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Space Ranger with Segmentation Pipeline Specification

Pipeline Details

  • Name: Space Ranger with Segmentation Pipeline
  • Pipeline UUID: bff8a8ee47284e70923b1ddf6a200b19
  • Version: 1.4.1
  • View Pipeline:

Overview

Space Ranger with Segmentation Pipeline is designed for high-resolution spatial transcriptomics analysis using Visium HD data, specifically the 2μm bin size. The pipeline integrates Space Ranger processing with a custom Jupyter notebook for downstream cell segmentation, quality control, and exploratory spatial analysis.

Key Use cases:

  • Visium HD Spatial Transcriptomics Analysis: Processing high-resolution spatial gene expression data with 2μm bin resolution for detailed tissue architecture analysis.
  • Cell Segmentation and Spatial Mapping: Automated cell segmentation using StarDist-based methods with mapping of segmentation labels onto spatial transcriptomics spots.
  • Single-Cell Approximation from Spatial Data: Converting spatial bins to single-cell approximations through sophisticated binning and filtering algorithms.

Features

  • Multi-Resolution Bin Support: Supports 2μm, 8μm, and 16μm bin sizes for flexible spatial resolution analysis.
  • Integrated Cell Segmentation: Implements destriping and StarDist-based cell segmentation with nucleus expansion capabilities.
  • Comprehensive Quality Control: Performs cell- and gene-level filtering with customizable thresholds and generates detailed QC visualizations.
  • Cross-Platform Compatibility: Outputs processed data in both Python (.h5ad) and R (.rds) formats for seamless integration with downstream analysis tools.
  • Interactive Visualization: Generates spatial plots, QC histograms, PCA/UMAP embeddings, and clustering visualizations in HTML reports.
  • Containerized Workflow: Utilizes Docker containers including Space Ranger 4.0.1, custom spatial analysis environments, and schard for format conversion.

Input/Output Specification

Inputs

Required

FASTQ Files

  • Description: Raw sequencing reads from Visium HD spatial transcriptomics experiments, containing both spatial barcode and gene expression information.
  • Format: .fastq.gz (paired-end reads)
  • Example File Path: /path/to/reads/sample_S1_R1_001.fastq.gz, /path/to/reads/sample_S1_R2_001.fastq.gz

Microscope Image

  • Description: High-resolution H&E stained tissue image corresponding to the spatial transcriptomics capture area.
  • Format: .tiff or .tiff.gz
  • Example File Path: /path/to/images/tissue_hires_image.tiff

Reference Genome

  • Description: 10x Genomics compatible reference genome for read alignment and gene quantification.
  • Format: .tar.gz (Space Ranger reference package)
  • Example File Path: /path/to/reference/refdata-gex-GRCh38-2020-A.tar.gz

Optional Inputs

Sample Sheet

  • Description: Metadata file containing sample information and processing parameters.
  • Format: Tab-separated values (.tsv) or comma-separated values (.csv)
  • Required Columns: Sample ID, FASTQ paths, image paths
  • Example File Path: /path/to/metadata/samplesheet.csv

Segmentation Mask

  • Description: Pre-computed cell segmentation mask for custom cell boundary definition.
  • Format: .geojson or .tiff
  • Example File Path: /path/to/masks/segmentation_mask.geojson

Outputs

Reported Outputs

  • Processed AnnData Object:
  • Description: Comprehensive spatial single-cell data object containing gene expression, spatial coordinates, cell metadata, and analysis results
  • Format: .h5ad
  • Example File Path: /output/processed_spatial_data.h5ad
  • Visualization App: Python (scanpy, squidpy), Jupyter notebooks

  • Location: Main output folder

  • Seurat-Compatible Object:

  • Description: R-compatible spatial transcriptomics data object for downstream analysis in Seurat
  • Format: .rds
  • Example File Path: /output/spatial_seurat_object.rds
  • Visualization App: R (Seurat, ggplot2)

  • Location: Main output folder

  • Interactive HTML Report:

  • Description: Comprehensive analysis report with QC metrics, spatial visualizations, and clustering results
  • Format: .html
  • Example File Path: /output/spatial_analysis_report.html
  • Visualization App: Web browser
  • Location: Reports folder

Supporting Outputs

  • Space Ranger Output Archive:
  • Description: Complete Space Ranger count output including gene expression matrices, spatial coordinates, and QC metrics
  • Format: .tar.gz

  • Example File Path: /output/spaceranger_output.tar.gz

  • Cloupe File:

  • Description: Interactive visualization file for 10x Genomics Loupe Browser
  • Format: .cloupe

  • Example File Path: /output/sample_analysis.cloupe

  • Gene Expression Matrices:

  • Description: Filtered and raw feature-barcode matrices in HDF5 format
  • Format: .h5
  • Example File Path: /output/filtered_feature_bc_matrix.h5

Associated Processes

References & Additional Documentation