Question: The probe set documentation talks about gene coverage in the human probe sets for Single Cell Gene Expression Flex or Spatial Gene Expression (probe-based) or In Situ Gene Expression assays.
Is this coverage directly proportional to the number of probes used? What exactly does the fold of coverage in these assays refer to?
The gene coverage in the Single Cell Gene Expression Flex, Spatial Gene Expression (probe-based), and In Situ Gene Expression products, simply refers to how many times the probe panel can cover all the transcripts of a specific gene, and the products have the following different gene coverages:
- Single Cell Gene Expression Flex (up to 3X)
- Probe-based Spatial Gene expression (up to 3X for human probe set V2, 1X for human probe set V1 and mouse probe set V1)
- In Situ Gene Expression (up to 8X)
Coverage cannot be strictly interpreted as the number of probes used for the specific gene. For instance, for Single Cell Gene Expression Flex, genes with 3X coverage may have more than 3 probes designed. Similarly the idea applies to other products listed above.
For each round of probe design (coverage round), we tried to design probes so that all transcripts of a gene can be covered at least one time to achieve 1X coverage. For most of the genes in the Flex panel and Spatial Gene Expression human probe set V2, we went through 3 rounds to achieve 3X coverage. Similarly for Spatial Gene expression probe set V1 (for mouse and human) it is 1X and for In Situ Gene Expression it is up to 8X.
In an ideal scenario, we may only need one probe to cover all the transcripts of a gene to achieve 1X coverage in one coverage round.
For example, Gene A below has two transcripts: the first transcript has 3 exons and the second transcript has two exons. The colored blocks on top of the exons denote probes. The orange probe hybridizes with the exon 1, which is present in both transcripts. Therefore, with the orange probe, we are able to achieve 1X coverage because it hybridizes with all the transcripts. Similarly, the yellow probe is able to provide 1X coverage because it hybridizes with all the transcripts. In total, with both the orange and yellow probes, we are able to achieve 2X coverage in this example because all the transcripts are covered twice.
However, for gene B below, we see a gene with three transcripts. It is not possible to design just one probe to hybridize with all transcripts since transcript 3 has an exon that is not present in the other two transcripts. In this case, we would need to design at least two probes to provide 1X coverage. This hypothetical case which may seem extreme, is presented only to illustrate the concept.
Products: Single Cell Gene Expression Flex, Spatial Gene Expression for FFPE, CytAssist Spatial Gene Expression for FFPE, CytAssist Spatial Gene Expression for Fixed Frozen, CytAssist Spatial Gene Expression for Fresh Frozen and In Situ Gene Expression
Last updated: June 2023