Transcriptional output, cell-type densities, and normalization in spatial transcriptomics

Spatial transcriptomics measures mRNA at hundreds of 100 micrometer-diameter spots evenly spread across 6.5x6.9 mm2 histological slices. Gene expression within each spot is commonly normalized by total read counts. However we show that the transcriptional output of individual spots reflects the number of cells they contain, hence total read counts per spot reflect relevant biology. Although per-spot read-count normalization reveals important enrichment trends, it may heavily distort cell-type-related absolute local expression and conceal important biological information.