Optics-free reconstruction of shapes, images and volumes with DNA barcode proximity graphs

Spatial genomics technologies include imaging- and sequencing-based methods. Sequencing-based spatial methods typically require surfaces coated with coordinate-associated DNA barcodes, but the physical registration of these barcodes to spatial coordinates is challenging, necessitating either high density printing of oligonucleotides or in situ sequencing/probing of randomly deposited, DNA-barcode-bearing beads. As a consequence, the surface areas available to sequencing-based spatial genomic methods are constrained by the time, labor, cost, and instrumentation required to either print or decode a coordinate-tagged surface. To address this challenge, we developed SCOPE (Spatial reConstruction via Oligonucleotide Proximity Encoding), an optics-free, DNA microscopy-inspired method. With SCOPE, the relative positions of DNA-barcoded beads within a 2D shape, 2D image or 3D volume are inferred from the ex situ sequencing of chimeric molecules formed from diffusing “sender” and tethered “receiver” oligonucleotides. To demonstrate the potential of this approach, we applied SCOPE to reconstruct 2D shapes, 2D images or 3D volumes defined by 104-106 x 20-100 µm DNA barcoded beads, including an asymmetric “swoosh” resembling the Nike logo (44 mm2), a “color” Snellen eye chart (704 mm2) and the surface topology of 3D molds of a teddy bear, star, butterfly or block letter (75-100 mm3). Each of the resulting “DNA barcode proximity graphs” was computationally reconstructed in an automated fashion, across fields of view and at resolutions that were determined by sequencing depth, bead size, and diffusion kinetics, rather than by microarray or microscope instrument time. Because the ground truth shapes are known, these datasets may be particularly useful for the further development of computational algorithms by this nascent field. Overall design: The SCOPE reaction involves subjecting the hydrogel-encased monolayer of polyacrylamide beads to a temperature-controlled, two-phase reaction mediated by a single reaction volume containing two enzymes. In the first phase (37°C; 15'), sender-messengers are released from beads by the USER enzyme, which cleaves at the dU present at the stem of a subset of DNA barcodes. Sender-messengers diffuse away and hybridize to receiver-messengers, of the same bead type but opposite strand, tethered to adjacent beads. Because the complementary regions reside in the functional cap (Fig. 1A), senders and receivers are expected to hybridize at their 3' ends. In the second phase (55°C; 15'), reverse transcriptase, which can mediate both RNA- and DNA-templated DNA polymerization, drives overlap extension and the generation of a chimeric molecule that includes both the sender-messenger and receiver-messenger derived DNA barcodes (or, alternatively, chimeras of decoders and proximate hashing oligos). Sender-receiver chimeras are then PCR amplified and subjected to massively parallel sequencing, which yields a sparse matrix of interaction counts between a large number of DNA barcode-defined beads.