Table of contents

• Panel design overview
• Xenium Panel Designer
• Standard workflow: Generate a standalone or add-on custom panel with Xenium Panel Designer
• Advanced workflow: Request species standalone or advanced custom panels
• Panel design input information
• Review recommendations
• Interpreting your panel design summary
• Finalize panel design
• Using the custom panel
• Acknowledgements

Panel design overview

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. The following types of panels are available:

1. 10x Genomics pre-designed Xenium panels are designed to cover all major cell types found in a tissue for specific species and tissue types (e.g., human brain). If you are only using 10x Genomics pre-designed panels, no need to use the Xenium Panel Designer! Learn more here: Xenium v1 panels or Xenium Prime panels.
2. Researchers may also 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.
3. Researchers can request standalone custom panel designs for mouse or human that are not dependent on pre-designed panels.
4. 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 or advanced custom options.

The table below summarizes the custom panel design options that the Xenium Panel Designer tool enables by Xenium In Situ Gene Expression assay chemistry.

• "Xenium v1" refers to experiments customizing v1 panels and following either the Xenium In Situ Gene Expression - Probe Hybridization, Ligation & Amplification User Guide (CG000582) or the Xenium In Situ Gene Expression with Cell Segmentation Staining User Guide (CG000749).
• "Xenium Prime" refers to experiments customizing Xenium Prime panels and following the Xenium Prime In Situ Gene Expression with optional Cell Segmentation Staining User Guide (CG000760).

Xenium Panel Designer

The following sections walk you through the design process using the Xenium Panel Designer on the 10x Genomics Cloud (available globally). First-time users of the 10x Cloud will be asked to create a new account. At any point during the design process, you can save ("Finish later") and resume work on the panel design later if needed. We provide examples of common design workflows here.

Click "Start a New Panel Request" to begin.

Standard workflow: Generate a standalone or add-on custom panel with Xenium Panel Designer

You can finalize designs on your own in the Xenium Panel Designer if you are designing for a standalone custom gene panel or an add-on custom gene panel that complements a 10x pre-designed panel for:

• Human or mouse
• Standard genes (defined as genes in the human GRCh38 or mouse GRCm38 transcriptome references)

Follow the design tool steps to add sample and input information. Click on the links to learn more about each step in the Xenium Panel Designer:

1. Add sample information
2. Select panel type
3. Provide reference
4. Provide genes
5. Once the input steps are completed, review recommendations provided by the design tool for your panel design.
6. The panel design summary will display suggestions for optimizing the design. When you are satisfied with the results, finalize the design.

Advanced workflow: Request species standalone or advanced custom panels

The Xenium Panel Designer tool enables you to submit a request for species standalone or advanced custom panel designs. Start by contacting your sales representative to order an Advanced Panel upgrade and generate a unique design ID to track your panel through the design process. If you do not know your sales representative, please contact [email protected]. Our 10x Support teams will work with you to review and finalize the panel design; you will not be able to finalize these panel design types within the Xenium Panel Designer on your own. These designs include:

• Non-human / non-mouse species (including multi-species samples)
• Advanced custom targets: SNVs, gene isoforms, gene fusions, viral sequences, bacterial sequences, fluorescent reporters, transgenes, CRISPR guides, CDR3 clonotypes, protein tags, barcodes (e.g. lineage tracing), 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)

Follow the design tool steps to add sample and input information. Click on the links to learn more about each step:

1. In the Xenium Panel Designer:

   • Add sample information. Enter your design ID and select the "Other (including multi-species samples)" (for Xenium v1 panels only) and/or "Advanced targets" options.
     The selections above ensure that your panel design request is directed to our 10x support team. Do not use the Standard workflow for advanced custom submissions.
   • Select panel type
   • Provide reference
   • Provide genes
   • Optional: Provide RNA-binding domain information. This step only applies to Xenium v1 species standalone custom or advanced custom panel designs.

2. After you submit the request in the design tool, our support team will be notified. Provide advanced custom sequence information to your 10x bioinformatics representative.

3. Next, 10x bioinformaticians will share and discuss the panel design summary results with you to fine-tune custom designs before they are finalized. A few rounds of review and discussion are typically required to finalize a high quality panel.

4. When you are satisfied with the results, 10x support will finalize the design. You will be able to go to the Xenium Panel Designer tool entry associated with your request to view the final design and download output files.

Panel design input information

The following inputs are part of the design process for both designing your own custom panel or submitting a panel request workflows.

