Methods and compositions for efficient base calling in sequencing reactions
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences. In particular, the present invention provides methods and compositions for improving the efficiency of sequencing reactions by using fewer labels to distinguish between nucleotides and by detecting nucleotides at multiple detection positions in a target sequence.
1. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:
(i) first dNTPs comprising a first label;
(ii) second dNTPs comprising a second label;
(iii) third dNTPs comprising both the first label and the second label; and
(iv) fourth dNTPs comprising neither the first label nor the second label;
wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and
wherein the first label and the second label are different labels and are distinguishable.
2. The composition of claim 1 wherein the first label and the second label are fluorescent dyes.
3. The composition of claim 2 wherein at least one of the first label and the second label comprises rhodamine.
4. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:
(i) first dNTPs comprising a first label;
(ii) second dNTPs comprising a second label;
(iii) third dNTPs wherein some of the third dNTPs comprise the first label but not the second label and some of the third dNTPs comprise the second label but not the first label; and
(iv) fourth dNTPs comprising neither the first label nor the second label;
wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and
wherein the first label and the second label are different labels and are distinguishable.
5. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:
(i) a plurality of first dNTPs labeled with a first label;
(ii) a plurality of second dNTPs comprising a second label;
(iii) a plurality of third dNTPs, wherein some of the third dNTPs are labeled with the first label but not the second label and some of the third dNTPs are labeled with the second label but not the first label; and
(iv) a plurality of fourth dNTPs labeled with neither the first label nor the second label;
wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and
wherein the first label and the second label are different labels and are distinguishable.
6. The composition of claim 5 wherein the first label and the second label are fluorescent dyes.
7. The composition of claim 6 wherein at least one of the first label and the second label comprises rhodamine.
8. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:
(i) a plurality of first dNTPs labeled with a first label;
(ii) a plurality of second dNTPs comprising a second label;
(iii) a plurality of third dNTPs, wherein the third dNTPs are labeled with both the first label and the second label; and
(iv) a plurality of fourth dNTPs labeled with neither the first label nor the second label;
wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and
wherein the first label and the second label are different labels and are distinguishable.
9. The composition of claim 8 wherein the first label and the second label are fluorescent dyes.
10. The composition of claim 9 wherein at least one of the first label and the second label comprises rhodamine.