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Space Ranger with Bin2Cell Segmentation Pipeline Specification

Pipeline Details

  • Name: Space Ranger with Bin2Cell Segmentation Pipeline
  • Pipeline UUID: 2826623e9d904863bae6f79fd139c751
  • Version: 1.2.8
  • View Pipeline:

Overview

Space Ranger with Bin2Cell Segmentation Pipeline is designed for high-resolution spatial transcriptomics analysis using Visium HD data, specifically the 2um bin size. The pipeline integrates Space Ranger processing with a custom Jupyter notebook for downstream cell segmentation, quality control, and exploratory spatial analysis using the bin2cell package.

Key Use cases:

  • High-Resolution Spatial Cell Segmentation: Transforms Visium HD 2um binned data into cell-level resolution using StarDist-based segmentation.
  • Quality Control and Filtering: Performs comprehensive cell- and gene-level filtering with customizable thresholds for minimum genes, cells, and bins per cell.
  • Spatial Transcriptomics Visualization: Generates spatial plots, QC histograms, PCA/UMAP, and clustering visualizations for exploratory analysis.

Features

  • Visium HD 2um Resolution Support: Specifically designed for processing high-resolution Visium HD data at 2 micrometer bin size.
  • Integrated Cell Segmentation: Uses StarDist-based cell segmentation with H&E image processing and destriping capabilities.
  • Customizable Parameters: Allows user control over microns_per_pixel, minimum genes/cells, and target image paths for different sample types.
  • Multi-Format Output: Produces both .h5ad files for Python analysis and .rds files for R/Seurat compatibility via SeuratDisk.
  • Comprehensive Quality Control: Implements filtering based on mitochondrial genes, ribosomal genes, and spatial bin quality metrics.
  • Interactive Visualization: Generates HTML reports with spatial plots, dimensionality reduction, and clustering results.

Input/Output Specification

Inputs

Required

FASTQ Files

  • Description: Raw sequencing reads from Visium HD spatial transcriptomics experiment, following 10x Genomics naming convention.
  • Format: .fastq.gz
  • Example File Path: gs://bucket/folder1/SAMPLE1_A1_S1_L001_R1_001.fastq.gz

Reference Genome

  • Description: Space Ranger compatible reference genome archive for read alignment.
  • Format: .tar.gz
  • Example File Path: /path/to/reference/refdata-gex-GRCh38-2020-A.tar.gz

Probe Set

  • Description: Probe set file defining gene targets for Visium HD assay.
  • Format: .csv
  • Example File Path: gs://bucket/folder1/probe_set.csv

CytAssist Image

  • Description: High-resolution tissue image captured during CytAssist processing.
  • Format: .tiff
  • Example File Path: gs://bucket/folder1/cytassist_image.tiff

Optional Inputs

Microscope Image

  • Description: High-resolution H&E stained tissue image for enhanced cell segmentation.
  • Format: .tiff
  • Example File Path: gs://bucket/folder1/micr_image.tiff

Samplesheet CSV

  • Description: Metadata file linking sample IDs to their corresponding data files and images.
  • Required Columns: sample_id, sample_folder, cyt_assist_image, microscope_image
  • Format: .csv
  • Example:
sample_id sample_folder cyt_assist_image microscope_image
SAMPLE1_A1 gs://bucket/folder1 gs://bucket/folder1/cytassist_image.tiff gs://bucket/folder1/micr_image.tiff

Outputs

Reported Outputs

  • Processed AnnData File (.h5ad):
  • Description: Quality-controlled spatial transcriptomics data with cell-level resolution, including normalized expression, dimensionality reduction, and spatial coordinates
  • Format: .h5ad
  • Example File Path: /output/directory/SAMPLE1_A1.processed.h5ad
  • Visualization App: scanpy, squidpy, napari

  • Location: Analysis Folder

  • Seurat Object (.rds):

  • Description: R-compatible Seurat object containing spatial transcriptomics data for downstream analysis
  • Format: .rds
  • Example File Path: /output/directory/SAMPLE1_A1.rds
  • Visualization App: Seurat, Loupe Browser

  • Location: Analysis Folder

  • Analysis Report (HTML):

  • Description: Comprehensive HTML report with QC metrics, spatial visualizations, and analysis results
  • Format: .html
  • Example File Path: /output/directory/SAMPLE1_A1.cell_binning_and_qc.html
  • Location: Report Folder

Supporting Outputs

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

  • Example File Path: /output/directory/SAMPLE1_A1_cloupe.cloupe

  • Feature Matrices:

  • Description: Gene expression count matrices at different resolutions (filtered and raw)
  • Format: .h5

  • Example File Path: /output/directory/SAMPLE1_A1_filtered_feature_bc_matrix.h5

  • Spatial Data Directory:

  • Description: Spatial coordinate files and tissue position information
  • Format: Directory with multiple files

  • Example File Path: /output/directory/SAMPLE1_A1_spatial/

  • Web Summary Report:

  • Description: Space Ranger generated summary report with alignment and QC metrics
  • Format: .html
  • Example File Path: /output/directory/SAMPLE1_A1_web_summary.html

Associated Processes

References & Additional Documentation