Standardized metrics for assessment and reproducibility of imaging-based spatial transcriptomics datasets

Our Spatial Touchstone (ST) dataset, generated from six tissue types across three global sites (n=76 assays), provides a comprehensive evaluation of reproducibility, sensitivity, dynamic ranges, signal-to-noise ratio, false discovery rates, cell type annotation, and congruence with single-cell profiling. Using single-nucleus RNA sequencing (snRNA-seq) on the same formalin-fixed paraffin-embedded tissues, we created a reference for transcriptional profiles to assess cellular organization and biomarker localization. In the absence of an independent 'gold standard’ in the field, this study provides an objective assessment of technical performance through standardized protocols, Spatial Touchstone Protocols (STSOP). Our open-source software, SpatialQC, allows users to evaluate samples across all technical metrics and directly impute cell annotations from single-cell datasets. This study features the largest imaging-based spatial transcriptomics data repository available, with a total of 203 spatial profiles. It incorporates both public (n=127) and ST datasets (n=76) through the Spatial Touchstone Portal (STP), available at www.spatialtouchstone.org. This resource offers users easy access to analyze and compare their own data against an extensive collection of imaging-based datasets. We show that the ST datasets demonstrate high reproducibility (r=0.93 to 1.0) and set a new benchmark for the field. Finally, we establish metrics to evaluate and integrate imaging based multi omics data from single cell into spatial transcriptomics to spatial proteomics. The ST project provides a comprehensive, reproducible assessment across platforms and sites, establishing essential standards for imaging-based spatial multi omics (transcriptomics and proteomics) and paving the way for future research and technological advancements.