Before you begin your Space Ranger run, you will need to prepare your input files. These files will vary depending on the parameters of your experiment, the assay chosen, and which Space Ranger pipeline you are using.
- Visit the Download Center to download the following:
- Pre-built references are available for human and mouse. Alternatively, you can create a custom reference.
- Probe sets are necessary for probe-based assays, including Visium HD and Visium v2 (CytAssist-enabled assays). Probe sets come bundled with the Space Ranger pipeline, but are also offered for convenience here.
- Slide layout files must be downloaded if the machine you are running Space Ranger on is not connected to the internet. You will need to know the slide serial number to retrieve the correct file.
- How are you generating your FASTQ files? If you are starting with BCL files, you can use spaceranger mkfastq, or you can demultiplex directly with Illumina software. If you already have FASTQ files, but are unsure how to specify them to
spaceranger count, visit Specifying Input FASTQs. - Space Ranger requires a microscope image (brightfield or fluorescence) to use as an anatomical map for expression data. CytAssist-enabled workflows also have a CytAssist image. Consider the image inputs that are required for your use case.
- Space Ranger requires the slide ID and capture area. If you are providing a CytAssist image, this information can be retrieved automatically from the image metadata, and no further inputs are required. The
--slideand--areaparameters are required when using--slidefile, which in turn is required when the pipeline is not connected to the internet. For more information, see the Slide Parameters page. - Manual alignment is necessary for fluorescence images (Visium v1, direct placement), for cases where the fiducial markers are obstructed or tissue boundaries are unclear, or when automatic alignment fails. Specify the resulting JSON file to
spaceranger countwith the--loupe-alignmentoption. - To analyze Protein Expression data (not supported with Visium HD), you must set up a Feature Reference CSV following these instructions.
- The
spaceranger aggrpipeline requires a CSV file that specifies paths to themolecule_info.h5files (output fromspaceranger count) representing the libraries you wish to aggregate. Visium HD is not supported inspaceranger aggr.