Spatial analysis toolkits for RNA in situ sequencing

Abstract Spatial transcriptomics (ST) is featured by high‐throughput gene expression profiling within their native cell and tissue context, offering a means to investigate gene regulatory networks in tissue microenvironment. In situ sequencing (ISS) is an imaging‐based ST technology that simultaneously detects hundreds to thousands of genes at subcellular resolution. As a highly reproducible and robust technique, ISS has been widely adapted and undergone a series of technical iterations. As the interest in ISS‐based spatial transcriptomic analysis grows, scalable and integrated data analysis workflows are needed to facilitate the applications of ISS in different research fields. This review presents the state‐of‐the‐art bioinformatic toolkits for ISS data analysis, which covers the upstream and downstream analysis workflows, including image analysis, cell segmentation, clustering, functional enrichment, detection of spatially variable genes and cell clusters, spatial cell–cell interactions, and trajectory inference. To assist the community in choosing the right tools for their research, the application of each tool and its compatibility with ISS data are reviewed in detailed. Finally, future perspectives and challenges concerning how to integrate heterogeneous tools into a user‐friendly analysis pipeline are discussed. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico