10x Genomics Support/Visium CytAssist Spatial Gene Expression/Documentation/Probe Sets/

Visium Probe Sets Overview

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

Probe setCompatible Assay
Human Probe Set v2Visium HD Spatial Gene Expression
Visium CytAssist Spatial Gene and Protein Expression
Visium CytAssist Spatial Gene Expression (FFPE, Fresh Frozen)
Human Probe Set v1Visium Spatial Gene Expression for FFPE
Mouse Probe Set v2Visium HD Spatial Gene Expression
Mouse Probe Set v1Visium CytAssist Spatial Gene Expression (FFPE, Fresh Frozen, Fixed Frozen)
Visium Spatial Gene Expression for FFPE

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 probe set?

Below are the genes excluded from the human probe set:

  • TCR joining and 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

2. Which genes have 1-fold coverage versus 3-fold coverage in the v2 human probe set?

Most genes have 3-fold coverage. 7.5% genes have 1-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. About 5% 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.

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. There are about 12k out of 55k probes that span splice junctions to different extents and these probes 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. Note that most genes have multiple probes (3-fold coverage, see question 2), and not all probes for one gene will cover the splice junction.

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

We are not supporting non-human species at this time. 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 cannot be guaranteed for other species.

5. Can custom probes be used with Visium Spatial Gene Expression for FFPE or Visium CytAssist Spatial Gene Expression assays?

Refer to the Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex Technical Note for guidance.

Document Type

Last Modified
March 20, 2024