed.

10X Downsampled Images¶

Location: results/images/{batch}/images_corrected_stitched_10X/painting/{plate}/{plate}-{well}/

Files: *.tiff

Description: Downsampled versions of stitched images for quick visualization (every 10th pixel in STITCH_ALIGN_CROP).

Barcoding Outputs¶

Aligned Images¶

Location: results/images/{batch}/images_aligned/barcoding/{plate}/{plate}-{well}/

Files: *.tiff

Description: Illumination-corrected and aligned barcoding images.

Preprocessed Images¶

Location: results/images/{batch}/images_corrected/barcoding/{plate}/{plate}-{well}-{site}/ (default, NO_STITCH) or results/images/{batch}/images_preprocessed/barcoding/{plate}/{plate}-{well}-{site}/ (STITCH_ALIGN_CROP - this entrypoint preprocesses after stitch/align/crop, so it’s kept under its own folder name rather than images_corrected)

Files: Plate_{plate}_Well_{well}_Site_{site}_Cycle{cycle}_{channel}.tiff, plus an optional ..._FociObjects.tiff (a per-site Foci object/label image, produced only if the barcoding preprocessing pipeline includes a module that saves it; STITCH_ALIGN_CROP only)

Description: Barcode-called and color-compensated images. In the default and NO_STITCH entrypoints these are ready for stitching/combined analysis; in STITCH_ALIGN_CROP they are produced from the already stitched, aligned and cropped tiles. In STITCH_ALIGN_CROP, the optional FociObjects.tiff is also made available to combined analysis (see below).

Stitched Images¶

Location: results/images/{batch}/images_corrected_stitched/barcoding/{plate}/{plate}-{well}/

Files: *.tiff, TileConfiguration.txt

Description: Default entrypoint: stitched preprocessed barcoding images, generated only if qc_barcoding_passed == true. STITCH_ALIGN_CROP: always generated (not gated by qc_barcoding_passed), stitched with ashlar from the illumination-corrected images, one file per well, cycle and channel named Plate_{plate}_Well_{well}_Site_1_Stitched_Cycle{NN}_{channel}.tiff (no TileConfiguration file).

Cropped Images¶

Location: results/images/{batch}/images_corrected_cropped/barcoding/{plate}/{plate}-{well}/ (default) or results/images/{batch}/images_aligned_cropped/barcoding/{plate}/{plate}-{well}/ (STITCH_ALIGN_CROP)

Files: *.tiff

Description: Cropped barcoding images with overlap regions removed.

Combined Analysis Outputs¶

This is where the final, most valuable outputs live. The combined analysis merges Cell Painting phenotype data with barcoding genotype data.

Location: results/workspace/analysis/{batch}/{plate}-{well}-{site}/

Segmentation Masks¶

Files:

segmentation_masks/
  {plate}_{well}_{site}_Nuclei.tiff
  {plate}_{well}_{site}_Cells.tiff
  {plate}_{well}_{site}_Cytoplasm.tiff

Description: Binary masks for segmented objects:

• Nuclei: Nuclear segmentation

• Cells: Whole cell segmentation

• Cytoplasm: Cytoplasm only (Cells - Nuclei)

Feature Measurements¶

Files:

Description: CSV files containing extracted features and measurements.

Image.csv¶

Per-image measurements, including:

Nuclei.csv / Cells.csv¶

Per-object measurements, including:

Experiment.csv¶

Pipeline metadata and run parameters.

STITCH_ALIGN_CROP Intermediate Images¶

Only produced by the STITCH_ALIGN_CROP entrypoint, between stitching and cropping.

Cross-cycle Aligned Images¶

Location: results/images/{batch}/images_aligned_intraarm/{arm}/{plate}/{plate}-{well}/

Files: full-well stitched *.tiff, one per cycle and channel

Description: Each cycle is registered to the first cycle of its arm (phase cross-correlation on the painting_channame / barcoding_channame reference channel), and the same shift is applied to every channel in that cycle. If only one cycle is present the images pass through unchanged. Alignment stats and QC overlays are written to qc_reports/{batch}/4_alignment/painting/ and 8_alignment/barcoding/.

Cross-arm Aligned Images¶

Location: results/images/{batch}/images_aligned_combined/{painting|barcoding}/{plate}/{plate}-{well}/

Files: full-well *.tiff for each arm

Description: Painting and barcoding images are scaled to a common pixel size (painting_scalingstring, barcoding_scalingstring), then painting is registered to barcoding cycle 1. The shift is applied to every painting image; barcoding images are scaled but not shifted. Stats and a QC overlay are written to qc_reports/{batch}/9_alignment/combined/.

Quality Control Outputs¶

QC reports are numbered to reflect the pipeline execution order, making it easy to review them sequentially. The numbering differs by entrypoint - the default and NO_STITCH entrypoints share one scheme, while STITCH_ALIGN_CROP uses a different one (alignment happens after stitching there, and preprocessing/segmentation happen much later, after crop).

Location: results/workspace/qc_reports/{batch}/

Default and NO_STITCH entrypoints¶

STITCH_ALIGN_CROP entrypoint¶

Stitching/alignment happen immediately after illumination correction here (before segmentation/preprocessing, which run later on cropped tiles), so this entrypoint numbers its qc_reports subfolders differently:

MultiQC Reports¶

Location: results/multiqc/

Files:

• multiqc_report.html: Interactive HTML report aggregating all QC metrics

• multiqc_data/: Raw data and plot data

• multiqc_plots/: Exported plot files

Description: Aggregated summary of pipeline execution metrics and QC results.

Pipeline Information¶

Location: results/pipeline_info/

The pipeline automatically generates execution reports and metadata in this folder.

Execution Reports¶

Files:

• execution_report_*.html: Resource usage and task statistics

• execution_timeline_*.html: Timeline visualization of task execution

• execution_trace_*.txt: Detailed execution log with per-task metrics

• pipeline_dag_*.html: Interactive DAG visualization of the workflow

Description: These files are automatically generated by Nextflow and include timestamps in their filenames. They provide detailed insights into pipeline performance, resource consumption, and task dependencies.

Pipeline Metadata¶

Files:

• params_*.json: JSON file containing all parameters used for the run

• nf-pooled-cellpainting_software_mqc_versions.yml: Software versions used by the pipeline

• pipeline_report.html / pipeline_report.txt: Summary report of the pipeline run

Description: These metadata files help with reproducibility by recording the exact configuration and software versions used.