Systems and methods for analyzing spatially-linked biological samples

A method for biological analysis, comprising:

• (a) disposing a spatially-linked biological sample on a planar surface, wherein:
• the spatially-linked biological sample comprises a tissue sample,
• the tissue sample comprises a plurality of cells,
• the planar surface comprises glass, plastic, or a combination thereof,
• the planar surface further comprises a plurality of capture sites,
• the plurality of capture sites are arranged in a two-dimensional pattern on the planar surface,
• a capture site of the plurality of capture sites comprises a dimension of about 1 micron to about 20 microns,
• each capture site of the plurality of capture sites comprises a plurality of single-stranded oligonucleotides,
• the plurality of single-stranded oligonucleotides are coupled to the planar surface,
• at least a subset of the plurality of single-stranded oligonucleotides comprise:
• i) a capture sequence comprising a plurality of thymine bases that are configured to bind to poly-A sequences,
• ii) a unique molecular identifier,
• iii) a barcode associated with a location on the planar surface, and
• iv) a sequencing primer, and
• each oligonucleotide of the subset of the plurality of single-stranded oligonucleotides comprises at least 50 nucleotides;
• (b) imaging at least a portion of the spatially-linked biological sample, wherein the imaging comprises bright-field imaging, phase-contrast imaging, fluorescence imaging, or a combination thereof;
• (c) releasing nucleic acids from the spatially-linked biological sample;
• (d) capturing at least a subset of the nucleic acids on at least the subset of the single-stranded oligonucleotides, thereby obtaining the captured nucleic acids, wherein the captured nucleic acids are immobilized on the planar surface;
• and
• (e) extending at least a subset of the captured nucleic acids using at least portions of the single-stranded oligonucleotides as templates, thereby obtaining the extended nucleic acids;
• (f) amplifying the extended nucleic acids, thereby generating amplicons, wherein:
• the amplification is performed using primers that comprise sequencing adaptors, and
• the sequencing adaptors comprise dual-indexed sequencing adaptors;
• (g) sequencing at least a portion of an amplicon of the amplicons, a derivative of the amplicon, a complement of the amplicon, or a combination thereof, and
• (h) associating a sequence obtained from the sequencing in (g) and imaging data from the imaging in (b) with a cell of the plurality of cells.