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.
| Probe set | Compatible Assay |
|---|---|
| Human Probe Set v2 | Visium HD Spatial Gene Expression Visium CytAssist Spatial Gene and Protein Expression Visium CytAssist Spatial Gene Expression |
| Human Probe Set v1 | Visium Spatial Gene Expression for FFPE |
| Mouse Probe Set v2 | Visium HD Spatial Gene Expression |
| Mouse Probe Set v1 | Visium CytAssist Spatial Gene Expression Visium Spatial Gene Expression for FFPE |
| Rat Probe Set v2 | Visium HD Spatial Gene Expression |
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. 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.
7. 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.