Methods, compositions, and systems for capturing probes and/or barcodes

A method for processing analytes in different biological samples, the method comprising:

• (a) hybridizing a first probe and a second probe to a nucleic acid analyte in a first biological sample mounted on a first substrate, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences of the nucleic acid analyte, wherein the second probe comprises a first capture probe binding domain, and wherein the first probe or the second probe comprises a first sample index sequence that identifies the first biological sample;
• (b) coupling the first probe and the second probe, thereby generating a first connected probe;
• (c) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the first biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a first capture probe of the plurality of capture probes comprises:
• (i) a first spatial barcode and (ii) a first capture domain;
• (d) releasing the first connected probe from the nucleic acid analyte when at least a portion of the first biological sample is aligned with at least a portion of the array;
• (e) hybridizing the first connected probe to the first capture probe of the array;
• (f) aligning a third substrate with the second substrate comprising the array, such that at least a portion of a second biological sample mounted on the third substrate is aligned with at least a portion of the array;
• and
• (g) analyzing a second analyte in the second biological sample mounted on the third substrate using the array.