Open-ST: High-resolution spatial transcriptomics in 3D

Abstract

Spatial transcriptomics (ST) methods have been developed to unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary tumor and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal and tumor populations in space. Distinct cell states were organized around cell-cell communication hotspots in the tumor, but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. We anticipate Open-ST to accelerate the identification of spatial molecular mechanisms in 2D and 3D.

Nova-ST: Nano-Patterned Ultra-Dense platform for spatial transcriptomics

“…Open-ST 15 is another independently developed spatial transcriptomics technique based on the NovaSeq 6000 S4 flow cell and is very comparable to the Nova-ST workflow. Nevertheless, there are some key differences between the two implementations.…”

“…At a bin size of 200, Nova-ST detects a median of 6318 genes compared to 4092 genes with the Stereo-seq platform at the same bin size. Open-ST 12 , another spatial sequencing technique developed independently by another group also demonstrates superior sensitivity compared to Stereo-seq. For Nova-ST, we detect a median UMI count of 294 per 100 µm 2 at a sequencing depth of ∼15 million reads per mm 2 of total sequenced surface (deeply sequenced sample), which compares to the sensitivity reported by the Open-ST platform 12 .…”

“…Open-ST 12 , another spatial sequencing technique developed independently by another group also demonstrates superior sensitivity compared to Stereo-seq. For Nova-ST, we detect a median UMI count of 294 per 100 µm 2 at a sequencing depth of ∼15 million reads per mm 2 of total sequenced surface (deeply sequenced sample), which compares to the sensitivity reported by the Open-ST platform 12 . Thus, Nova-ST obtains more genes and UMIs than Stereo-seq at similar bin sizes, allowing us to decrease bin sizes in Nova-ST to achieve increased resolution (Fig.…”

New tools to study renal fibrogenesis

“…Overlaying HE staining, Visium HD is capable of identifying cell types in fine anatomical structures, as well as immune cells that are difficult to detect by other spatial sequencing approaches. A more cost-effective assay could be the use of Illumina flow cells themselves, which have been already leveraged by several groups to generate highresolution spatial transcriptomics at a fraction of the Visium HD costs [32,33].…”

Characterization of immune cell populations in the tumor microenvironment of colorectal cancer using high definition spatial profiling

“…Several commercial technologies are currently available for discovery-based spatial transcriptomics, including Visium CytAssist Spatial Gene Expression (“Visium v2”, 10x Genomics), STOmics (BGI), and Curio Seeker (Curio Bioscience). Other published methods include Seq-Scope 17 , Nova-ST 18 , Open-ST 19 , HDST 20 , DBiT-seq 21 , Pixel-seq 22 , and XYZeq 23 . These methods have enabled the localization of cell types within tissues, which is critical for understanding the interaction between cells in the TME of CRC 24–27 .…”