Spatial transcriptomics: Technologies, applications and experimental considerations

Abstract

The diverse cell types of an organ have a highly structured organization to enable their efficient and correct function. To fully appreciate gene functions in a given cell type, one needs to understand how much, when and where the gene is expressed. Classic bulk RNA sequencing and popular single cell sequencing destroy cell structural organization and fail to provide spatial information. However, the spatial location of gene expression or of the cell in a complex tissue provides key clues to comprehend how the neighboring genes or cells cross talk, transduce signals and work together as a team to complete the job. The functional requirement for the spatial content has been a driving force for rapid development of the spatial transcriptomics technologies in the past few years. Here, we present an overview of current spatial technologies with a special focus on the commercially available or currently being commercialized technologies, highlight their applications by category and discuss experimental considerations for a first spatial experiment.

Keywords: 10× Visium; BMKMANU S1000; CosMx SMI; GeoMx DSP; MERFISH; RNA sequencing; Spatial transcriptomics; Stereo-seq; Xienum.

Fig. 1.

Schematic depiction of different probe hybridization and imaging strategies of 4 imaging-based spatial transcriptomics technologies. A) Molecular Cartography technology-a three-probe hybridization strategy. B) MERSCOPE-a two-probe hybridization strategy. The secondary probes are only fluorescently labeled in some of the hybridization rounds. C) Xenium-a Padlock probe-based hybridization strategy. Gene-specifc ligation increases in hybridization specificity. D) CosMx SMI-a modified two-probe hybridization strategy. The primary probe has 16 read-out domain and the secondary probe contains multiple dyes to increase signal intensity.

Fig. 2.

Core technologies of 4 sequencing-based platforms. Visium (A), S1000 (B) and Stereo-sEq. (C) use probes mounted on the array to capture RNA for library construction and sequencing. The key difference among those 3 platforms is spot size (A > B > C), which determines spatial resolution. GeoMx DSP (D) uses a probe hybridization strategy. Each probe is linked with a gene-specifc barcode through a UV cleavable linker. The barcodes are cleaved from the selected region of interest, and collected for library construction and sequencing. UMI: Unique Molecule Identifier. CID: Coordinate Identity. The pink box located in the upper right corner of B indicates a probe comprising Read1 handle, a spatial barcode, UMI, and PolyT.