Reagents for massively parallel nucleic acid sequencing

Abstract

A method of analysing a nucleic acid is claimed comprising: (a) bringing a primed nucleic acid sequence into contact with a fluorescently-labelled nucleotide conjugate under conditions sufficient to f

Description

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 108,207, filed October 30, 2020, which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Sequences of nucleic acid molecules may be determined using massively parallel sequencing. Massively parallel sequencing may be performed using sequencing by synthesis in which, in a primer extension reaction, a polymerizing enzyme adds nucleotides sequentially to a growing strand to yield a strand that is complementary to a template strand, and the nucleotides being incorporated are detected. Such sequences may be used in various applications, such as, for example, disease (e.g., cancer) diagnostics. SUMMARY

[0003] Aspects disclosed herein provide methods of analyzing a nucleic acid, the method comprising: (a) bringing a primed nucleic acid sequence into contact with a fluorescently-labeled nucleotide conjugate under conditions sufficient to form a binding complex comprising a first nucleotide of the primed nucleic acid molecule bound to a second nucleotide of the fluorescently-labeled nucleotide conjugate; (b) contacting the binding complex with an imaging reagent; and (c) obtaining an image of the binding complex in a presence of the imaging reagent, thereby reducing a risk of photo-bleaching of the fluorescently-labeled nucleotide conjugate as compared to a risk of photo-bleaching under like conditions in absence of the imaging reagent.

[0004] In some embodiments, the method further comprises identifying the first nucleotide by analyzing the image obtained in (c). In some embodiments, the imaging reagent comprises ascorbic acid. In some embodiments, the ascorbic acid comprises sodium ascorbate. In some embodiments, the concentration of the ascorbic acid is at least 20 mM. In some embodiments, the concentration of the ascorbic acid is between about lOmM and about 100 mM. In some embodiments, the concentration of the ascorbic acid is about 50 mM. In some embodiments, the fluorescently-labeled nucleotide conjugate comprises: (i) a core, (ii) a plurality of the second nucleotide coupled thereto, and (ii) one or more fluorophores directly coupled to the core. In some embodiments, the fluorescently-labeled nucleotide conjugate 18 08 25 further comprises a core attachment moiety linking the plurality of the second nucleotide to the core. In some embodiments, the second nucleotide comprises between about 3 and about 10 phosphate groups. In some embodiments, the second nucleotide is a nucleotide triphosphate comprising a removable chain terminating moiety.

[0005] In some embodiments, the method further comprises bringing the primed nucleic acid sequence into contact with a polymerase under conditions sufficient to form the binding complex, wherein the binding complex further comprises the polymerase. In some embodiments, the polymerase lacks a detectable label. In some embodiments, the polymerase comprises a detectable label. In some embodiments, the binding complex is immobilized to a support. In some embodiments, the support comprises a surface, and wherein the surface comprises a hydrophilic coating layer coupled thereto. In some embodiments, the hydrophilic coating layer comprises a water contact angle of less than 50 degrees.

[0006] In some embodiments, the method further comprises bringing the primed nucleic acid sequence into contact with a second fluorescently-labeled nucleotide conjugate under conditions sufficient to form a second binding complex comprising the next nucleotide of the primed nucleic acid molecule bound to a third nucleotide of the fluorescently-labeled nucleotide conjugate, wherein the third nucleotide is different than the second nucleotide. In some embodiments, the primed nucleic acid sequence is comprised in a concatemer nucleic acid molecules comprising tandem repeats of the primed nucleic acid sequence. In some embodiments, the imaging reagent comprises a non-catalytic divalent cation that inhibits incorporation of the second nucleotide, wherein the non-catalytic divalent cation comprises strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium ions.

[0007] In some aspects, the present disclosure describes a formulation for reducing photo-bleaching of a biological entity during imaging, the formulation comprising: at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and ascorbic acid. In some embodiments, the pH buffering agent comprises Tris-HCl. In some embodiments, the pH of the Tris-HCL is about 8.8. In some embodiments, the chelating agent comprises EDTA. In some embodiments, the monovalent cation comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. In some embodiments, the non-catalytic divalent cation comprises strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium ions, or a combination thereof. In some embodiments, the formulation lacks a catalytic divalent cation which 18 08 25 comprises magnesium or manganese, or a combination thereof. In some embodiments, the detergent comprises Triton X-100 (RTM). In some embodiments, the formulation further comprises a sugar. In some embodiments, the formulation further comprises a viscosity agent comprising glycerol. In some embodiments, the formulation further comprises 1,3,5,7 cyclooctatetraene (COT). In some embodiments, the COT has a concentration of about 2 micromolar (mM). In some embodiments, the ascorbic acid comprises sodium ascorbate. In some embodiments, the concentration of the ascorbic acid is at least 10 mM. In some embodiments, the concentration of the ascorbic acid is at least 20 mM. In some embodiments, the concentration of the ascorbic acid is between about 10 m M and about 100 mM. In some embodiments, the concentration of the ascorbic acid is about 50 mM. In some embodiments, the formulation further comprises Trolox. In some embodiments, the concentration of the Trolox is about 2 mM. In some embodiments, the formulation further comprises 3-nitrobenzoic acid (NBA). In some embodiments, the formulation further comprises cysteamine.