1. Select panel type

Select either the pre-designed panel you wish to build upon with add-on custom genes or standalone custom panel, as well as the number of custom genes you would like to include in your custom panel.

2. Provide reference: single cell reference data and cell type annotation format

You have three options to specify up to five reference datasets:

1. Select from a collection of publicly available reference datasets with similar tissue type and condition to the samples you plan to run from our provided list of pre-built references.
2. Upload your own annotated single cell gene expression data in HDF5, MEX, or CLOUPE format. See Creating Single Cell References for Xenium Custom Panel Design from Seurat or AnnData for additional guidance on reference file format conversion.
3. Choose a combination of public reference datasets and your own single cell reference datasets.

The design tool uses single cell data to create a model of the expression profiles of the cell types present in your samples. It then uses this model to evaluate the risk of optical crowding, check for highly expressed genes, and assign codewords to the genes you selected in an optimal fashion. Here are important considerations for panel design and sample preparation that can affect detection budget:

• Mismatched panels: It is important that panels are designed and used with tissues that are representative of the reference data included in the panel design. More than one reference can be used in custom panel designs to represent the diversity of samples that may be studied using a single panel. Detection budget may be exceeded if a panel designed for one tissue is used on another with relatively higher expression in certain genes or cell types (i.e., healthy vs. tumor tissue). This affect may be prevented by including a tumor reference in panel design.
• High utilization panels: Panels that are designed with very high utilization are more likely to exceed the detection budget described in the Xenium Add-on Panel Design Tech Note. In addition, panels that are designed using reference data that are not representative of the samples being analyzed may be designed with higher than calculated utilization. It is important to review recommendations during custom panel design to avoid panels with utilization over the detection budget.
• Abnormally large tissue thicknesses: We recommend a section thickness of 5 µm for FFPE and 10 µm for fresh frozen as described in Xenium In Situ for FFPE - Tissue Preparation Guide and Xenium In Situ for Fresh Frozen Tissues - Tissue Preparation Guide. Sections that are thicker than this will be more likely to exceed the detection budget.

The single cell reference must be accompanied by cell type annotations for the barcodes. In the design process, the expression levels are aggregated across each cell type. This information is used to assign codewords that minimize optical crowding, as well as ensure that cell type clusters match the broad, expected categories. See general guidelines for panel gene selection in the Xenium Add-On Panel Design Technical Note.

The information the design tool needs is a gene list and a measure of expected gene expression in the sample stratified by cell type.

If providing your own reference data, the Xenium Panel Designer will accept one of the following formats:

• One or more unnormalized whole transcriptome filtered feature-barcode matrices with cell type annotations for each matrix. The matrix and annotation files should be bundled as a .zip, .tar, or tar.gz file (one matrix + one annotation file per bundle).

  • The feature-barcode matrix can be in either Cell Ranger HDF5 or Matrix Exchange (MEX) format. The HDF5 matrix is a single file, while the MEX format is a folder containing three files (matrix.mtx.gz, barcodes.tsv.gz, features.tsv.gz).
  • The cell type annotations file can be in CSV or TSV format. It is a two-column file and headers are required. The first column must be "barcode". For example:

  Copy
  barcode,annotation
  ATGCATTGCGTAAGTG-1,fibroblast
  TTGCAAAGCCGAAGTG-1,fibroblast
  CATCATTGCGTAATTG-1,T cell
  ...
  

  Important

  It is critical that barcode suffixes and prefixes in the annotations file exactly match those for barcodes in the matrix file.

• A single uncompressed CLOUPE file generated by Cell Ranger. The Xenium Panel Designer uses the graph-based clustering results for cell annotations, so no additional annotation file is needed for this input format.

If looking for rare cell types, providing matrix files for multiple samples may yield better results. We recommend providing a matrix file per sample; it does not need to be aggregated. If multiple matrices are provided, the cell type information across all of the matrices will be evaluated.

An error message will be shown for these input file issues:

• Gene IDs and/or gene symbols do not match between the matrix, gene list, and the 2020-A reference.
• Gene names contain spaces/blanks in the gene name or have typos.
• Files have missing column headers or headers with unexpected or misspelled names.
• Matrix and annotation CSV files do not have exactly the same barcodes. This is often seen when barcodes in the annotation file have an extra sample suffix after aggregation, but the matrix itself does not.

