Generating capture probes for spatial analysis
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.
1. A method of generating a spatial array comprising:
(a) immobilizing a first plurality of primers and a second plurality of primers on a substrate, wherein:
(i) a first primer of the first plurality of primers comprises one or more functional domains and a sequence complementary to a hybridization domain, wherein the one or more functional domains of the first primer comprise a sequencing specific site or a primer binding site; and
(ii) a second primer of the second plurality of primers comprises a sequence complementary to a capture domain;
(b) providing a template oligonucleotide comprising the hybridization domain capable of hybridizing to the sequence complementary to the hybridization domain of the first primer, a complement of a spatial barcode, and a complement of the capture domain;
(c) hybridizing the hybridization domain of the template oligonucleotide to the sequence complementary to the hybridization domain of the first primer;
(d) extending the first primer using the template oligonucleotide as a template to generate a capture probe comprising the first primer, the spatial barcode, and the capture domain; and
(e) releasing the template oligonucleotide from the capture probe, thereby generating the spatial array.
2. The method of claim 1 , further comprising amplifying the capture probe to create a clonal cluster of capture probes.
3. The method of claim 2 , wherein the amplifying comprises:
(a) hybridizing the capture domain of the capture probe to the sequence complementary to the capture domain of the second primer;
(b) extending the second primer using the capture probe as a template to generate a copy of the template oligonucleotide immobilized on the substrate;
(c) releasing the copy of the template oligonucleotide from the capture probe;
(d) hybridizing the copy of the template oligonucleotide to an unextended first primer; and
(e) extending the unextended first primer using the copy of template oligonucleotide as a template to generate a second capture probe immobilized on the substrate, thereby generating the clonal cluster of capture probes.
4. The method of claim 3 , further comprising extending the template oligonucleotide using the first primer as a template.
5. The method of claim 3 , wherein the second primer further comprises a cleavage domain.
6. The method of claim 5 , wherein releasing the copy of the template oligonucleotide comprises cleaving the cleavage domain of the second primer and denaturing the copy of the template oligonucleotide from the capture probe.
7. The method of claim 1 , wherein the template oligonucleotide further comprises a unique molecular identifier (UMI) and one or more functional domains, wherein the one or more functional domains of the template oligonucleotide comprise a sequencing specific site or a primer binding site.
8. The method of claim 7 , wherein the UMI is disposed between the complement of the spatial barcode and the complement of the capture domain.
9. The method of claim 1 , wherein the first primer and the second primer are immobilized to the substrate at their 5′ ends.
10. The method of claim 1 , wherein the first primer and the second primer are at a ratio between 1:1.5 and 1.5:1.
11. The method of claim 2 , wherein the first primer and the second primer are less than 5 μm apart.
12. The method of claim 1 , wherein the sequence complementary to the capture domain of the second primer comprises a poly(A) sequence.
13. The method of claim 1 , wherein the template oligonucleotide is attached to a bead.
14. The method of claim 13 , wherein the bead is a gel bead.
15. The method of claim 13 , wherein the bead is 2 microns or less in diameter.
16. The method of claim 15 , wherein the bead is 1 micron or less in diameter.
17. The method of claim 13 , wherein the bead is removed after the step of extending the first primer.
18. The method of claim 13 , wherein the bead is removed between steps (c) and (d).
19. The method of claim 13 , wherein the bead is comprised in a plurality of beads dispersed on the substrate such that a single layer of beads is disposed on the substrate.
20. The method of claim 1 , wherein the capture domain comprises a poly(T) sequence.
21. The method of claim 20 , wherein the poly(T) sequence hybridizes to an analyte.
22. The method of claim 21 , wherein the analyte is mRNA.
23. The method of claim 22 , further comprising extending the capture probe using the mRNA as a template to generate an extended capture probe.
24. The method of claim 1 , wherein the substrate comprises a glass slide.
25. The method of claim 24 , wherein the glass slide is a treated glass slide.
26. The method of claim 1 , further comprising determining the sequence of the spatial barcode of the generated capture probe.
27. The method of claim 26 , wherein determining the sequence of the spatial barcode of the generated capture probe is performed after the template oligonucleotide is released.
28. The method of claim 27 , wherein determining the sequence of the spatial barcode of the generated capture probe comprises sequencing.
29. The method of claim 28 , wherein the sequencing comprises sequencing-by-synthesis (SBS), sequencing-by-hybridization (SBH), or sequencing-by-ligation (SBL).