Spatial analysis to detect sequence variants

A method of determining presence of a genetic variant in a nucleic acid at a spatial location in a biological sample, the method comprising:

• (a) contacting the biological sample with a first probe and a second probe, wherein:
• (i) the first probe comprises a sequence substantially complementary to a sequence 3′ of a target sequence in the nucleic acid,
• (ii) the second probe comprises:
• a sequence of non-complementary nucleotides;
• a sequence substantially complementary to a sequence 5′ of the target sequence in the nucleic acid;
• and
• a capture probe binding domain, and
• (iii) the target sequence is at least one nucleotide;
• (b) hybridizing the first probe and the second probe to the nucleic acid;
• (c) extending the first probe to generate an extended first probe;
• (d) cleaving the sequence of non-complementary nucleotides of the second probe when the genetic variant is present;
• (e) ligating the extended first probe and the second probe, thereby creating a ligated probe;
• (f) releasing the ligated probe from the nucleic acid;
• (g) hybridizing the ligated probe to a capture probe on a substrate comprising a plurality of capture probes, wherein the capture probe is included in the plurality of capture probes, and wherein the capture probe comprises a spatial barcode and a capture domain;
• and
• (h) determining (i) all or a part of the sequence of the ligated probe hybridized to the capture domain, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the presence of the genetic variant in the nucleic acid in the biological sample.