Xenium In Situ measures gene expression in tissue sections derived from either formalin fixed & paraffin embedded (FFPE) or fresh frozen (FF) tissue samples placed on Xenium slides. Gene expression is measured with panels of probes that target genes of interest.

10x Genomics offers several customization options for Xenium In Situ Gene Expression panel designs. The pre-designed and custom panel types are described below.

• If you have decided to use pre-designed panels, please see the pre-designed panel information and part number pages for Xenium v1 and Xenium Prime 5K options.
• Custom design options are implemented by the Xenium Panel Designer tool. If you have decided to pursue a custom panel for your experiment, please see the Xenium Panel Designer Workflow Guide page for guidance on assay chemistry compatibility and tool workflow steps.

For new Xenium users, 10x Genomics recommends starting a pilot experiment with pre-designed panels. There are several advantages to this approach:

• Pre-designed panels have been extensively tested by 10x for sensitivity and specificity on the specific tissue types.
• Pre-designed panels are in inventory, so researchers can get started right away. No need to wait to design and manufacture a custom panel.
• Pre-designed panels are well-suited for exploratory analyses that identify major cell types in the selected tissue.
• Researchers can ensure their lab is comfortable with the entire workflow, and their first datasets can be compared to 10x Genomics public data demonstrations.
• It is easier to reproduce experiments across labs or research groups.
• If there is any experimental optimization or troubleshooting to be done with the 10x Genomics Support Team, starting with pre-designed panels may help by first addressing any issues unrelated to specific panels.

If you are only using 10x Genomics pre-designed panels, no need to use the Xenium Panel Designer! Learn about pre-designed panels here:

• Xenium v1 panels
• Xenium Prime panels

10x Genomics offers several types of custom panel design:

1. Researchers may be interested in selecting markers for specific subpopulations of cells, or genes expressed under specific conditions (e.g., disease). 10x Genomics supports add-on custom panels (with up to 100 custom genes) to supplement the pre-designed panels on Xenium v1 and Xenium Prime 5K chemistries.
2. Researchers can request standalone custom panel designs for mouse or human that are not dependent on pre-designed panels for Xenium v1 chemistry.
3. If your experimental goals do not align with the above options (e.g., you want to target another species or specific isoforms) please contact your sales representative for information about species standalone (Xenium v1) or advanced custom (Xenium v1, Xenium Prime 5K) options.

Add-on custom panel designs offer the flexibility to include more genes to available pre-designed panels (see Xenium Add-on Panel Design Technical Note). The add-on custom configuration is only compatible with pre-designed panels; it is not compatible with a standalone custom base.

Review the curated pre-designed panel gene lists carefully. The lists are designed to comprehensively cell type a sample. Do the genes adequately cover the biology you are exploring? If not, consider adding additional genes to complement it.

To test a biological hypothesis, researchers may want to select markers for certain subpopulations of cells or genes expressed under specific conditions (e.g., diseased vs. healthy). For example, 10x scientists designed an add-on panel to better understand breast cancer biology in this publication: "High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis". They analyzed data from 313 genes total: 280 genes from common cell types using the pre-designed Xenium Human Breast Panel and 33 additional genes, like invasive myoepithelial marker genes. The add-on genes were selected and curated primarily based on single cell reference data for both healthy and tumorigenic human breast tissues (Pal et al. 2021, Bhat-Nakshatri et al. 2021, Karlsson et al. 2021) (see Supp. Fig. 1).

Standalone custom panels can be designed for human or mouse. Standalone custom panels may be useful when the tissue being analyzed has a very different expression signature compared to the specific tissues covered by pre-designed panels.

We support standalone custom panels up to 480 genes. However, depending on the desired gene list and tissue(s) being analyzed, the final number of genes may be reduced in our design process to ensure high sensitivity across all biology of interest. Thus, we recommend submitting ~10-20 additional genes that could be used in the design process to replace genes in the main set as needed.

Advanced custom panels are designed against species besides human or mouse, or panels designed against non-standard targets (see list below). For information about advanced customizations, please see the Species Standalone Custom and Advanced Custom Panel Design for Xenium In Situ Technical Note (CG000683) for general guidance.

Advanced targets may be included in add-on custom (for human/mouse references only), standalone custom (for human/mouse references only), or species standalone custom design options. Possible advanced targets include:

• SNVs
• Gene isoforms
• Gene fusions
• Viral sequences
• Bacterial sequences
• Fluorescent reporters
• Transgenes
• CRISPR guides
• CDR3 clonotypes
• Protein tags
• Barcodes (e.g. lineage tracing)
• Or other (use this category for requesting targets that are not listed, i.e., xenografts, which we define as adding human genes to mouse or vice versa)