Space Ranger provides the following pipelines for analyzing Visium experiments:

• spaceranger mkfastq demultiplexes raw data from Illumina sequencers into FASTQ files. Alternatively, Illumina's BCL Convert or bcl2fastq can be used. Often, users obtain FASTQ files directly from a sequencing facility, allowing them to skip this step.

• spaceranger count is the main pipeline, compatible with most Visium data, including Visium HD. The pipeline inputs a reference, a microscope slide image, and FASTQ files to generate feature-barcode matrices, identify clusters, and perform differential gene expression.

• spaceranger aggr combines data from multiple samples into an experiment-wide feature-barcode matrix and analysis. Aggregation of Visium HD data is not supported.

Space Ranger uses a microscope slide image as an anatomical reference for visualizing gene expression. It accepts brightfield images, usually H&E-stained, and fluorescence images. Brightfield input involves a single image, whereas fluorescence input includes multiple channels. For detailed guidelines, refer to the Space Ranger Image Recommendations.

In most cases, Space Ranger will automatically align microscope images with CytAssist images and expression data captured on the Visium slide. For Visium v1 experiments with fluorescence images, or for cases where automatic alignment fails, Loupe Browser can be used to manually align the data layers.

Space Ranger requires an annotated reference transcriptome. 10x Genomics offers pre-built references to download for human (GRCh38) and mouse (mm10). Alternatively, users can create a custom reference. Visium probe-based assays also use probe sets targeting protein coding genes in the human and mouse transcriptomes. Probe sets are bundled with the Space Ranger software, but can also be downloaded directly.

Libraries supported by different versions of Space Ranger are summarized below.