Spatial sequencing

Patent metadata

Field	Value
Publication number	US-11680288-B2
Application number	US-202217815542-A
Country	US
Kind code	B2
Filing date	Jul 27, 2022
Priority date	Aug 6, 2020
Publication date	Jun 20, 2023
Grant date	Jun 20, 2023

Abstract

Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a sample comprising a cell.

First claim

What is claimed is:

1. A method of sequencing an endogenous nucleic acid of a cell, said method comprising:
i) contacting the cell with a polynucleotide probe comprising a first region and a second region, hybridizing the first region of the polynucleotide probe to a first sequence of said endogenous nucleic acid, and hybridizing the second region of the polynucleotide probe to a second sequence of said endogenous nucleic acid, thereby forming a complex comprising the polynucleotide probe hybridized to said endogenous nucleic acid, wherein said endogenous nucleic acid comprises a target sequence between the first sequence and the second sequence, and wherein the polynucleotide probe comprises one or more locked nucleic acid (LNA) nucleotides;
ii) extending the polynucleotide probe with deoxynucleotide triphosphates (dNTPs) along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming a circular oligonucleotide;
iii) amplifying the circular oligonucleotide by extending a first amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase to generate a first extension product comprising one or more copies of the target sequence, and hybridizing the first extension product to a second amplification primer and extending with a polymerase to generate a second extension product comprising one or more complements of the first extension product; and
iv) sequencing the one or more copies of the target sequence in said cell, wherein sequencing comprises extending one or more sequencing primers hybridized to the first extension product.

2. A method of sequencing an endogenous RNA molecule in a cell, said method comprising:
i) contacting the cell with a polynucleotide probe and hybridizing a 3′ end of the polynucleotide probe to a first complementary sequence of the endogenous RNA molecule, and hybridizing a 5′ end of the polynucleotide probe to a second complementary sequence of the endogenous RNA molecule, wherein said first complementary sequence and second complementary sequence are separated by 1 or more nucleotides, and wherein the polynucleotide probe comprise one or more locked nucleic acid (LNA) nucleotides;
ii) extending the 3′ end of the polynucleotide probe along the endogenous RNA molecule to generate a complement of the endogenous RNA molecule, and ligating the complement of the endogenous RNA molecule to the 5′ end of the polynucleotide probe to form a circular oligonucleotide;
iii) amplifying the circular oligonucleotide by extending a first amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase to generate a first extension product comprising one or more copies of the endogenous RNA molecule, and hybridizing the first extension product to a second amplification primer and extending with a polymerase to generate a second extension product comprising one or more complements of the first extension product; and
iv) sequencing the one or more copies of the endogenous RNA molecule in said cell, wherein sequencing comprises extending one or more sequencing primers hybridized to the first extension product.

3. The method of claim 2 , wherein extending consists of incorporating deoxyribonucleotides into the 3′ end of the polynucleotide probe.

4. The method of claim 1 , wherein said polynucleotide probe comprises DNA, RNA, or a combination thereof.

5. The method of claim 1 , wherein said target sequence is at least 5 nucleotides in length.

6. The method of claim 1 , wherein the target sequence is an oncogene, or a cancer-associated gene; a bacterial nucleic acid sequence, a fungal nucleic acid sequence, or a viral nucleic acid sequence; a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, or any fragment thereof; a B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof; or a gene sequence corresponding to an immunoglobulin light chain polypeptide, or a gene sequence corresponding to an immunoglobulin heavy chain polypeptide.

7. The method of claim 1 , wherein the circular oligonucleotide comprises DNA, RNA, or a combination thereof.

8. The method of claim 1 , wherein said sequencing in said cell is performed in situ.

9. The method of claim 1 , wherein the endogenous nucleic acid is an RNA molecule.

10. The method of claim 2 , wherein the first complementary sequence and the second complementary sequence are separated by 5 or more nucleotides.

11. The method of claim 2 , wherein the first complementary sequence and the second complementary sequence are separated by 10 or more nucleotides.

12. The method of claim 1 , wherein the cell is a bacterial cell, a fungal cell, a plant cell, a mammalian cell, a stem cell, an immune cell, a cancer cell, a viral-host cell, or a cell that selectively binds to a desired target.

13. The method of claim 2 , wherein the endogenous RNA molecule comprises a CDR3 sequence.

14. The method of claim 1 , wherein the cell is a neuronal cell, an endothelial cell, epithelial cell, germ cell, plasma cell, a muscle cell, peripheral blood mononuclear cell (PBMC), a myocardial cell, cancer cell, or a retina cell.

15. The method of claim 1 , wherein the cell is permeabilized and immobilized to a solid support.

16. The method of claim 15 , wherein the solid support comprises a patterned surface suitable for immobilization of a plurality of cells in an ordered pattern.

17. The method of claim 1 , wherein the polynucleotide probe is about 50 to about 500 nucleotides in length.

18. The method of claim 1 , wherein the polynucleotide probe is a single-stranded polynucleotide comprising at least one amplification primer binding sequence, at least one sequencing primer binding sequence, or both one amplification primer binding sequence and one sequencing primer binding sequence.

19. The method of claim 1 , wherein the circular oligonucleotide is about 100 to about 1000 nucleotides in length, about 100 to about 300 nucleotides in length, about 300 to about 500 nucleotides in length, or about 500 to about 1000 nucleotides in length.

20. The method of claim 1 , wherein amplifying comprises rolling circle amplification (RCA).

21. The method of claim 1 , wherein the extension product comprises three or more copies of the circular oligonucleotide.

22. The method of claim 1 , wherein said sequencing comprises sequencing-by-synthesis.

23. The method of claim 1 , wherein sequencing comprises extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue, and detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue, wherein the sequencing primer is hybridized to the extension product.

24. The method of claim 2 , wherein two or more polynucleotide probes hybridize to different complementary sequences of the same endogenous RNA molecule.

25. The method of claim 1 , further comprising identifying a protein in the cell, wherein detecting the protein comprises:
i) contacting the cell with a specific binding reagent and binding said specific binding reagent to the protein, wherein said specific binding reagent comprises an oligonucleotide;
ii) hybridizing a first region of a second polynucleotide probe to a first sequence of said oligonucleotide, and hybridizing a second region of the second polynucleotide probe to a sec

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N

Classifications

• C12Q1/6841 Primary

  In situ hybridisation · CPC title