High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue

SUMMARY Spatial gene expression heterogeneity plays an essential role in a range of biological, physiological and pathological processes but it remains a scientific challenge to conduct high-spatial-resolution, genome-wide, unbiased biomolecular profiling over a large tissue area. Herein, we present a fundamentally new approach – microfluidic Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq). Parallel microfluidic channels (10μm, 25μm, or 50μm in width) are used to deliver molecular barcodes to the surface of a formaldehyde fixed tissue slide in a spatially confined manner. Crossflow of two sets of barcodes A1-A50 and B1-B50 followed by ligation in situ yields a 2D mosaic of tissue pixels, each containing a unique combination of full barcode AiBj (i=1-50, j=1-50). It permits simultaneous barcoding of mRNAs, proteins, or even other omics on a fixed tissue slide, enabling the construction of a high-spatial-resolution multi-omics atlas by NGS sequencing. Applying it to mouse embryo tissues revealed all major tissue types in early organogenesis, distinguished brain microvascular networks, discovered new developmental patterning in forebrain, and demonstrated the ability to detect a single-cell-layer of melanocytes lining an optical vesicle and asymmetric expression of Rorb and Aldh1a1 within it, presumably associated with the onset of retina and lens, respectively. Automated feature identification using spatial differential expression further identified dozens of developmental features. DBiT-seq is a highly versatile technology that may become a universal method for spatial barcoding and sequencing of a range of molecular information at a high resolution and the genome scale. It can be readily adopted by biologists with no experience in microfluidics or advanced imaging, and could be quickly disseminated for broader impacts in a variety of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.