[0008] Aspects disclosed herein provide formulations comprising (i) at least one solvent, (ii) a pH buffering agent, (iii) a chelating agent, (iv) at least one monovalent cation, (v) a non-catalytic divalent cation, (vi) a detergent, (vii) a plurality of multivalent molecules and (viii) a sequencing polymerase enzyme, wherein each of the plurality of multivalent molecules comprises (1) a core, and (2) a plurality of nucleotides and a plurality of detectable moi eties coupled to the core, and wherein the sequencing polymerase enzyme comprises an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 2-5. In some embodiments, each of the plurality of multivalent molecules further comprises a linker that couples the plurality of nucleotides to the core, and wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits. In some embodiments, the core is spheroidal. In some embodiments, the plurality of nucleotides are of the same type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of the multivalent molecules comprises two or more different types of multivalent molecules, wherein each of the two or more different types of the multivalent molecules comprise a different plurality of nucleotides. In some embodiments, the sequencing polymerase comprises a mutation in the amino acid sequence comprising a substitution at one or more positions relative to SEQ ID NO: 1 comprising Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2-5. 18 08 25

[0009] Aspects disclosed herein provide kits comprising one or more containers containing a formulation described herein. In some embodiments, the kit further comprises instructions for reducing photo-damage to the biological entity while imaging the biological entity during a biochemical reaction in the presence of the formulation. In some embodiments, the biochemical reaction comprises a sequencing reaction. In some embodiments, the instructions comprise submerging the biological entity in the formulation during the biochemical reaction prior to imaging the biological entity.

[0010] Aspects disclosed herein provide kits comprising: a trap reagent comprising at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent, a plurality of fluorescently-labeled nucleotide conjugates and a first sequencing polymerase enzyme; a post-trap reagent comprising at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and a first sequencing polymerase enzyme; an imaging reagent comprising at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and ascorbic acid; and a stepping reagent comprising at least one solvent, at least one pH buffering agent, at least one monovalent cation, a catalytic divalent cation, a detergent, a second sequencing polymerase enzyme and a plurality of nucleotides, wherein each nucleotide of the plurality of nucleotides comprises a cleavable terminator moiety attached to a 3’ sugar position.

[0011] In some embodiments, the kit further comprises instructions for using the trap reagent, wherein the instructions comprise contacting a plurality of immobilized template nucleic acid molecules with (i) the trap reagent and (ii) a plurality of sequencing primers under conditions sufficient to form a plurality of immobilized fluorescently-labeled ternary complexes without incorporating the plurality of fluorescently-labeled nucleotide conjugates into the sequencing primer.

[0012] In some embodiments, the kit further comprises instructions for using the posttrap reagent, wherein the instructions comprise contacting the plurality of immobilized fluorescently-labeled ternary complexes with a post-trap reagent under conditions sufficient for preserving the plurality of immobilized fluorescently-labeled ternary complexes without incorporation of the plurality of immobilized fluorescently-labeled ternary complexes into the sequencing primer.

[0013] In some embodiments, the kit further comprises instructions for using the stepping reagent, wherein the instructions comprise contacting the plurality of immobilized template 18 08 25 nucleic acid molecules with the stepping reagent under conditions sufficient to extend the immobilized template nucleic acid molecule.

[0014] In some embodiments, the kit further comprises a first amplification reagent comprising at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions, a plurality of nucleotides and an amplification polymerase enzyme; and a second amplification reagent comprising at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions and a plurality of nucleotides.

[0015] In some embodiments, the kit further comprises instructions for using the first amplification reagent and the second amplification reagent, wherein the instructions comprise: contacting the immobilized template nucleic acid molecules with a first amplification reagent under conditions that inhibit activity of the amplification polymerase enzyme; and contacting the immobilized template nucleic acid molecules with a second amplification reagent under a condition suitable for reviving the activity of the amplification polymerase enzyme to perform a plurality of nucleic acid amplification reactions.

[0016] In some embodiments, the kit further comprises a wash-removal reagent comprising at least one solvent, a pH buffering agent, a chelating agent, a detergent and a chaotropic agent.

