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 a mixture of labeled and unlabeled reversible terminators in solution, said terminators comprising nucleotide analogues of dCTP, dGTP and dATP, wherein the concentration of the unlabeled nucleotide analogue of dCTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dCTP, wherein the concentration of the unlabeled nucleotide analogue of dGTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dGTP, wherein the concentration of the unlabeled nucleotide analogue of dATP is present in said solution at a higher concentration than the labeled nucleotide analogue of dATP, and wherein each labeled and unlabeled nucleotide analogue contains a removable chemical moiety capping the 3′-OH group.
2. The composition of claim 1 , wherein said terminators further comprise nucleotide analogues of dTTP, wherein the concentration of the unlabeled nucleotide analogue of dTTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dTTP, wherein each labeled and unlabeled nucleotide analogue of dTTP contains a removable chemical moiety capping the 3′-OH group.
3. The composition of claim 1 , wherein said unlabeled reversible terminators are present at a concentration between 1 uM and 100 uM.
4. The composition of claim 1 , wherein said first plurality of nucleotide analogues is present at a concentration between 1 nM and 1 uM.
5. The composition of claim 1 , wherein the labeled reversible terminators comprise a label selected from the group consisting of BODIPY, Rhodamine, Carboxyrhodamine, and Cyanine.
6. The composition of claim 1 , wherein said removable chemical moiety comprises a disulfide bond.
7. The composition of claim 1 , wherein said removable chemical moiety comprises an azido group.
8. The composition of claim 1 , wherein said removable chemical moiety comprises an azidomethyl ether.
9. The composition of claim 5 , wherein said label is attached via a cleavable linker.
10. The composition of claim 9 , wherein said cleavable linker comprises a disulfide bond.
11. The composition of claim 9 , wherein said removable chemical moiety comprises an azido group and said cleavable linker comprises a disulfide bond.
12. The composition of claim 1 , further comprising polymerase.