Question: Can cell multiplets be filtered out when using the 3’ CellPlex Kit for Cell Multiplexing?
Answer: Some, but not all, cell multiplets can be identified and filtered out in Cell Ranger. The fraction of cell multiplets that can be filtered out will depend on two factors: the number of pooled samples and the pooling ratios of these samples.
Cell Multiplet filtering
When analyzing data in Cell Ranger, cell multiplets containing cells labeled with different CMO tags can be identified and filtered out.
In contrast, cell multiplets containing cells labeled with the same CMO tag will not be identified as multiplets in Cell Ranger and will remain in the final data.
Number of pooled samples / CMO tags
As the number of pooled samples (each with a unique CMO tag) increases, it becomes more likely that a multiplet will contain cells labeled with different tags and be filtered out.
The expected percentage of cell multiplets that will be identified and filtered out in Cell Ranger is:
- ~50% of multiplets if 2 tags are used
- ~75% of multiplets if 4 tags are used
- ~88% of multiplets if 8 tags are used
- ~92% of multiplets if 12 tags are used
Note: these percentages assume that the CMO-labeled samples are pooled at equal ratios.
Sample pooling ratios
Pooling cell samples at equal ratios are recommended as it allows for optimal multiplet detection.
If samples are pooled at unequal ratios, it becomes more likely that cell multiplets will contain cells labeled with the same tag and therefore not be filtered out in Cell Ranger.
For example, if two CMO-labeled samples are pooled together, the expected percentage of cell multiplets that will be identified and filtered out in Cell Ranger is:
- ~50% of multiplets if cells are pooled at a 1:1 ratio
- ~37% of multiplets if cells are pooled at a 3:1 ratio
- ~18% of multiplets if cells are pooled at 9:1 ratio
For additional details on cell multiplet identification and filtering, please refer to our Technical Note on Cell Multiplexing.
Products: Single Cell Gene Expression, CellPlex