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 labeled reversible terminator comprising a nucleotide analogue of dUTP having a 3′-O group and a label, wherein the label is Rhodamine 6G and is attached to the nucleotide analogue of dUTP though a cleavable linker comprising a disulfide bond, and wherein a removable chemical moiety caps the 3′-O group, wherein said labeled reversible terminator is in a mixture in solution with an unlabeled reversible terminator comprising a nucleotide analogue of dUTP, wherein the concentration of the unlabeled nucleotide analogue of dUTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dUTP.
2. The labeled reversible terminator of claim 1 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dGTP.
3. The labeled reversible terminator of claim 2 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.
4. The labeled reversible terminator of claim 3 , wherein said labeled nucleotide analogues are present at a concentration between 1 nM and 1 uM.
5. A labeled reversible terminator comprising a nucleotide analogue of dGTP having a 3′-O group and a label, wherein the label is Cy5 and is attached to the nucleotide analogue of dGTP though a cleavable linker comprising a disulfide bond, and wherein a removable chemical moiety caps the 3′-O group, wherein said labeled reversible terminator is in a mixture in solution with an unlabeled reversible terminator comprising a nucleotide analogue of dGTP, 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.
6. The labeled reversible terminator of claim 5 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dATP.
7. The labeled reversible terminator of claim 6 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.
8. The labeled reversible terminator of claim 7 , wherein said labeled nucleotide analogues are present at a concentration between 1 nM and 1 uM.