Space Ranger provides the following pipelines:

• spaceranger count is the main pipeline, compatible with most Visium data, including Visium HD and Visium HD 3'. Expected pipeline inputs are a reference, a microscope slide image, and FASTQ files to generate feature-barcode matrices, identify clusters, and perform differential gene expression. Cell segmentation for Visium HD data is included in Space Ranger v4.0 and later.

• spaceranger segment runs Space Ranger's nucleus segmentation algorithm on an H&E image. You can use this pipeline to evaluate segmentation performance on specific tissues before running the Visium HD assay.

• 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 v4.0 and later supports automatic nucleus and cell segmentation in the spaceranger count pipeline for Visium HD and Visium HD 3' Spatial Gene Expression data with corresponding H&E microscopy images. Users can also use the spaceranger segment pipeline to run nucleus segmentation on an H&E image prior to running the Visium HD or Visium HD 3' assays.

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.