Space Ranger Processing

Processing Visium spatial transcriptomics data

Overview

Space Ranger is the 10x Genomics pipeline for processing Visium Spatial Gene Expression data. It performs alignment, tissue detection, fiducial alignment, and barcode/UMI counting.

Download Reference

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# Human reference
curl -O https://cf.10xgenomics.com/supp/spatial-exp/refdata-gex-GRCh38-2024-A.tar.gz
tar -xzf refdata-gex-GRCh38-2024-A.tar.gz

# Mouse reference
curl -O https://cf.10xgenomics.com/supp/spatial-exp/refdata-gex-GRCm39-2024-A.tar.gz

Run Space Ranger Count

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spaceranger count \
    --id=sample_1 \
    --transcriptome=/path/to/refdata-gex-GRCh38-2024-A \
    --fastqs=/path/to/fastqs \
    --sample=Sample1 \
    --image=/path/to/image.tif \
    --slide=V19J01-001 \
    --area=A1 \
    --localcores=16 \
    --localmem=64

Parameters

Parameter	Description
`--image`	High-resolution tissue image (TIFF or JPEG)
`--slide`	Slide serial number
`--area`	Capture area (A1, B1, C1, D1)
`--cytaimage`	(Optional) CytAssist image for enhanced alignment

Output Structure

sample_1/
├── outs/
│   ├── filtered_feature_bc_matrix/
│   ├── raw_feature_bc_matrix/
│   ├── spatial/
│   │   ├── aligned_fiducials.jpg
│   │   ├── detected_tissue_image.jpg
│   │   ├── scalefactors_json.json
│   │   ├── tissue_hires_image.png
│   │   ├── tissue_lowres_image.png
│   │   └── tissue_positions.csv
│   ├── web_summary.html
│   └── cloupe.cloupe

Quality Metrics

Key metrics from web_summary.html:

Metric	Expected	Concern
Spots under tissue	1,000-5,000	<500
Mean reads/spot	>25,000	<10,000
Median genes/spot	>2,000	<500
Sequencing saturation	>50%	<30%

Image Alignment Issues

If tissue detection is poor: - Ensure image is properly oriented - Check slide serial number and area - Try manual alignment in Loupe Browser

Next Steps

With Space Ranger complete, proceed to Visium Analysis in R/Python.