Spatial analysis to detect sequence variants
Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample.
1. A method for determining the presence of a genetic variant in a nucleic acid at a spatial location in a tissue section, the method comprising:
(a) contacting the tissue section with a first probe and a second probe, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to a sequence of the nucleic acid, wherein the first probe and the second probe form an invasive cleavage structure in the presence of the genetic variant, and wherein:
(i) the first probe further comprises at least one nucleotide that is complementary to a wild-type sequence of the genetic variant; and
(ii) the second probe further comprises:
a sequence of non-complementary nucleotides;
a nucleotide complementary to the genetic variant; and
a capture probe binding domain;
(b) hybridizing the first probe and the second probe to the nucleic acid in the tissue section;
(c) cleaving the sequence of the non-complementary nucleotides of the second probe when the genetic variant is present;
(d) ligating the first probe and the second probe, thereby creating a ligated probe;
(e) releasing the ligated probe from the nucleic acid in the tissue section prior to step (f);
(f) contacting the ligated probe with a substrate, wherein the substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;
(g) hybridizing the ligated probe to the capture domain; and
(h) determining (i) all or a part of the sequence of the ligated probe hybridized to the capture domain, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the presence of the genetic variant in the nucleic acid at the spatial location in the tissue section.
2. The method of claim 1 , wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of the nucleic acid.
3. The method of claim 1 , wherein the first probe comprises a sequence that is substantially complementary to a sequence 3′ of the genetic variant, or wherein the second probe comprises a sequence substantially complementary to a sequence 5′ of the genetic variant.
4. The method of claim 1 , wherein the second probe comprises from 5′ to 3′: the sequence of non-complementary nucleotides, one or more nucleotides complementary to the genetic variant, a sequence substantially complementary to a sequence 5′ of the genetic variant, and the capture probe binding domain, wherein the capture probe binding domain is capable of hybridizing to a capture domain of a capture probe on the substrate.
5. The method of claim 1 , wherein the cleaving step comprises use of an endonuclease that cleaves the invasive cleavage structure.
6. The method of claim 5 , wherein the endonuclease is Flap endonuclease 1 (FEN1).
7. The method of claim 5 , wherein the endonuclease is selected from a Thermus aquaticus (Taq) DNA polymerase, a Thermus thermophilus (Tth) polymerase, Xeroderma pigmentosum group G (XPG) protein, Rad2 from budding yeast, AtRAD2 from Arabidopsis , or Rad13 in fission yeast, or derivatives thereof.
8. The method of claim 1 , further comprising cleaving the first probe, and wherein:
the first probe further comprises a first moiety, wherein the first moiety is a first fluorophore, and wherein the first fluorophore emits a first signal when the first probe is cleaved.
9. The method of claim 8 , wherein the second probe further comprises a second moiety, wherein the second moiety is a second fluorophore, and wherein the second fluorophore emits a second signal when the second probe is cleaved.
10. The method of claim 9 , wherein the first signal and the second signal emit at different wavelengths.
11. The method of claim 1 , wherein the ligating comprises ligating the first probe and the second probe using enzymatic or chemical ligation.
12. The method of claim 11 , wherein the enzymatic ligation utilizes a ligase.
13. The method of claim 12 , wherein the ligase is one or more of a T4 RNA ligase (Rn12), a PBCV-1 DNA ligase, a Chorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.
14. The method of claim 12 , wherein the ligase is a pre-activated T4 DNA ligase.
15. The method of claim 1 , wherein the genetic variant is a single nucleotide variant.
16. The method of claim 1 , further comprising extending the first probe to generate an extended first probe before the ligating in step (d).
17. The method of claim 1 , further comprising contacting the tissue section with a plurality of gap probes, wherein a gap probe of the plurality of gap probes comprises a sequence substantially complementary to the nucleic acid, and further comprising ligating (i) the first probe and the 5′ end of the gap probe and (ii) the second probe and the 3′ end of the gap probe.
18. The method of claim 1 , wherein the tissue section is a formalin-fixed, paraffin-embedded section, a frozen section, or a fresh section.
19. The method of claim 1 , wherein the nucleic acid is RNA or DNA.
20. The method of claim 19 , wherein the RNA is an mRNA.
21. The method of claim 1 , wherein the first probe further comprises a functional sequence, wherein the functional sequence is a primer sequence, and wherein the capture probe binding domain comprises a poly(A) sequence.
22. The method of claim 1 , wherein the releasing step comprises contacting the ligated probe hybridized to the nucleic acid in the tissue section with an endoribonuclease.
23. The method of claim 22 , wherein the endoribonuclease is RNAseH.
24. The method of claim 23 , wherein the RNase H comprises RNase H1, RNase H2, or RNase H1 and RNase H2.
25. The method of claim 1 , wherein the method further comprises contacting the tissue section with a permeabilization reagent, wherein the permeabilization reagent comprises an endopeptidase.
26. The method of claim 1 , wherein the determining step comprises amplifying all or part of the ligated probe hybridized to the capture domain of the capture probe.
27. The method of claim 1 , wherein the determining step comprises sequencing.
28. The method of claim 11 , wherein the chemical ligation utilizes click chemistry.
29. The method of claim 1 , wherein the determining step further comprises extending the capture probe using the ligated probe as a template to generate an extended capture probe.