A Complete Guide to 10x Xenium Probe Customization

10x Xenium is a fluorescence in situ hybridization–based spatial imaging platform. Currently, 10x offers 11 predesigned probe panels, divided into two categories: Xenium V1 and Xenium Prime 5K. Both allow researchers to add up to 100 custom genes on top of the predesigned panels, enabling flexibility for specialized applications.

For more complex studies, 10x Genomics also offers a fully custom probe design service that supports up to 480 genes, including non-human and non-mouse species. This is especially valuable in comparative biology, veterinary studies, plant research, or cross-species immunology.

While 10x provides the Xenium Panel Designer software to streamline design, the learning curve can be challenging. Below we break down each step—from data preparation, to probe design, to final order submission—so you can avoid common pitfalls and achieve successful outcomes.

Figure 1 Schematic illustration of Xenium probes (source: 10x Genomics) 

1. Two Probe Design Workflows

Depending on your species and experimental design, 10x Genomics supports two distinct workflows:

1.1 Standard Workflow

• Who it’s for:

          Extending an existing V1 or Prime 5K panel with up to 100 extra genes.

          Designing custom probes (≤480 genes) for human or mouse reference panels.

• Process:

        Use Xenium Panel Designer to upload data and gene lists.

        Generate a Design ID

        Place the order through your distributor.

1.2 Advanced Workflow

• Who it’s for:

           Non-human and non-mouse species.

           Cross-species or mixed-species samples.

           Highly specific needs (e.g., SNVs, allelic variants, gene fusions, viral sequences, CRISPR guides, fluorescent proteins, protein tags, CDR3 clonotypes).

• Process:

         Contact a 10x sales representative.

         Collaborate with the 10x bioinformatics team to refine design.

         Submit via Panel Designer once the requirements are confirmed.

2. Preparing Data for Probe Design

The quality of your input single-cell data is critical. Xenium Panel Designer relies on annotated single-cell expression profiles to identify highly expressed genes that may cause optical crowding (strong fluorescent signals masking weaker ones).

2.1 Accepted Input Formats

• CLOUPE file

        Generated by Cell Ranger.

        Contains clustering and annotations.

        No separate annotation file required.

• HDF5 or MEX matrix

         Must be unnormalized, unfiltered, with cell barcodes.

         Requires an annotation file (CSV/TSV).

         Package as .zip, .tar, or .tar.gz.

2.2 Annotation File Requirements

• Two-column format with headers:

• File must align perfectly with barcodes in the matrix.

•  Inconsistent suffixes or prefixes between matrix and annotation files often cause upload errors.

2.3 File Size

• Recommended: <500 MB per dataset.

• If >500 MB, reduce the number of cells or samples before upload.

2.4 Using Public Data

• If you don’t have your own dataset, 10x provides publicly available single-cell datasets.

• You may combine public and proprietary datasets to strengthen design.

3. Preparing the Gene List

The gene list is the foundation of customization.

3.1 Accepted Formats

• Gene symbols or Ensembl IDs.

• Optional probe group number.

Examples:

         EGFR

         ENSG00000146648

         EGFR,5

         ENSG00000146648,5

         EGFR, ENSG00000146648,7

         ENSG00000146648, EGFR,7

3.2 Strict Formatting Rules

• Gene IDs must match the human/mouse 2020-A reference GTF.

• Do not include transcript IDs.

• Do not include Ensembl decimal versions (e.g., ENSG0000010404.3 ❌).

  
  

3.3 Gene List Strategy

• Sort genes from most important to least important.

• Provide 10–20 backup genes in case replacements are required.

• When adding probes to an existing panel, avoid duplicating genes already present.

4.Designing Probes with Xenium Panel Designer

4.1 Access

• Register an account on the 10x Genomics.

• Log in to launch the Xenium Panel Designer tool.

4.2 Submission Workflow

1. Select the panel type (V1, Prime 5K, or custom).

2. Upload single-cell data.

3. Upload the gene list.

4. Generate a panel summary with design warnings.

4.3 Common Error Messages

• Mismatched gene IDs between gene list and 2020-A reference.

• Typos or spaces in gene names.

• Missing column headers in annotation files.

• Barcode mismatches (annotation suffixes not matching matrix barcodes).

   

4.4 Reviewing the Summary

• The tool may flag:

         High-expression genes → risk of optical crowding.

         Insufficient probe groups → poor detection.

         Low cell-type resolution → clustering issues.

• Adjust gene list and re-run until warnings are cleared.

4.5 Final Submission

• Once satisfied, click Finish to generate a Design ID.

• Provide this ID to your 10x distributor to complete the purchase.

Why Collaborate with Omics Empower?

At Omics Empower, we specialize in delivering end-to-end Xenium services, supporting researchers from sample processing through to advanced data analysis.

• Extensive project experience: Over 20,000 processed samples across neuroscience, oncology, immunology, developmental biology, plant sciences, and agriculture.

• Proven research impact: More than 370 publications by our partners in high-impact journals such as Nature, Cell, and the European Heart Journal.

• Comprehensive Xenium expertise: Optimized workflows across multiple Xenium platforms, ensuring reliable performance for both human/mouse and non-human projects.

• Global laboratory network: Facilities in Europe, Asia, and the United States provide consistent service standards, local project support, and faster turnaround times.

• Advanced bioinformatics: Dedicated teams offering end-to-end spatial data processing, integrative analysis, and expert interpretation.

Get in touch with us today to discuss your project and see how Omics Empower can accelerate your next spatial discovery.