[0017] In some embodiments, the kit further comprises a nucleic acid hybridization reagent, comprising: at least one solvent, a pH buffering agent, and at least one monovalent cation; and a detergent, a reducing agent, a chaotropic agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol or a crowding agent, or a combination thereof. INCORPORATION BY REFERENCE

[0018] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls. DESCRIPTION OF THE DRAWINGS

[0019] Some novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present 18 08 25 inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0020] FIG. 1 is an exemplary schematic illustration of one embodiment of alternating layers of hydrophilic coatings which are covalently or non-covalently adhered to a support structure (e.g., glass, plastic or other polymer material), where the coatings comprise chemically-reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., surface capture primers).

[0021] FIG. 2A is an exemplary schematic showing a nucleotide arm of a multivalent molecule.

[0022] FIG. 2B is an exemplary schematic of a multivalent molecule comprising a core attached to a plurality of nucleotide arms where each nucleotide arm comprises (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit.

[0023] FIG. 2C is a schematic of an exemplary multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0024] FIG. 2D is a schematic of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0025] FIG. 3 is an amino acid sequence (SEQ ID NO: 1) of a wild type DNA polymerase from Candidatus altiarchaeales archaeon.

[0026] FIG. 4 is an amino acid sequence (SEQ ID NO: 2) of a mutant DNA polymerase from Candidatus altiarchaeales archaeon comprising amino acid substitution mutations.

[0027] FIG. 5 is an amino acid sequence (SEQ ID NO: 3) of a mutant DNA polymerase from Candidatus altiarchaeales archaeon comprising amino acid substitution mutations.

[0028] FIG. 6 is an amino acid sequence (SEQ ID NO: 4) of a mutant DNA polymerase from Candidatus altiarchaeales archaeon comprising amino acid substitution mutations.

[0029] FIG. 7 is an amino acid sequence (SEQ ID NO: 5) of a mutant DNA polymerase from Candidatus altiarchaeales archaeon comprising amino acid substitution mutations.

[0030] FIG. 8 is an amino acid sequence SEQ ID NO: 6) of a wild type (9°N DNA polymerase.

[0031] FIG. 9 is an amino acid sequence (SEQ ID NO: 7) of a mutant 9°N DNA polymerase.

[0032] FIG. 10 is an amino acid sequence (SEQ ID NO: 8) of a variant 9°N DNA polymerase.

[0033] FIG. 11 is an amino acid sequence (SEQ ID NO: 9) of a Vent DNA polymerase. 18 08 25

[0034] FIG. 12 is an amino acid sequence (SEQ ID NO: 10) of a Deep Vent DNA polymerase.

[0035] FIG. 13 is an amino acid sequence (SEQ ID NO: 11) of a Pfu DNA polymerase.

[0036] FIG. 14 is an amino acid sequence (SEQ ID NO: 12) of a Pyrococcus abyssi DNA polymerase.

[0037] FIG. 15 is an amino acid sequence (SEQ ID NO: 13) of an RB69 DNA polymerase.

[0038] FIG. 16 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain either no compounds that reduce photo damage or different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 1-4 comprise Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate and glycerol. Formulation 1 contains no photo-damage reducing compound. Formulation 2 comprises Trolox (2 mM) and sodium ascorbate (50 mM). Formulation 3 comprises Trolox (2 mM) and methyl viologen (2 mM). Formulation 4 comprises Trolox (2 mM), methyl viologen (2 mM) and sodium ascorbate (50 mM).

[0039] FIG. 17 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 5-8 comprise Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate and glycerol. Formulation 5 comprises 1,3,5,7 cyclo-octatetraene (COT) (2 mM). Formulation 6 comprises COT (2 mM) and sodium ascorbate (50 mM). Formulation 7 comprises COT (2 mM), sodium ascorbate (50 mM) and Trolox (2 mM). Formulation 8 comprises sodium ascorbate (50 mM) and Trolox (2 mM).

[0040] FIG. 18 is a series of images of dye-labeled nucleotides where the nucleotides are joined to a dye via a cleavable moiety (CM). The images show no residual fluorescent signals after a 90 second exposure to high laser power. The imaging reagent comprises Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate, glycerol, 1,3,5,7 cyclooctatetraene (COT) (2 mM), sodium ascorbate (50 mM) and Trolox (2 mM).

