Generating capture probes for spatial analysis

A method of generating a spatial array comprising:

• (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein a first acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the first acceptor oligonucleotide is attached to the substrate;
• (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle;
• (c) ligating a first plurality of donor oligonucleotides, wherein a first donor oligonucleotide of the first plurality of donor oligonucleotides comprises the second ligation handle and a first capture domain, to the 3′ end of a first acceptor oligonucleotide to generate a first capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle;
• and
• (d) ligating a second plurality of donor oligonucleotides, wherein a second donor oligonucleotide of the plurality of second donor oligonucleotides comprises the second ligation handle and a second capture domain, to the 3′ end of a second acceptor oligonucleotide to generate a second capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.