The XOA output files analysis_summary.html and metrics_summary.csv report negative control metrics.

The "Negative control probe counts per control per cell" metric is derived first from calculating the absolute number of negative control probe counts and then calculating the metric per control probe per cell. The "Estimated number of false positive transcripts per cell" is derived from this metric. Derived values may differ slightly from those reported in summary outputs due to using rounded values.

They are calculated using the following inputs:

• Total high quality decoded transcripts: displayed on the Summary and Decoding tabs, includes genes (Q-Score ≥ 20) and negative controls
• Percent of transcripts within cells: displayed on the Cell Segmentation tab
• Adjusted negative control probe rate: displayed on the Decoding tab
• Number of target genes: the sum of genes in the pre-designed panel and any add-on custom genes, displayed on the Summary tab
• Number of negative control probes in the panel: in Xenium v1, each pre-designed and add-on panel contains 20 negative control targets (except runs that use the Xenium Mouse Brain base panel, which have 27 negative targets). In Xenium Prime, each pre-designed and add-on panel contains 40 negative control targets.
• Number of cells detected: hero metric on the Summary tab

1. The Negative control probe count is calculated as:

   $\footnotesize{\text{Q20 decoded transcripts in cells}\times{\text{Adjusted negative control probe rate}}\times{\frac{\text{Number of negative control probes}}{\text{Number of target genes}}}}$

   where Q20 decoded transcripts in cells is calculated as:

   $\footnotesize{\text{Total high quality decoded transcripts}\times{\text{Percent of transcripts within cells}}}$

2. The Negative control probe count per control per cell is then calculated as:

   $\footnotesize{{\text{Negative control probe count}}\div{\text{Number of negative control probes}}\div{\text{Number of cells detected}}}$

3. The Estimated number of false positive transcripts per cell is calculated as:

   $\footnotesize{{\text{Negative control probe counts per control per cell}}\times{\text{Number of target genes}}}$

The calculations for "Genomic control probe counts per control per cell" and "Estimated number of false positive transcripts per cell including genomic counts" are analogous to the calculations shown above. Both metrics are available in outputs generated with Xenium Onboard Analysis v3.0.

The inputs are:

• Total high quality decoded transcripts
• Percent of transcripts within cells
• Adjusted genomic control probe rate
• Number of target genes
• Number of genomic control probes in the panel (21 for Xenium Prime 5K pre-designed panels)
• Number of cells detected