Spatially encoded biological assays

A substrate, comprising:

• a plurality of capture areas, wherein each capture area of the plurality of capture areas is at a different location on the substrate;
• and
• within one or more of the plurality of capture areas, a pair of probes independently immobilized on the substrate, wherein the pair of probes comprises:
• a first probe comprising, in a 5′ to 3′ direction, a first primer region comprising an oligonucleotide sequence that is complementary to a first sequencing primer, and a first capture region comprising an oligonucleotide sequence that is complementary to a target nucleic acid sequence;
• and
• a second probe comprising, in a 5′ to 3′ direction, a second primer region comprising an oligonucleotide sequence that is complementary to a second primer different from the first primer, and a ligation region comprising a terminal group that can be ligated to an extension product of the first probe,
• wherein at least one of the first and second probes individually, or the first and second probes together, comprise an oligonucleotide sequence that is unique to the capture area within which the pair of probes are immobilized;
• and
• wherein one or more of the plurality of capture areas comprises more than one pair of first and second probes comprising the oligonucleotide sequence that is unique to the capture area within which the more than one pair of first and second probes is independently immobilized.