A comprehensive review of spatial transcriptomics data alignment and integration

Spatial data acquisition technologies enable high-throughput quantification of molecular expression in tissue sections maintaining spatial context information. However, performing downstream analysis on a whole tissue section requires the alignment and integration of multiple tissue slices. This is a nontrivial task due to tissue heterogeneity and plasticity. Although manual solutions exist, they are time-consuming and require technical expertise. Hence, automated and robust alignment and integration of multiple slices within and across datasets, individuals, and experiments becomes essential. This study aims to (i) present a comprehensive review of methodologies for spatial transcriptomics (ST) data alignment and integration, (ii) explain the problem, its scope and challenges, and (iii) propose a general pipeline. We review 24 tools addressing multi-slice ST alignment and integration, and tackling key challenges through downstream validation. Tools focusing solely on single-slice ST analyses or multi-omics integration are excluded. We categorize these approaches by methodology (statistical mapping, image processing and registration, and graph-based) in accordance with the generalized pipeline. We evaluate their strengths, limitations, and real-world applications based on task scope and their potential to advance biological insights. Despite improved spatial resolution and 3D tissue reconstruction, significant challenges persist in achieving robust alignment and integration across heterogeneous tissue slices.