Question:
In the custom probe design technical note, there is a design criterion on the 25th nucleotide of the probe: “The 25th nucleotide of the probe (3' most nucleotide of the LHS probe) must be a T. The complementary nucleotide in the target RNA must be an A.” However, I see that some probes in the whole transcriptome probe sets for Flex Gene and Visium Spatial Gene Expression assays do not follow this rule. Why is that?
Answer:
In the technical note, we use the 25th nucleotide T as a design constraint because using a T at position 25 is "safe". Most 10x probes follow this rule and it is a straightforward guideline to communicate and for people to follow. In our prebuilt whole transcriptome probe sets, we do use other motifs as well. This means that “TN” at the ligation junction is preferred but not strictly required, and motifs other than “TN” at the ligation junction may also work. However, the exact parameters of design beyond what are provided in the technical note are proprietary.
Please note that the use of custom probes is not supported or validated by 10x Genomics. 10x Genomics cannot guarantee that custom probes will yield data comparable to that from the whole transcriptome probe sets.
Products: Flex Gene Expression, Visium Spatial Gene Expression for FFPE, Visium CytAssist-enabled applications
Last Updated: July 2024