Output Reference - nf-pooled-cellpainting Documentation

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¶

results/
├── images/
│   └── {batch}/
│       ├── illum/                          # Illumination functions
│       ├── images_corrected/               # Illumination-corrected images
│       ├── images_aligned/                 # Aligned barcoding images
│       ├── images_segmentation/            # Segmentation check outputs
│       ├── images_corrected_stitched/      # Stitched images (full resolution)
│       ├── images_corrected_cropped/       # Cropped images
│       └── images_corrected_stitched_10X/  # 10X downsampled stitched images
├── workspace/
│   ├── analysis/                           # Combined analysis outputs
│   └── qc_reports/                         # Quality control reports
│       ├── 1_illumination_painting/
│       ├── 3_segmentation/
│       ├── 4_stitching_painting/
│       ├── 5_illumination_barcoding/
│       ├── 6_alignment/
│       ├── 7_preprocessing/
│       └── 8_stitching_barcoding/
├── 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. Generated only if qc_painting_passed == true. Includes the FIJI TileConfiguration file for reference.

Cropped Images¶

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

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: 10X downsampled versions of stitched images for quick visualization.

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}/

Files: Plate_{plate}_Well_{well}_Site_{site}_Cycle{cycle}_{channel}.tiff

Description: Barcode-called and color-compensated images, ready for stitching and combined analysis.

Stitched Images¶

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

Files: *.tiff, TileConfiguration.txt

Description: Stitched preprocessed barcoding images. Generated only if qc_barcoding_passed == true.

Cropped Images¶

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

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:

File	Description
`Image.csv`	Per-image measurements (counts, metadata)
`Nuclei.csv`	Per-nucleus measurements (location, intensity, texture)
`Cells.csv`	Per-cell measurements
`Cytoplasm.csv`	Per-cytoplasm measurements
`Experiment.csv`	Pipeline metadata

Description: CSV files containing extracted features and measurements.

Image.csv¶

Per-image measurements, including:

Column	Description
`ImageNumber`	Unique image identifier
`Metadata_Plate`	Plate identifier
`Metadata_Well`	Well position
`Metadata_Site`	Site number
`Count_Nuclei`	Number of nuclei detected
`Count_Cells`	Number of cells detected

Nuclei.csv / Cells.csv¶

Per-object measurements, including:

Column	Description
`ObjectNumber`	Unique object ID within image
`ImageNumber`	Reference to Image.csv
`Metadata_Barcode`	Assigned barcode sequence
`Location_Center_X`	X coordinate of object center
`Location_Center_Y`	Y coordinate of object center
`AreaShape_Area`	Object area in pixels
`Intensity_MeanIntensity_*`	Mean intensity per channel
`Texture_*`	Texture features
`Granularity_*`	Granularity features

Experiment.csv¶

Pipeline metadata and run parameters.

Quality Control Outputs¶

QC reports are numbered to reflect the pipeline execution order, making it easy to review them sequentially.

Location: results/workspace/qc_reports/

Directory	Description	Files
`1_illumination_painting/`	Painting illumination correction montages	`*.png` montages
`3_segmentation/`	Segmentation QC with overlays	`*.png` montages
`4_stitching_painting/`	Painting stitching QC	`*.png` montages
`5_illumination_barcoding/`	Barcoding illumination correction montages	`*.png` montages
`6_alignment/`	Barcode alignment reports	`.html`, `.ipynb`, `*.png`
`7_preprocessing/`	Barcoding preprocessing QC	`.html`, `.ipynb`, `*.png`
`8_stitching_barcoding/`	Barcoding stitching QC	`*.png` montages

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.

File	Description
`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
`params_*.json`	Parameters used for the run
`nf-pooled-cellpainting_software_mqc_versions.yml`	Software versions

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.