Question: The adjusted negative control probe rate appears elevated, what does this mean?
Answer: The adjusted negative control probe rate measures both erroneous decoding and off-target binding, relative to the total number of high-quality transcripts detected. A high value may be due to non-specific binding, but could also be due to a low number of detected gene transcripts. The metric is an estimate of what fraction of the observed Q20 transcripts are false positives. It is important to understand that this alert does not necessarily imply that negative control probe counts are elevated (see below). Please refer to the Support Webpage for additional information on general overview of Xenium Analysis Summary metrics.
Small variation in the adjusted negative control probe rate is of little concern. An alert (warning) is raised when this metric exceeds 4%. A stronger alert (error) is raised when this metric exceeds 10%. These alerts will be replaced with a new metric that more accurately/directly estimates non-specific binding Onboard Analysis Software v1.6+. In the meantime, if the adjust negative control probe rate appears high, it is important to determine whether:
1) The negative control probe rate appears elevated because there was an elevated number of negative control probe counts observed, indicating non-specific binding.
2) The negative control probe rate appears elevated because of lower gene transcript detection.
In the absence of obvious workflow deviations in either probe hybridization or post-hybridization washes, an impact assessment can be made by surveying the ‘absolute number of negative control probe counts’ and then calculate the ‘negative control probe count per control per cell.
Absolute number of negative control probe counts
The negative control probe count is the total count of negative control probes with high quality in the sample (Q20 or above). This can be calculated by:
- Total high quality decoding transcripts
- Can be found in the ‘Decoding’ tab of analysis summary and includes genes and negative controls.
- Adjusted negative control probe rate
- Can be found in the ‘Decoding’ tab of analysis summary.
- Number of target genes
- This is the sum of genes in base panel + any add-on genes and can be found on the first tab of the analysis summary.
- 20: the number of negative controls in the panel. This number is 20 for human pre-designed and add-on panels, and 27 for mouse brain pre-designed and add-on panels.
Negative control probe count per control per cell
Building off the total negative control probe count, we can now further survey the negative control probe counts on a per control per cell basis.
- This metric will be added in Xenium Onboard Analysis Software v1.6.
If the Negative control probe counts per control per cell is smaller than 0.025, the absolute level of negative controls observed is in the expected range.. The “high adjusted negative control probe rate” analysis summary alert is triggered because there are not a lot of actual gene transcripts detected. This could be due to an issue with sample quality, sample preparation, panel design (e.g. many targeted genes are not expressed), or something else.
Alternatively, if the negative control probe counts per control per cell is greater than 0.025, then we actually see an elevated number of counts. Please contact firstname.lastname@example.org to further discuss how to perform these calculations and how high counts might impact datasets.
The negative control probe count per control per cell metric will be added to v1.6 Analysis Summaries. This will reduce the burden of having to calculate whether elevated negative probe control counts are likely due to a low number of transcripts (less than 0.025) or likely due to small deviations in the assay protocol (more than 0.025). Please see:
Products: Xenium In Situ Gene Expression