Methods of quantifying target nucleic acids and identifying sequence variants

1. A method for identifying nucleotides in a template nucleic acid, comprising

(a) providing a solid support comprising a template nucleic acid comprising a first region and a second region, wherein the template nucleic acid is hybridized to a first primer having a 3′ match upstream of the first region, and wherein the template nucleic acid is hybridized to a second primer having a 3′ mismatch upstream of the second region;

(b) after step (a), forming a first stabilized ternary complex that comprises the template nucleic acid hybridized to the first primer, a polymerase and a first cognate nucleotide, wherein the 3′ mismatch prevents the second primer from forming a stabilized ternary complex;

(c) after steps (a) and (b), detecting the first stabilized ternary complex to identify the first cognate nucleotide, wherein formation and detection occurs without incorporation of the first cognate nucleotide into the first primer, and wherein the first primer comprises a terminal nucleotide with a chemical modification that inhibits incorporation;

(d) after steps (a)-(c), removing the 3′ mismatch from the second primer;

(e) after steps (a)-(d) after step (d), forming a second stabilized ternary complex comprising the template nucleic acid hybridized to the second primer, a polymerase and a second cognate nucleotide; and

after steps (a)-(e), detecting the second stabilized ternary complex to identify the second cognate nucleotide, wherein formation and detection occurs without incorporation of the second cognate nucleotide into the second primer, and wherein the second primer comprises a terminal nucleotide with a chemical modification that inhibits incorporation.

2. The method of claim 1 , wherein the template nucleic acid comprises a concatemer of target sequences.

3. The method of claim 1 , wherein the template nucleic acid is covalently attached to the solid support.

4. The method of claim 1 , wherein the template nucleic acid is attached to the solid support via hybridization a primer.

5. The method of claim 1 , wherein the second cognate nucleotide complements a base in a barcode sequence.

6. The method of claim 5 , wherein the first cognate nucleotide complements a sequence variant in a target sequence.

7. The method of claim 1 , wherein the first cognate nucleotide comprises a label that is detected in step (c).

8. The method of claim 1 , wherein the second cognate nucleotide comprises a label that is detected in step (f).

9. The method of claim 1 , wherein the chemical modification on the first primer comprises a reversible blocking moiety.

10. The method of claim 9 , wherein step (c) further comprises (ii) deblocking the first primer and (iii) adding a reversibly terminated nucleotide to the deblocked primer, thereby producing a reversibly terminated, extended primer.

11. The method of claim 1 , further comprising, prior to step (d), incorporating the first cognate nucleotide into the primer.

12. The method of claim 11 , wherein the chemical modification on the second primer comprises a reversible blocking moiety.

13. The method of claim 12 , wherein step (f) further comprises (ii) deblocking the second primer and (iii) adding a reversibly terminated nucleotide to the deblocked second primer, thereby producing a reversibly terminated, extended, second primer.

14. The method of claim 1 , further comprising, after step (f), incorporating the second cognate nucleotide into the primer.

15. The method of claim 1 , wherein the polymerase in the first stabilized ternary complex comprises a label that is detected in step (c).

16. The method of claim 1 , wherein the polymerase in the second stabilized ternary complex comprises a label that is detected in step (f).

17. The method of claim 1 , wherein step (d) comprises removing the 3′ mismatch from the second primer using a polymerase with a 3′-5′ exonuclease activity.

18. The method of claim 1 , wherein a non-naturally occurring nucleotide is present at the 3′ end of the second primer.

19. The method of claim 1 , further comprising, prior to step (d), replacing the first cognate nucleotide with a nucleotide that is incorporated into the primer.

20. The method of claim 1 , further comprising, after step (f), replacing the second cognate nucleotide with a nucleotide that is incorporated into the primer.