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 false positive transcript detections 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 likely to be false positives.
The number of detected transcripts is dependent on sample complexity and quality. Less than anticipated detected transcripts may indicate poor sample quality or low RNA content or problems with sample handling.
Non-specific binding can be the result of performing probe hybridization or probe washes incorrectly (e.g. incorrect temperature or leaving behind liquid during wash steps). Lower temperature can result in higher negative control probe binding.
Some tissues seem to be more susceptible to higher counts in negative control probes. We have noticed internally and with other users in the field an increased occurrence of this phenotype in fresh frozen human brain and mouse spleen.
If negative controls are high, this could indicate a higher level of noise in the data. We recommend investigating the possible cause(s) of high negative control probes. A few scenarios are outlined here:
- The negative control probe rate appears elevated because there was an elevated number of negative control probe counts observed, indicating non-specific binding.
- The negative control probe rate appears elevated because of lower gene transcript detection.
To investigate further, see guidance in the following article:
- What is the Xenium ‘Negative control probe counts per control per cell’ metric?
- Is Xenium data usable despite a few high NCPs?
In any datasets with high negative control probes alerts, we recommend exercising caution in data interpretation and confirming biological conclusions with alternate methods.
Products: Xenium In Situ Gene Expression