Methods and compositions for incorporating nucleotides
The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.
1. A composition comprising i) a first plurality of labeled reversible terminators comprising nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′-OH group; and ii) a second plurality of unlabeled reversible terminators comprising nucleotide analogues wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′-OH group, wherein the second plurality of nucleotide analogues is present in said solution at a higher concentration than said first plurality of nucleotide analogues.
2. The composition of claim 1 , wherein said nucleotide analogues are in solution.
3. The composition of claim 1 , wherein said second plurality of nucleotide analogues is present at a concentration between 1 μM and 100 μM.
4. The composition of claim 1 , wherein said first plurality of nucleotide analogues is present at a concentration between 1 μM and 1 μM.
5. The composition of claim 1 , wherein said first plurality of nucleotide analogues comprises four different nucleotide analogues.
6. The composition of claim 5 , wherein said second plurality of nucleotide analogues comprises four different nucleotide analogues.
7. The composition of claim 5 , wherein each of said four different nucleotide analogues comprises a unique label, said label selected from the group consisting of BODIPY, Rhodamine, Carboxyrhodamine, and Cyanine.
8. The composition of claim 1 , wherein said removable chemical moiety comprises a disulfide bond.
9. The composition of claim 1 , wherein said removable chemical moiety comprises an azido group.
10. The composition of claim 1 , wherein said removable chemical moiety comprises an azidomethyl ether.
11. The composition of claim 1 , wherein said label is attached via a cleavable linker.
12. The composition of claim 10 , wherein said cleavable linker comprises a disulfide bond.
13. The composition of claim 10 , wherein said removable chemical moiety comprises an azido group and said cleavable linker comprises a disulfide bond.
14. The composition of claim 1 , further comprising polymerase.
15. A composition comprising i) a first plurality of labeled reversible terminators comprising nucleotide analogues comprising four different nucleotide analogues, wherein each different nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′-OH group; and ii) a second plurality of unlabeled reversible terminators comprising nucleotide analogues comprising four different nucleotide analogues, wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′-OH group, wherein the second plurality of nucleotide analogues is present in said solution at a higher concentration than said first plurality of nucleotide analogues.
16. The composition of claim 15 , wherein said nucleotide analogues are in solution.
17. The composition of claim 15 , wherein said second plurality of nucleotide analogues is present at a concentration between 1 μM and 100 μM.
18. The composition of claim 15 , wherein said first plurality of nucleotide analogues is present at a concentration between 1 nM and 1 μM.
19. The composition of claim 15 , further comprising a polymerase.