Methods and compositions for probe detection and readout signal generation

A method for analyzing a biological sample, comprising:

• a) contacting the biological sample with a plurality of encoding probes, wherein:
• the plurality of encoding probes comprises a first encoding probe and one or more second encoding probes,
• the first encoding probe and each second encoding probe are capable of hybridizing to a first target sequence and a second target sequence, respectively, in a target nucleic acid in the biological sample,
• the first encoding probe is circularizable and comprises an interrogatory region for interrogating a region of interest in the first target sequence, and
• each second encoding probe is circular or circularizable;
• b) circularizing the first encoding probe to generate a circularized first encoding probe, and optionally circularizing the one or more second encoding probes;
• c) contacting the biological sample with one or more primary detectable probes that hybridize to the first encoding probe and/or the second encoding probe;
• d) detecting a signal associated with the one or more primary detectable probes;
• e) generating a rolling circle amplification (RCA) product of the circularized first encoding probe;
• and
• f) detecting a signal associated with the RCA product.