Three-dimensional spatial analysis

A method for determining abundance or location of a nucleic acid in three-dimensional space in a biological sample, the method comprising:

• (a) immobilizing the biological sample in a hydrogel matrix on an array;
• (b) providing a plurality of spatially-programmed capture probes, wherein a spatially-programmed capture probe in the plurality of spatially-programmed capture probes comprises:
• a programmable migration domain;
• (ii) a detectable moiety;
• and
• (iii) a capture domain that hybridizes to a sequence in the nucleic acid;
• (c) migrating the spatially-programmed capture probe into the hydrogel matrix towards the array;
• (d) hybridizing the spatially-programmed capture probe to the nucleic acid at a location on a z-axis;
• (e) detecting the detectable moiety at the location on the z-axis where the spatially-programmed capture probe hybridized to the nucleic acid, thereby determining the location of the spatially-programmed capture probe on the z-axis;
• extending the spatially-programmed capture probe using the nucleic acid as a template at the location on the z-axis, thereby generating an extension product;
• (g) migrating the extension product to the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises
• a spatial barcode and a capture domain that hybridizes to a sequence in the extension product;
• and
• (h) determining (i) all or part of the sequence in the extension product, or a complement thereof, and (ii) all or part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii), and the determined location on the z-axis in step (e), to identify the abundance and the location of the nucleic acid in the three-dimensional space in the biological sample.

A method for determining abundance or location of a nucleic acid in three-dimensional space in a biological sample, the method comprising:

• (a) applying the biological sample to an array and immobilizing the biological sample in a hydrogel matrix;
• (b) providing a plurality of pairs of spatially-programmed capture probes, wherein a pair of spatially-programmed capture probes in the plurality of pairs of spatially-programmed capture probes comprises a first spatially-programmed capture probe and a second spatially-programmed capture probe, wherein:
• at least one of the first spatially-programmed capture probe and the second spatially-programmed capture probe comprises a detectable moiety;
• the first spatially-programmed capture probe and the second spatially-programmed capture probe each comprise sequences that are complementary to adjacent sequences of the nucleic acid;
• and
• each of the first and the second spatially-programmed capture probes comprise a programmable migration domain,
• (c) migrating the pair of spatially-programmed capture probes into the hydrogel matrix towards the array;
• (d) hybridizing the pair of spatially-programmed capture probes to the adjacent sequences on the nucleic acid at a location on a z-axis;
• (e) detecting the detectable moiety at the location on the z-axis where the pair of spatially-programmed capture probes hybridized to the nucleic acid, thereby determining the location of the pair of spatially-programmed capture probes on the z-axis;
• ligating the pair of spatially-programmed capture probes, thereby generating a ligation product;
• (g) migrating the ligation product to the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain that hybridizes to a sequence in the ligation product;
• and
• (h) determining (i) all or part of the sequence in the ligation product, or a complement thereof, and (ii) all or part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii), and the determined location on the z-axis in step (e), to identify the abundance and the location of the nucleic acid in the three-dimensional space in the biological sample.

A system for determining a location of an analyte in three-dimensional space in a biological sample, the system comprising:

• (a) a plurality of spatially-programmed capture probes comprising, wherein a spatially-programmed capture probes of the plurality of spatially-programmed capture probes comprises:
• a programmable migration domain comprising a domain selected from a charged domain, a size-specific domain, and electromagnetic domain, a metallic nanoparticle, or a polymer;
• (ii) a detectable moiety;
• and
• (iii) a capture domain that binds specifically to a sequence within a nucleic acid;
• and
• (b) an array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain that binds specifically to a sequence that is not present in the spatially-programmed capture probe.