Methods and compositions for reducing nucleotide impurities
Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.
1. A method of sequencing, said method comprising:
(a) contacting a sequencing solution comprising a plurality of labeled nucleotides comprising a free 3′-OH and a plurality of labeled nucleotides lacking a free 3′-OH with depleting reagents to generate a refined solution comprising a reduced amount of labeled nucleotides comprising a free 3′-OH, wherein the depleting reagents comprise
a depletion primer hybridized to a depletion polynucleotide and a depletion polymerase capable of incorporating the nucleotides comprising a free 3′-OH into the depletion polynucleotide and incapable of incorporating labeled nucleotide triphosphates comprising a 3′ reversible terminator moiety, wherein the depletion polynucleotide is free in solution;
(b) contacting a sequencing primer annealed to a target polynucleotide with the refined solution, incorporating with a sequencing polymerase a labeled nucleotide lacking a free 3′-OH into the sequencing primer, and detecting the label of the incorporated labeled nucleotide; and
(c) repeating (b).
2. The method of claim 1 , wherein (b) comprises heating the refined solution to about 55° C. to about 65° C.
3. The method of claim 1 , wherein the depletion polymerase comprises a Klenow fragment or mutant thereof.
4. The method of claim 1 , wherein the depletion polynucleotide comprises a homopolymer sequence, or a single polynucleotide comprising a hairpin structure and a 5′ overhang.
5. The method of claim 1 , wherein (a) occurs at about 1° C. to about 45° C.
6. The method of claim 5 , further comprising, after (a), increasing the temperature to about 55° C. to about 65° C.
7. The method of claim 1 , wherein each of the labeled nucleotides lacking a free 3′-OH comprise a reversible terminator moiety.
8. The method of claim 7 , wherein the reversible terminator moiety comprises an azido moiety, a disulfide moiety, or an alkoxyalkyl moiety.
9. The method of claim 1 , wherein the labeled nucleotide lacking a free 3′-OH 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.
10. The method of claim 1 , wherein the labeled nucleotide 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.
11. The method of claim 10 , wherein L 100 is a cleavable linker.
12. The method of claim 1 , wherein the depletion polynucleotide comprises 2 to 30 consecutive identical nucleotides.
13. The method of claim 1 , wherein the depletion polynucleotide comprises a depletion primer hybridized to a depletion template.
14. The method of claim 1 , wherein the depletion polynucleotide comprises poly(A), poly(G), poly(C), and poly(T) nucleotides.
15. The method 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: 5.
16. The method of claim 1 , wherein the labeled nucleotides comprising a free 3′-OH and the labeled nucleotides lacking a free 3′-OH each comprise:
a first plurality of labeled adenosine triphosphates;
a second plurality of labeled thymidine triphosphates;
a third plurality of labeled guanosine triphosphates; and
a fourth plurality of labeled cytosine triphosphates.
17. The method of claim 1 , wherein sequencing polymerase is mutated to favor incorporating nucleotides lacking a free 3′-OH over nucleotides comprising a free 3′-OH.
18. The method of claim 4 , wherein the depletion polynucleotide is a polynucleotide comprising a hairpin structure and a 5′ overhang.
19. A method of incorporating a labeled nucleotide into a sequencing primer, said method comprising:
(i) forming a refined solution comprising a reduced amount of labeled nucleotides comprising a free 3′-OH from an unrefined solution, wherein said unrefined solution comprises a labeled nucleotide comprising a 3′-OH and a labeled nucleotide comprising a 3′-reversible terminator moiety, wherein forming the refined solution comprises incorporating with a depletion polymerase the labeled nucleotide comprising a 3′-OH into a depletion primer hybridized to a depletion polynucleotide, wherein,
a) the depletion polymerase is capable of incorporating a labeled nucleotide comprising a 3′-OH into the depletion polynucleotide and incapable of incorporating a labeled nucleotide comprising a 3′-reversible terminator moiety; and
b) the depletion polynucleotide comprises a hairpin structure and a 5′ overhang; and
(ii) contacting a target polynucleotide hybridized to the sequencing primer with the refined solution and incorporating with a sequencing polymerase the labeled nucleotide sequencing comprising the 3′-reversible terminator moiety into the sequencing primer.