What is Space Ranger?

Space Ranger is a set of analysis pipelines that process Visium spatial RNA-seq output and brightfield and fluorescence 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 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.

• 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.

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.

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.