Methods for determining a location of a feature on an array include: (a) providing a first array with a first plurality of features immobilized on a first substrate; (b) providing a second array with a second plurality of features immobilized on a second substrate; (c) aligning the first array with the second array; (d) hybridizing a first barcoded oligonucleotide of the first array to a second barcoded oligonucleotide of the second array, thereby producing a combined nucleic acid that includes first and second spatial barcodes; (e) determining all or a portion of the sequence of the combined nucleic acid; and (f) identifying the second barcoded oligonucleotide associated with the first barcoded oligonucleotide in the combined nucleic acid, and determining the location of a second feature in the second array.

The present disclosure relates to compositions and methods for generating capture probes on a substrate for identifying the location of analytes in a biological sample.

A sample holder includes a first member featuring a first retaining mechanism configured to retain a first substrate that includes a sample, a second member featuring a second retaining mechanism configured to retain a second substrate that includes a reagent medium, and an alignment mechanism connected to at least one of the first and second members, and configured to align the first and second members such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned.

A sample holder includes a first member featuring a first retaining mechanism configured to retain a first substrate that includes a sample, a second member featuring a second retaining mechanism configured to retain a second substrate that includes a reagent medium, and an alignment mechanism connected to at least one of the first and second members, and configured to align the first and second members such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned.

Methods for determining a location of a feature on a spatial array include (a) providing an array of features on a substrate, where a feature of the array includes a barcoded oligonucleotide having, in a 5 to 3 direction, a spatial barcode, a cleavage domain, and a constant sequence; (b) hybridizing a priming oligonucleotide to the constant sequence; (c) extending the priming oligonucleotide using the barcoded oligonucleotide as a template; and (d) determining all or a portion of a sequence of the extended priming oligonucleotide corresponding to the spatial barcode, or a complement thereof, and a location of the extended priming oligonucleotide, and using the location of the extended priming oligonucleotide to determine the location of the feature on the spatial array.