Question: In the probe set for Fixed RNA profiling (FRP), most genes (~90%) have 3-fold coverage. However, there are ~10% of genes with 1 to 2 fold coverage based on the FAQ. Does Cell Ranger normalize UMI counts among genes based on the coverage in FRP data analysis?
Cell Ranger does not normalize UMI counts among genes based on the coverage in the FRP probe set. For genes targeted by more than one probe pair, the UMI counts of all probe pairs targeting that gene are summed up and output as the UMI count for that gene in the feature barcode matrix.
If you are interested in normalizing the data based on the difference in coverage per gene, you may find the coverage for each gene in the probe set by looking into the Probe set metadata TSV file. The TSV file can be downloaded here. Note that even with the same coverage, the UMI counts for different genes may not be compared to each other directly because the signal can also be impacted by other technical factors, such as some variation in the probe hybridization efficiency of the probes. In addition, when it comes to gene expression comparison, the common comparison to make is the expression of the same genes between clusters or samples. In this case, the differential expression analysis should not be biased by the difference in coverage and hybridization efficiency because all the data are produced using the same probe set.