Question: Why can’t we design Xenium probes or enough probes for certain genes?
Answer:
Generally, Xenium v1 panels are designed to include up to 8 probe sets per gene. For Xenium Prime panels, the base panel usually includes around 3 probe sets per gene, and 5 probe sets per custom add-on genes. During the panel design process, you cannot always add genes with enough probes for your panel.
There are several reasons why it can be challenging to design probes or enough probes for certain genes:
- GC Content: For genes with very high or very low GC (guanine-cytosine) content, it is difficult to pick probes because the melting temperature (Tm) of the probe must be within an optimal range for proper hybridization. If the Tm is too high or too low, the probe may not bind efficiently or specifically.
- Homology: Genes that have high sequence similarity to other genes (homologous genes) pose a challenge for probe design because it is difficult to find unique sequences that specifically target the desired gene without cross-reacting with others.
- Gene Length: Xenium probes are non-overlapping, so short genes may not have enough sequence length to accommodate multiple probes.
- Deprecated Probes: Some genes may have probes that were previously designed but later deprecated due to issues such as non-specific binding or contamination. These problems render the probes unreliable and unsuitable for use.
Other than the reasons above, the fact whether you can add certain genes with enough probes into a panel varies case by case.
- Expression Levels: Some genes predicted to have very high expression levels can lead to issues such as optical crowding in assays, Xenium Panel Designer will reduce the number probe sets accordingly.
- Cross ligation screening: Cross-ligation events may occur between padlock probes. To screen probes, XPD aligns padlock probe sequences to each other to ensure the custom panel design does not include any probes that may interact. Please refer to https://www.10xgenomics.com/support/software/xenium-panel-designer/latest/tutorials/xenium-panel-design-concepts-terms#cross-ligation-screen for more information.
You can find the list of genes for which 10x Genomics can design probes here: Xenium Panel Designer Gene List. Please note that due to chemistry differences, some genes can be successfully added into Xenium Prime but not in Xenium v1, and vice versa.