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# What is Space Ranger?

Space Ranger is a set of analysis pipelines that process Visium spatial RNA-seq output and brightfield microscope images in order to detect tissue, align reads, generate feature-spot matrices, perform clustering and gene expression analysis, and place spots in spatial context on the slide image. Space Ranger includes two pipelines relevant to spatial gene expression experiments:

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

• spaceranger count takes a bright field 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.

These pipelines combine Visium-specific algorithms with the widely used RNA-seq aligner STAR. Output is delivered in standard BAM, MEX, CSV, HDF5, TIFF, PNG, JPEG and HTML formats that are augmented with spatial information.

## Automatic and Manual Image Processing Workflows

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

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

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

These image processing tasks are described further in the Image Processing section.

The automated alignment and tissue identification processes require images taken with proper tissue placement and image exposure. Samples where these conditions cannot be met can still 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 as input in order to prepare data for visualzation within Loupe Browser. Note that both Loupe and Space Ranger must be given the same image for a given sample.