Question: What is the Xenium Negative control probe counts per control per cell metric?
Answer: The Negative control probe counts per control per cell metric captures a standardized rate of high quality (>= Q20) negative control probe (NCP) transcript counts. High NCP levels trigger the High negative control probe counts per control per cell alert in the Xenium run summary report and can indicate nonspecific probe binding assay conditions or low transcripts. If a few NCP counts are high in contrast to most of the NCPs, see Is Xenium data usable despite a few high NCPs?
- The Negative control probe counts per control per cell metric calculation is as follows
(Q20+ negative control probe counts)/(# control probe sets)/(# cells)
- A similar alert High adjusted negative control probe rate indicates high NCPs in Xenium Onboard Analyzer (XOA) v1.5 and prior versions.The underlying metrics that trigger the two high NCP alerts differ in their calculations. If you encounter the High adjusted negative control probe rate alert, run xeniumranger-v1.6+ relabel. Updating to the ‘per control per cell’ metric can resolve High adjusted negative control probe rate alerts from older software.
NCPs are present as physical molecules in the probe pool. All panels except the Xenium Mouse Brain panel have twenty negative control probe sets per panel. The Mouse Brain panel has 27 negative control probe sets. Each probe set can contain up to eight probes. Like gene target probes, probes in a probe set will share a codeword and thus are not differentiable. Across panels, negative control probes overlap.
NCPs do not factor in transcript Quality Values. Instead, QV scores are based on negative control codewords.
- Is Xenium data usable despite a few high NCPs?
- The adjusted negative control probe rate appears elevated, what does this mean?
Product: Xenium In Situ Gene Expression
Last modified: February 15, 2024