Methods and compositions for reducing nucleotide impurities
Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.
1. A composition comprising:
(a) labeled nucleotide triphosphates comprising a free 3′-OH,
(b) labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety, and
(c) a non-immobilized depletion polynucleotide comprising a depletion primer hybridized to a depletion template; and
(d) a depletion polymerase that is active to selectively incorporate the nucleotide triphosphates comprising a free 3′-OH into the depletion primer and incapable of incorporating the labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety;
(e) a sequencing primer hybridized to an immobilized polynucleotide; and
(f) a sequencing polymerase capable of incorporating the nucleotide triphosphates comprising a free 3′-OH and capable of incorporating the labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety.
2. The composition of claim 1 , wherein the composition is stored for one week at about 2° ° C.-8° C., about 20° ° C.-30° ° C., or about 4° C.-37° C.
3. The composition of claim 1 , wherein the depletion polynucleotide comprises a homopolymer sequence.
4. The composition of claim 1 , wherein the reversible terminator comprises an azido moiety, a disulfide moiety, or an alkoxyalkyl moiety.
5. The composition of claim 1 , wherein the labeled nucleotide triphosphates comprising the 3′ reversible terminator moiety has the formula:
wherein
R 1 is a triphosphate moiety;
R 2 is hydrogen or —OH;
R 3 is a reversible terminator moiety;
R 4 is a detectable moiety;
B is a divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof; and
L 100 is a divalent linker.
6. The composition of claim 1 , wherein the labeled nucleotide triphosphate comprising a free 3′-OH has the formula:
wherein
R 1 is a triphosphate moiety;
R 2 is hydrogen or —OH;
R 4 is a detectable moiety;
B is a divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof; and
L 100 is a divalent linker.
7. The composition of claim 5 or 6 , wherein L 100 is a cleavable linker.
8. The composition of claim 1 , wherein the depletion polynucleotide comprises 2 to 30 consecutive identical nucleotides.
9. The composition of claim 1 , comprising a first plurality of depletion polynucleotides comprising a homopolymer sequence of poly(dA) nucleotides; and a second plurality of depletion polynucleotides comprising a homopolymer sequence of poly(dC) nucleotides.
10. The composition of claim 9 , further comprising a third plurality of depletion polynucleotides comprising a homopolymer sequence of poly(dT) nucleotides; and a fourth plurality of depletion polynucleotides comprising a homopolymer sequence of poly(dG) nucleotides.
11. The composition of claim 1 , wherein the depletion polymerase comprises an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 6.
12. The composition of claim 1 , wherein the number of labeled nucleotide triphosphates comprising a free 3′-OH is less than the number labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety.
13. The composition of claim 1 , wherein the depletion polynucleotide comprises a hairpin structure and a 5′ overhang.
14. A composition comprising:
a labeled nucleotide comprising a 3′ reversible terminator moiety,
a depletion primer hybridized to a depletion polynucleotide;
a depletion polymerase, wherein the depletion polymerase is capable of incorporating 3′-OH nucleotides and incapable of incorporating the labeled nucleotide comprising a 3′ reversible terminator moiety;
a sequencing primer hybridized to a target polynucleotide; and
a sequencing polymerase, which is different than the depletion polymerase.
15. The composition of claim 14 , wherein the sequencing polymerase is capable of incorporating 3′-OH nucleotides, and capable of incorporating the labeled nucleotide comprising a 3′ reversible terminator moiety.
16. The composition of claim 14 , further comprising a nucleotide comprising a free 3′-OH.
17. The composition of claim 1 , comprising 4 labeled nucleotide triphosphates comprising a free 3′-OH, and 2,000 labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety.