Visium probe-based assays target protein-coding genes in the human, mouse, or rat transcriptomes. Each probe consists of a pair of oligonucleotides hybridized to the targeted transcript and are subsequently ligated.

Gene IDs and probe sequences are defined in the probe set reference CSV input file for spaceranger count. Three supporting files are provided: a BED file listing the reference genome coordinates for each probe, a CSV file listing probes with predicted off-target activity, which are excluded from analysis by default, and a TSV file that contains gene name and description for each of the probes. These files are described in detail on the Probe Set Files Description page.

Frequently asked questions

1. What genes are excluded from the human, mouse, and rat probe sets?

Below are the genes excluded:

• TCR joining and majority of variable regions^*
• IG joining and variable regions
• Ribosomal proteins
• Mitochondrial ribosomal proteins
• Readthrough genes
• KIR and HLA genes (due to their inherent allelic diversity)
• Non-coding RNA

^* Refer this Knowledge Base Article for details about variable region genes included in the rat probe set.

2. Which genes have 1-fold coverage versus 3-fold coverage in the probe sets?

Most genes have 3-fold coverage. The genes with 1-fold coverage include highly expressed genes and mitochondrial protein coding genes. This design dampens the reads on highest expression genes. A very small fraction of genes with specificity issues during probe design have 1 to 2-fold coverage.

• Human probe set: ~7.5% genes have 1-fold coverage. ~5% of genes have more than three probes to cover all possible isoforms.
• Mouse probe set: ~6.6% genes have 1-fold coverage. ~1.7% of genes have more than three probes to cover all possible isoforms.
• Rat probe set: ~8.1% of genes have 1-fold coverage. ~1.0% of genes have more than three probes to cover all possible isoforms.

The information on the coverage for genes can be found in the probe set metadata TSV file. Learn more about fold coverage in this Knowledge Base article.

3. Are these probes expected to work equally well on pre-spliced mRNA from nuclei?

Most of the probes do not overlap splice junctions, so they should work well with pre-spliced mRNA from nuclei.

• Human probe set: ~14% of 54k probes span a splice junction by at least 10 bp and would be less likely to detect pre-mRNA.
• Mouse probe set: ~16% of 55k probes span a splice junction by at least 10 bp and would be less likely to detect pre-mRNA.
• Rat probe set: ~13% of 62k probes span a splice junction by at least 10 bp and would be less likely to detect pre-mRNA.

Users can find out whether a probe spans a splice junction by looking at the probe set BED file. If the value in the 10th column (blockCount) is more than 1, it means the probe spans a splice junction.

4. Would the human probe sequences work on non-human primate samples?

The human probe sets are designed based on the GENCODE basic transcript annotations for human and validated with human samples. We have not tested the human probe set with samples from other species (including primates), and the performance of the human probe set for other species is unknown.

5. Can the mouse probe set be used on rat samples?

No. For optimal performance and accurate results, the officially supported rat probe set must be used on rat samples. Sequence divergence between the two genomes substantially reduces probe hybridization efficiency. Using a mouse probe set on rat tissue results in reduced sensitivity compared to the species-matched rat probe set.

• For new projects: We strongly recommend using the rat probe set to ensure maximum sensitivity and data integrity.
• For ongoing projects: For data comparisons within a single study, do not mix probe sets. Data generated using mouse probe sets cannot be directly compared to data generated with the rat probe set due to these differences in sensitivity.

6. Can I use any probe set with any transcriptome reference?

No, not all probe sets are compatible with every transcriptome reference. Compatibility depends on the version of Space Ranger you are using. Please refer to the table below for specific probe set and transcriptome reference compatibility.

Space Ranger versions are backward compatible with earlier probe set file formats.

7. What changed in probe set v2.1 compared to v2.0?

• The v2.1 human and mouse probe sets use the 2024 transcriptome reference builds: GRCh38-2024-A (human) and GRCm39-2024-A (mouse).
• The v2.0 human and mouse probe sets use the 2020 reference builds: GRCh38-2020-A (human) and mm10-2020-A (mouse).
• The v2.1 probe sets are available for human and mouse only. See the compatibility table above for the Space Ranger versions that support each probe set.

8. What changed in probe set v2.0 compared to v1.0?

• v2.0 adds a rat probe set, built on the mRatBN7.2-2024-A reference (the transcriptome reference is optional for analysis). The v1.0 probe sets cover human and mouse only.
• The human and mouse v2.0 probe sets use the same 2020 reference builds as v1.0 (GRCh38-2020-A and mm10-2020-A).
• Refer to the assay compatibility table at the top of this page for the assays supported by each probe set version.

9. What is the difference between the probe set version and the probe set file format version?

These are two independent version numbers, and they should not be confused:

• The probe set version (for example, v1.0, v2.0, or v2.1) identifies the content of the probe set — the probes, the genes they target, and the reference build used to design them. This is the version referenced throughout this page and the one you use to select the correct probe set for your assay.
• The probe_set_file_format version is a separate value found in the header of the probe set reference CSV (the #probe_set_file_format= line). It specifies only which version of the CSV file format specification the file conforms to, not the biological content of the probe set.

The two numbers are unrelated and do not track each other. When selecting or citing a probe set, always use the probe set version, not the file format version.

For more details, see the Probe Set Reference CSV and Supporting Files page.

10. Are Visium and Chromium Flex probe sets interchangeable?

No. Visium and Chromium Flex probe reagents and kits are workflow-specific and must not be substituted across assays. Always use the platform-specific probe set CSV downloaded from the corresponding Space Ranger (Visium) or Cell Ranger (Flex) page, even where the underlying probe content is equivalent.

11. Can custom probes be used with Visium assays?

Refer to the Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex Technical Note for guidance. This Technical Note applies to the Visium Spatial Gene Expression for FFPE (Visium v1), Visium CytAssist Spatial Gene Expression (Visium v2), and Visium HD Spatial Gene Expression assays.