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# What is Space Ranger?

Visium Spatial Gene Expression is a next-generation molecular profiling solution for classifying tissue based on total mRNA. Space Ranger is a set of analysis pipelines that process Visium Spatial Gene Expression data with brightfield and fluorescence microscope images. Space Ranger allows users to map the whole transcriptome in formalin fixed paraffin embedded (FFPE) and fresh-frozen tissues to discover novel insights into normal development, disease pathology, and clinical translational research.

Space Ranger includes five pipelines relevant to Visium Spatial Gene Expression experiments:

• spaceranger mkfastq wraps Illumina's bcl2fastq to demultiplex Visium-prepared sequencing runs and to convert barcode and read data to FASTQ files.

• spaceranger count takes a microscope slide image and FASTQ files from spaceranger mkfastq and performs alignment, tissue detection, fiducial detection, and barcode/UMI counting. The pipeline uses the Visium spatial barcodes to generate feature-spot matrices, determine clusters, and perform gene expression analysis. In addition to fresh-frozen tissues, spaceranger count is also used for FFPE and Targeted Gene Expression libraries. The pipeline uses the RNA-seq aligner STAR for fresh-frozen samples or a probe aligner algorithm for FFPE tissues. Outputs are delivered in BAM, MEX, CSV, HDF5, TIFF, PNG, JPEG and HTML formats.

• spaceranger aggr takes the output of multiple runs of spaceranger count from related samples and aggregates their output, normalizing those runs to the same sequencing depth, and then recomputing the feature-barcode matrices and the analysis on the combined data. The aggr pipeline combines data from multiple samples into an experiment-wide feature-barcode matrix and analysis. This pipeline is supported for both fresh-frozen and FFPE tissues, although the aggregated libraries must be of the same type.

• spaceranger targeted-compare compares a starting input library, referred to as the parent library, to its corresponding Targeted Gene Expression dataset. spaceranger targeted-compare can be used to assess targeting performance with greater accuracy than when only the targeted data are known. Quality control metrics are provided to verify the extent to which targeted genes were enriched and the parent sample data were recovered. This pipeline is only supported for fresh-frozen tissues.

• spaceranger targeted-depth summarizes a whole transcriptome analysis (WTA) dataset in the context of a hypothetical Targeted Gene Expression experiment. Given an existing WTA dataset and a target panel CSV file, spaceranger targeted-depth computes the fraction of reads mapped to targeted genes from the panel. This pipeline is only supported for fresh-frozen tissues.

## Brightfield and Fluorescence Imaging

Space Ranger takes a slide image as input to be used as an anatomical map on which gene expression measures are visualized. Space Ranger can take two styles of images: either a brightfield image stained with hematoxylin and eosin (H&E) with dark tissue on a light background or a fluorescence image with bright signal on a dark background. While brightfield input comprises a single image, fluorescence input comprises one or more channels of information generated by separate excitations of the sample.

## Automatic and Manual Image Processing Workflows

Space Ranger combines algorithms for processing Visium slide images with the robust gene expression analysis used in Space Ranger.

The spaceranger count pipeline performs the following automated image processing tasks:

• Alignment of the slide's barcoded spot pattern to the input slide image. (brightfield only)
• Discrimination between tissue and background in the slide image. (brightfield only)
• Preparation of the full-resolution slide image for visualization in Loupe Browser.

These image processing tasks are described further in the Image Processing section. We encourage you to read Input Recommendations for imaging and sequencing suggestions prior to data collection.

The automated alignment and tissue identification processes require images taken with proper tissue placement and image exposure and currently only works for brightfield images. Samples with fluorescence images or brightfield images where these conditions cannot be met can be processed by using Loupe Browser to manually align the spot pattern and identify tissue in the slide image. When this "manual workflow" is chosen, the spaceranger count pipeline will still take the full resolution image or images as input in order to prepare data for visualzation within Loupe Browser. Note that both the Loupe Manual Aligner and Space Ranger must be given the same set of images for a given sample.