Comparative analysis of multiplexed in situ gene expression profiling technologies

Austin Hartman, Rahul Satija
bioRxiv doi: 10.1101/2024.01.11.575135

The independent study, “Comparative analysis of multiplexed in situ gene expression profiling technologies” by Austin Hartman and Rahul Satija from the New York Genome Center concludes MERSCOPE data showed:

-2X more sensitivity than Xenium despite the larger panel size (p 6-7)
-Higher sensitivity with enrichment for very lowly expressed genes such as G-coupled protein receptors (p 3)
-Superior specificity compared to Xenium (p 4)


The burgeoning interest in in situ multiplexed gene expression profiling technologies has opened new avenues for understanding cellular behavior and interactions. In this study, we present a comparative benchmark analysis of six in situ gene expression profiling methods, including both commercially available and academically developed methods, using publicly accessible mouse brain datasets. We find that standard sensitivity metrics, such as the number of unique molecules detected per cell, are not directly comparable across datasets due to substantial differences in the incidence of off-target molecular artifacts impacting specificity. To address these challenges, we explored various potential sources of molecular artifacts, developed novel metrics to control for them, and utilized these metrics to evaluate and compare different in situ technologies. Finally, we demonstrate how molecular false positives can seriously confound spatially-aware differential expression analysis, requiring caution in the interpretation of downstream results. Our analysis provides guidance for the selection, processing, and interpretation of in situ spatial technologies.