Question: What metrics could be affected by using an unsupported thermal cycler to prepare single cell gene expression libraries?
Answer: Thermal cyclers vary in ramp rates, lid temperatures, temperature accuracy, temperature uniformity, maximum supported volume, and other factors. Due to these differences, the same program may not perform the same in all thermal cyclers. We recommend that only the thermal cyclers that we have validated be used for our assays. Please refer to the User Guide for each assay or to one of the following articles to see which thermal cyclers have been validated for each assay.
In limited testing of non-validated thermal cyclers we have observed small negative effects to some metrics. Below is a list of which metrics are known to potentially be affected by using non-validated thermal cyclers.
For Gene Expression libraries generated using the 3’ Gene Expression Solution, 5’ Immune Profiling Solution, or Multiome assays:
- Median genes per cell
- Median UMI counts per cell
- Valid barcodes
- Reads mapped confidently to the transcriptome
For Gene Expression libraries generated using the Fixed RNA Profiling assay:
- Median genes per cell*
- Median UMI counts per cell*
*Resulting in a decrease in library complexity. This may be exacerbated by the high cell loads in this assay.
Other metrics may also be affected. Since the effects are specific to each thermal cycler and can be sample dependent, this list of affected metrics is not exhaustive. In many cases the effects are minor and would not affect the overall data analysis. If you are using a non-supported thermal cycler and observing lower than expected values for these metrics, we recommend switching to a validated thermal cycler and/or contacting Support.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome, Fixed RNA Profiling