Methods for spatial analysis using RNA-templated ligation

A method for determining a location of a target nucleic acid in a biological sample comprising:

• (a) providing an array comprising a plurality of capture probes affixed to the array, wherein a capture probe of the plurality of capture probes comprises:
• (i) a spatial barcode and (ii) a capture domain;
• (b) contacting a plurality of first probes and second probes to a biological sample on the array, wherein the plurality of first probes and second probes target a plurality of nucleic acids in the biological sample and wherein a first probe and a second probe of the plurality comprise sequences that are substantially complementary to the target nucleic acid, and wherein the second probe comprises a capture probe capture domain sequence that is complementary to all or a portion of the capture domain of the capture probes affixed to the array;
• (c) hybridizing the first probe and the second probe to the target nucleic acid;
• (d) generating a ligation product by ligating the first probe and the second probe;
• (e) releasing the ligation product from the target nucleic acid;
• (f) hybridizing the ligation product to the capture domain of the capture probe affixed on the array;
• and
• (g) determining (i) all or part of the sequence of the ligation product bound to the capture domain, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the target nucleic acid in the biological sample.