Introduction Usage Parameters Output

Introduction

This document describes the output produced by the pipeline. Most of the output is contained within HTML reports created with Quarto, but there are also other files which you can either take and analyse further by yourself or explore interactively with e.g. TissUUmaps.

The directories listed below will be created in the results directory after the pipeline has finished. Results for individual samples will be created in subdirectories following the <OUTDIR>/<SAMPLE>/ structure. All paths are relative to the top-level results directory.

The pipeline is built using Nextflow and processes data using the following steps:

Space Ranger

Output files
  • <SAMPLE>/spaceranger/
    • outs/spatial/tissue_[hi/low]res_image.png: High and low resolution images.
    • outs/spatial/tissue_positions.csv: Spot barcodes and their array positions.
    • outs/spatial/scalefactors_json.json: Scale conversion factors for the spots.
    • outs/filtered_feature_bc_matrix/barcodes.tsv.gz: List of barcode IDs.
    • outs/filtered_feature_bc_matrix/features.tsv.gz: List of feature IDs.
    • outs/filtered_feature_bc_matrix/matrix.mtx.gz: Matrix of UMIs, barcodes and features.
    • outs/web_summary.html: Interactive summary report from Space Ranger.
    • outs/cloupe.cloupe: File for visualization in 10X Loupe Browser.

All files produced by Space Ranger are currently published as output of this pipeline, regardless if they’re being used downstream or not; you can find more information about these files at the 10X website.

FastQC

Output files
  • <SAMPLE>/fastqc/
    • *_fastqc.html: FastQC report.
    • *_fastqc.zip: FastQC data archive.

FastQC gives general quality metrics about your sequenced reads. It provides information about the quality score distribution across your reads and per base sequence content among other metrics.

Per-sample data

Output files
  • <SAMPLE>/data/
    • <SAMPLE>-raw.h5ad: Raw (unprocessed) data in AnnData format, as extracted from the SpatialData object before any downstream analysis.
    • <SAMPLE>.h5ad: Processed data in AnnData format, including QC metrics, clustering, differential expression, and spatial analysis results.
    • <SAMPLE>_svg.csv: List of spatially variable genes.

Data in .h5ad formats as processed by the pipeline, which can be used for further downstream analyses if desired; unprocessed data is also present in these files. It can also be used by the TissUUmaps browser-based tool for visualisation and exploration, allowing you to delve into the data in an interactive way. The list of spatially variable genes are added as a convenience if you want to explore them in e.g. Excel.

Per-sample reports

Output files
  • <SAMPLE>/reports/
    • report-<SAMPLE>.html: Analysis report including quality controls, clustering, differential expression, and spatial analysis results.
    • report.qmd: Quarto source notebook of the report.
    • report.yml: Parameters used by the report.
    • _extensions/: Quarto nf-core extension, common to all reports.
  • <SAMPLE>/plots/
    • *.png: Individual plots from the per-sample report, saved as PNG files for easy reuse.

The per-sample reports contain quality control metrics, clustering results, differential expression analysis, neighbourhood enrichment, cluster interaction matrices, and spatially variable gene analysis.

Integration

Integration outputs are only produced when both --skip_integration and --skip_downstream are unset (which is the default). This merges all per-sample data and applies batch correction using the selected integration method (Harmony or Scanorama).

Output files
  • integration/data/
    • merged.zarr: Merged SpatialData object containing all samples (before integration).
    • <METHOD>.zarr: Integrated SpatialData object with batch-corrected results.
    • <METHOD>.h5ad: Integrated AnnData object with batch-corrected embeddings and clustering.
  • integration/reports/
    • report-integrated.html: Integration report with cross-sample comparisons, batch mixing assessment, and integrated clustering results.
    • report-integrated.qmd: Quarto source notebook of the integration report.
    • report-integrated.yml: Parameters used by the integration report.
    • _extensions/: Quarto nf-core extension.
  • integration/plots/
    • *.png: Individual plots from the integration report.

Workflow reporting

Pipeline information

Output files
  • pipeline_info/
    • Reports generated by Nextflow: execution_report.html, execution_timeline.html, execution_trace.txt and pipeline_dag.dot/pipeline_dag.svg.
    • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and software_versions.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter’s are used when running the pipeline.
    • Reformatted samplesheet files used as input to the pipeline: samplesheet.valid.csv.
    • Parameters used by the pipeline run: params.json.
  • multiqc/
    • Report generated by MultiQC: multiqc_report.html. Includes FastQC results, Space Ranger metrics, per-sample filtering statistics (spots and genes removed at each QC step), and software versions.
    • Data and plots generated by MultiQC: multiqc_data/ and multiqc_plots/.

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.
On this page