Common input file issues that do not halt panel design but give poor results:

• The design tool will not error with normalized counts data, but results will be skewed. The design tool should be used with integer counts data.
• The design tool will not error with matrix files that filter many genes, but results will be skewed and consequently generate a suboptimal design.
• Matrix files that are missing genes in the gene list.
• Poorly matched expression data.
• Annotation CSV files where the first two columns are not "barcode,annotation". If hierarchical annotations are present in additional columns, they are ignored.

3. Provide genes

The Xenium Panel Designer will check your gene list and provide feedback if any problems or ambiguous genes are encountered for human or mouse samples. You can modify the list based on the feedback before moving to the next step.

Enter a list of gene names and/or Ensembl IDs. Sort the list from most to least important to help us prioritize genes to retain for the final design.

• For add-on custom panels, up to 100 genes can be added to a 10x pre-designed panel. The add-on panel list should not include genes that are already in the selected pre-designed panel.
• For standalone custom panels, up to 480 genes are supported.
• Recommended for any panel type: submit ~10-20 additional genes in case any in the main set need to be replaced during the design process.

For Xenium v1 panels, 10x Genomics can design probes for these human and mouse genes, which cover:

• 96.8% of the human transcriptome and 99.7% of the human coding transcriptome
• 85.4% of the mouse transcriptome and 99.8% of the mouse coding transcriptome

For Xenium Prime panels, 10x Genomics can design probes for these human and mouse genes, which cover:

• 99.5% of the human transcriptome and 99.4% of the human coding transcriptome
• 99.7% of the mouse transcriptome and 99.6% of the mouse coding transcriptome

The files below list the genes that are not covered by our existing probes per assay chemistry based on their respective human or mouse 2020-A reference transcriptomes (gene name, Ensembl ID, and gene type):

• Xenium v1: Human genes, Mouse genes
• Xenium Prime: Human genes, Mouse genes

It is important that the gene symbols and feature IDs match the features in the human or mouse 2020-A reference GTF file exactly. If provided, the Ensembl IDs should not include "version decimals" (e.g., "ENSG00000010404" is ok, while "ENSG00000010404.3" is not). The Ensembl IDs must be the gene ID (ENSG, ENSMUSG) not the transcript ID.

Review recommendations

After the input information is submitted, the Xenium Panel Designer will generate a custom panel for you. The page will update as soon as the design is complete (typically ~5 minutes).

During the process, the design tool:

• Screens the customer-provided gene list for genes that cannot have probes designed for them (given the reference data and maximum number of probe sets). It may also screen for low-probe targets before running the design tool. The designer will explicitly check for the following:
• Genes whose expression is predicted to be too high to assay using Xenium technology
• Genes that we cannot design probes for
• Combinations of probes that are predicted to cause issues
• Genes that are moderately expressed in many cell types and are unlikely to be beneficial in your panel
• Genes that we have previously assayed for and pose issues (in sensitivity, specificity, etc.)
• Creates a cell type model based on the reference data (customer-submitted or in-house).
• Generates the panel design summary to assess the design. The file includes a panel summary with panel metadata and plots to assess the design. If the tool detects any potential issues, alerts or warnings are also shown.

Interpreting your panel design summary

Next, review the panel design summary HTML file to determine whether to further modify or move forward with the current panel design. Plot interpretation guidance is available here.

Finalize panel design

You may edit the panel based on results in the panel design summary as many times as needed. When you are satisfied with the design, select the relevant option to finalize the design on the Review Design Recommendations page (10x support will do this step for the advanced workflow option).

For the standard workflow option, a 6-digit design ID will be generated. The design ID is used to order your first panel, as well as reorders of the same panel in the future. The output files associated with your panel will be generated for download.

On the finalized panel design screen, you can:

1. Contact your sales representative with the design ID to request a quote to start the panel ordering process.
2. Learn what is included in your panel(s) order
3. Upload the panel JSON file to the Xenium Analyzer instrument
4. Download panel design output files (see table below)

Using the custom panel

For add-on custom and standalone custom panel designs, after shipment has been initiated, your order will include a gene panel file (JSON format). You can download the finalized panel design JSON file from the Xenium Panel Designer.

The JSON file should be uploaded to the Xenium Analyzer instrument by inserting a USB into the Xenium Analysis Computer USB port (exFAT format). The Xenium Analyzer User Guide details the steps to start an instrument run.

Acknowledgements

Xenium Panel Designer leverages NUPACK in the panel design process, which was developed in part with funding from NSF and NIH.

Next Steps

• Return to panel design landing page
• Understanding Xenium Onboard Analysis outputs
• Getting Started with Xenium Explorer
• Continuing your journey after Xenium Analyzer