Methods of sequencing circular template polynucleotides

1. A method of sequencing, the method comprising:

(A) extending an amplification primer hybridized to a circular template polynucleotide with a strand-displacing polymerase to generate an immobilized extension product comprising one or more complements of the circular template polynucleotide, wherein said amplification primer is immobilized to a solid support;

(B) hybridizing a first sequencing primer to the immobilized extension product, and extending said first sequencing primer hybridized to said immobilized extension product and generating a first sequencing read; and

(C) hybridizing a second sequencing primer to said immobilized extension product and extending said second sequencing primer and generating a second sequencing read.

2. The method of claim 1 , wherein the circular template polynucleotide comprises primer binding sequences complementary to one or more additional amplification primers.

3. The method of claim 1 , wherein extending the amplification primer comprises rolling circle amplification (RCA), exponential rolling circle amplification (eRCA), hyperbranched rolling circle amplification (HRCA), loop-mediated isothermal amplification (LAMP), or multiple displacement amplification (MDA).

4. The method of claim 1 , wherein extending the amplification primer comprises rolling circle amplification (RCA) or exponential rolling circle amplification (eRCA).

5. The method of claim 1 , wherein the circular template polynucleotide is single-stranded DNA.

6. The method of claim 1 , wherein prior to hybridizing the first sequencing primer, said method further comprises amplifying the immobilized extension product or a complement thereof with a plurality of primers attached to the solid support, wherein the plurality of primers comprise a first plurality of primers comprising a complementary sequence to the immobilized extension product and a second plurality of primers comprising a complementary sequence to a complement of the immobilized extension product, and the amplifying comprises a plurality of cycles of strand denaturation, primer hybridization, and primer extension.

7. The method of claim 1 , prior to (A), the method further comprises forming the circular template polynucleotide by ligating two ends of a linear polynucleotide together.

8. The method of claim 1 , wherein extending the amplification primer comprises incubating the amplification primer with the strand-displacing polymerase (i) for about 10 seconds to about 30 minutes, and/or (ii) at a temperature of about 20° C. to about 50° C.

9. The method of claim 8 , wherein prior to extending the amplification primer, the strand-displacing polymerase contacts the amplification primer and circular template polynucleotide in the absence of dNTPs; and optionally, any excess strand-displacing polymerase is removed.

10. The method of claim 6 , wherein amplifying the immobilized extension product or a complement thereof comprises an initial denaturation at about 85° C.-95° C. for about 1 minutes to about 10 minutes.

11. The method of claim 6 , wherein the plurality of cycles comprises thermally cycling between (i) about 85° C.-95° C. for about 15-30 sec for denaturation, and (ii) about 65° C. for about 30 seconds for annealing/extension of the primer.

12. The method of claim 6 , wherein amplifying the immobilized extension product comprises a plurality of fluidic cycles of strand denaturation using a chemical denaturant, and a plurality of fluidic cycles of primer extension comprising an extension mixture.

13. The method of claim 1 , wherein the circular template polynucleotide 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.

14. The method of claim 1 , wherein the 3′ end of the immobilized extension product comprises a blocking moiety.

15. The method of claim 1 , wherein the solid support further comprises a polymer or hydrogel to which the amplification primer is attached.

16. A method of sequencing a circular template polynucleotide, the method comprising:

(A) hybridizing a first sequencing primer to a first polynucleotide immobilized to a solid support, and extending said first sequencing primer hybridized to said polynucleotide and generating a first sequencing read, wherein said first polynucleotide comprises one or more complements of a circular template polynucleotide, and wherein said first polynucleotide comprises a cleavable site;

(B) cleaving the cleavable site and removing the first polynucleotide;

(C) hybridizing a second sequencing primer to a second polynucleotide immobilized to the solid support, and extending said second sequencing primer hybridized to said polynucleotide and generating a second sequencing read, wherein said second polynucleotide comprises a complement of the first polynucleotide; and

(D) hybridizing a third sequencing primer to said second sequencing read and extending said third sequencing primer and generating a third sequencing read.

17. A method of detecting a circular template polynucleotide in or on a cell or tissue, the method comprising:

(A) extending an amplification primer hybridized to a circular template polynucleotide in or on a cell or tissue with a strand-displacing polymerase to generate an immobilized extension product comprising one or more complements of the circular template polynucleotide, wherein said amplification primer is attached to a cellular component in or on the cell or tissue;

(B) hybridizing a primer to the immobilized extension product, and incorporating a labeled nucleotide into the primer; and

(C) detecting the labeled nucleotide, thereby detecting the circular template polynucleotide.

18. The method of claim 17 , wherein the circular template polynucleotide comprises a barcode sequence.

19. The method of claim 17 , wherein said cellular component is a protein.

20. The method of claim 17 , wherein the labeled nucleotide comprises a 3′ blocking moiety.

21. The method of claim 1 , wherein the solid support further comprises a cell to which the amplification primer is attached.

22. The method of claim 1 , wherein generating a first sequencing read comprises determining the identity of a portion of the immobilized extension product.

23. The method of claim 1 , wherein said amplification primer is covalently attached to a cellular component in or on a cell, wherein the cell is immobilized to a flow cell surface.

24. The method of claim 17 , wherein the immobilized extension product comprises a barcode sequence.

25. The method of claim 17 , wherein the amplification primer is covalently attached to the cellular component.

26. The method of claim 25 , wherein said cellular component is a protein.

27. The method of claim 25 , wherein the cellular component is on the cell.

28. The method of claim 17 , wherein the amplification primer is attached to an organelle.

29. The method of claim 17 , wherein the circular template polynucleotide is attached to an RNA transcript.