Compositions and methods for improving sequencing signals
Embodiments of the present disclosure relate to compositions and kits for use in sequencing by synthesis to improve fluorescent signal intensity and reduce signal decay caused by short wavelength light source during the imaging events. Methods of sequencing using the compositions and kits described herein are also provided.
1 . A method for reducing light-induced sequencing signal decay during sequencing by synthesis, comprising:
(i) contacting a solid support with an incorporation mixture comprising DNA polymerase and four different types of nucleotides A. C. G, and T or U, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon, and sequencing primers that are complementary and hybridized to at least a portion of the target polynucleotides;
(ii) incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one of the four different types of nucleotides comprises a detectable label, and each of the four different types of nucleotides comprises a 3′ blocking group;
(iii) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides in an aqueous scan mixture to determine the identity of the incorporated nucleotides using one or more light sources operating at wavelengths between 450-460 nm and between 520-535 nm; and
(iv) removing the 3′ blocking groups and the detectable labels of the incorporated nucleotides;
wherein the aqueous scan mixture comprises one or more additives for reducing fluorescent signal decay caused by the fluorescent measurements, wherein the one or more additives comprise a salt or an ester of gallic acid, at least one triplet state quencher (TSQ), and one or more compounds selected from the group consisting of a radical scavenger, an oxygen scavenger, a reducing reagent, and combinations thereof, and wherein the method reduces signal decay by at least 10% after about 300 cycles when comparing to the method using an aqueous scan mixture without the at least one TSQ, and one or more compounds selected from the group consisting of an oxygen scavenger, a radical scavenger, a reducing reagent, and combinations thereof.
2 . The method of claim 1 , wherein the oxygen scavenger comprises an O 2 scavenger, and the radical scavenger comprises a reactive oxygen species (ROS) scavenger.
3 . The method of claim 1 , wherein the aqueous scan mixture further comprises at least one oxygen scavenger or at least one reducing reagent.
4 . The method of claim 1 , wherein the TSQ comprises a nickel (II) salt or complex, cyclooctatetraene (COT) or a substituted analog thereof, 2-mercaptoethylamine (MEA) or a salt thereof, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) or a salt thereof, or combinations thereof.
5 . The method of claim 1 , wherein the oxygen scavenger comprises an enzyme capable of reacting with oxygen, glucose oxidase, catalase, diethylhydroxylamine (DEHA), hydroquinone, or combinations thereof.
6 . The method of claim 1 , wherein the radical scavenger comprises 1,4-diazabicyclo [2.2.2] octane (DABCO), caffeine, mannitol, or combinations thereof.
7 . The method of claim 1 , wherein the reducing reagent comprises a phosphine or a salt thereof, sodium sulfite (Na 2 SO 3 ), a thiol containing compound, 2-mercaptoethanol (bME), cysteine or an analog thereof, or combinations thereof.
8 . The method of claim 1 , wherein the aqueous scan mixture comprises 2-hydroxylethyl gallate, a salt of MEA, Trolox, 2-mercaptoethanol, and cysteine or an analog thereof.
9 . The method of claim 1 , wherein the aqueous scan mixture has a basic pH of from about 7.2 to about 8.0.
10 . The method of claim 1 , wherein step (iii) comprises using two light sources operating at wavelengths between 450-460 nm and between 520-535 nm.
11 . The method of claim 1 , further comprising: step (v) contacting the solid support with an aqueous wash solution.
12 . The method of claim 11 , wherein steps (i) through (v) are repeated at least about 50 cycles to determine the target polynucleotides sequences.
13 . The method of claim 12 , wherein the method reduces sequencing error rate, percent phasing value, or percent prephasing value by at least 10% after about 300 cycles, when comparing to the method using an aqueous scan mixture without the one or more compounds selected from the group consisting of a TSQ, a radical scavenger, an oxygen scavenger, a reducing reagent, and combinations thereof.
14 . A kit for use with a sequencing apparatus, comprising a scan mixture composition, the scan mixture composition comprising:
2-hydroxylethyl gallate, a salt of 2-mercaptoethylamine (MEA), Trolox, 2-mercaptoethanol, and cysteine or an analog thereof.
15 . The kit of claim 14 , further comprising an incorporation mixture composition for incorporating 3′ blocked, labeled nucleotides into copy polynucleotide strands complementary to at least a portion of template polynucleotide strands, wherein the incorporation mixture composition comprises:
one or more different types of labeled nucleotides, wherein each of the labeled nucleotides comprises a 3′ blocking group; and
a DNA polymerase.
16 . The kit of claim 14 , wherein the concentrations of 2-hydroxylethyl gallate and Trolox in the scan mixture composition are independently from about 1 mM to about 50 mM, from about 5 mM to about 20 mM, or about 10 mM.
17 . The kit of claim 14 , wherein the concentration of the salt of MEA in the scan mixture composition is from about 10 mM to about 200 mM, or about 100 mM.
18 . The kit of claim 14 , wherein the concentration of 2-mercaptomethanol in the scan mixture composition is from about 1 mM to about 10 mM, or from about 2 mM to about 5 mM.
19 . The kit of claim 14 , wherein the concentration of cysteine or the analog thereof in the scan mixture composition is from about 1 mM to about 100 mM, from 5 mM to about 50 mM, or about 20 mM.
20 . The method of claim 1 , wherein the concentrations of the salt or ester of gallic acid in the scan mixture composition are independently from about 1 mM to about 50 mM, from about 5 mM to about 20 mM, or about 10 mM.
21 . The method of claim 1 , wherein the concentration of at least one TSQ in the scan mixture composition is from about 1 mM to about 100 mM.
22 . The method of claim 1 , wherein the concentration of the reducing reagent in the scan mixture composition is from about 1 mM to about 50 mM, or from about 2 mM to about 25 mM.