Flex assays use probes that target protein-coding genes in the human, mouse, or rat transcriptome. Each probe consists of a pair of oligonucleotides that hybridize to the targeted transcript and are subsequently ligated.

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

Frequently asked questions

1. Which probe set file version should I use for NextGEM or GEM-X Flex data?

• For NextGEM Flex v1, use the v1.1 probe sets.
• For GEM-X Flex v1, use the v1.1 probe sets.
• For GEM-X Flex v2, use the v2.0 probe sets.

2. Can the rat probe set be used with the Flex v2 Singleplex workflow?

No. Unlike the human and mouse probe sets, the rat probe set is only compatible with the Flex v2 Multiplex workflow and cannot be used with the Singleplex workflow. More details on library construction here.

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

The excluded genes are:

• TCR joining and majority of variable regions^*
• IG joining and variable regions
• Ribosomal proteins
• Mitochondrial ribosomal proteins
• Read-through 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.

4. Which genes have 1-fold coverage versus 3-fold coverage in the probe set?

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.

5. 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.

The region column of the probe set reference CSV file also indicates whether a probe spans a splice junction by at least 10 bp (spliced) or not (unspliced). 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.

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 Cell Ranger you are using. Please refer to the table below for specific probe set and transcriptome reference compatibility.

7. 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 cannot be guaranteed for other species.

8. 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 per-cell sensitivity compared to the species-matched rat probe set.

• For new projects: We strongly recommend using Flex v2/Apex with 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.

9. Can custom probes be used with Flex assays?

Refer to the Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex Technical Note for general guidance on designing and ordering custom probes.

These Knowledge Base articles provide additional help for designing custom probes.