Methods of determining a surgical margin and methods of use thereof

A method of determining a surgical margin of a tissue to be resected in a subject, the method comprising:

• (a) contacting a tissue section obtained from the subject to a first substrate;
• (b) contacting the tissue sample to a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises:
• an analyte binding moiety that binds specifically to an analyte,
• an analyte binding moiety barcode, and
• an analyte capture sequence;
• (c) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises:
• (i) a spatial barcode and (ii) a capture domain;
• (d) permeabilizing the tissue section and hybridizing the analyte capture sequence to the capture domain;
• (e) determining (i) all or a part of a nucleic acid sequence corresponding to the analyte capture sequence hybridized to the capture domain or a complement thereof, and (ii) the spatial barcode or a complement thereof, and using the determined nucleic acid sequences of (i) and (ii) to identify the presence of the analyte at a location in the tissue section;
• and
• (f) comparing the presence of the analyte at the location in the tissue section to presence of the analyte at one or more different locations in the tissue section, thereby determining the surgical margin of the tissue to be resected from the subject based on the comparison.