[0041] FIG. 19 is a graph showing the effect of various imaging reagents on residual signals from fluorescently-labeled multivalent molecules with repeat cycles. The imaging reagent comprises Tris-HCl (pH 8.8), EDTA, NaCl (100 mM), Triton X-100 (RTM), sucrose (1 M), strontium acetate, and glycerol. 18 08 25

[0042] FIG. 20 is a graph showing the effect of various imaging reagents on residual signals from fluorescently-labeled multivalent molecules with repeat cycles. The imaging reagent comprises Tris-HCl (pH 8.8), EDTA, NaCl (100 mM), Triton X-100 (RTM), sucrose (1 M), strontium acetate, glycerol, Trolox (2 mM) and sodium ascorbate (50 mM). Compare with the results shown in FIG. 19.

[0043] FIGS. 21A-21H schematically illustrates a method for sequencing a nucleic acid using reagents of the present disclosure, in accordance with some embodiments.

[0044] FIGS. 22A-22B illustrates kits comprising reagents of the present disclosure, in accordance with some embodiments.

[0045] FIG. 23 schematically illustrates on-surface splint ligation, in accordance with some embodiments.

[0046] FIG. 24 shows process diagrams for circularizing a nucleic acid library, in accordance with some embodiments.

[0047] FIG. 25 shows the chemical structure of an exemplary spacer (top), and the chemical structures of various exemplary linkers, including an 11-atom Linker, 16-atom Linker, 23-atom Linker and an N3 Linker (bottom).

[0048] FIG. 26 shows the chemical structures of various exemplary linkers, including Linkers 1-9.

[0049] FIG. 27 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0050] FIG. 28 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0051] FIG. 29 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0052] FIG. 30 shows the chemical structures of various exemplary linkers joined / attached to nucleotide units.

[0053] FIG. 31 shows the chemical structure of an exemplary biotinylated nucleotide-arm. In this example, the nucleotide unit is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0054] FIG. 32 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 9-12 comprise Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate and glycerol. 18 08 25 Formulation 9 comprises sodium ascorbate (50 mM). Formulation 10 comprises Trolox aged for 3 hours (2 mM). Formulation 11 comprises no photo-damage reducing compound. Formulation 12 comprises Trolox (non-aged) (2 mM).

[0055] FIG. 33 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 13-16 comprise Tris-HCl (pH 8 or 8.8), EDTA, NaCl, Triton X-100 (RTM), strontium acetate and glycerol (and no sucrose). Formulation 13 comprises Tris-HCl (pH 8), Trolox (2 mM) and sodium ascorbate (10 mM). Formulation 14 comprises Tris-HCl (pH 8), Trolox (2 mM) and sodium ascorbate (20 mM). Formulation 15 comprises Tris-HCl (pH 8), Trolox (2 mM) and sodium ascorbate (50 mM). Formulation 16 comprises Tris-HCl (pH 8.8), Trolox (2 mM) and sodium ascorbate (50 mM).

[0056] FIG. 34 is a series of images of micro crystal formation of sodium ascorbate (pH 8.8) with an increasing titration of NaCl. The micro crystal formation can be an indication of instability of sodium ascorbate. The formulation tested for all of the images comprises Tris-HCl (pH 8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate and glycerol (and no sucrose).

[0057] FIG. 35 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 17-20 comprise Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate and glycerol. Formulation 17 comprises cysteamine (10 mM). Formulation 18 comprises 3-nitrobenzoic acid (NBA) (2 mM). Formulation 19 comprises no photo-damage reducing compound. Formulation 20 comprises P-phenyl diamine (PPD) (1 mM).

[0058] FIG. 36 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 21-24 comprise Tris-HCl (pH 8), EDTA, NaCl, Triton X-100 (RTM), strontium acetate and glycerol (and with or without sucrose). Formulation 21 comprises sucrose (1 M), Trolox (2 mM) and sodium ascorbate (50 mM). Formulation 22 comprises no sucrose, Trolox (2 mM) and sodium ascorbate (50 mM). 18 08 25 Formulation 23 comprises no sucrose, Trolox (5 mM) and sodium ascorbate (50 mM). Formulation 24 comprises no sucrose, Trolox (8 mM) and sodium ascorbate (50 mM).

[0059] FIG. 37 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 25 and 26 comprise Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), sucrose (reduced from 1 M to 0.5 M), strontium acetate and glycerol. Formulation 25 comprises Trolox (2 mM) and sodium ascorbate (50 mM). Formulation 26 comprises Trolox quinone (2 mM) and sodium ascorbate (50 mM).

[0060] FIG. 38 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 27-30 comprise Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), strontium acetate and glycerol (and with or without sucrose). Formulation 27 comprises Trolox quinone (1 mM), Trolox (3 mM), sodium ascorbate (25 mM) and no sucrose. Formulation 28 comprises Trolox quinone (2 mM), sodium ascorbate (25 mM) and no sucrose. Formulation 29 comprises Trolox quinone (3 mM), sodium ascorbate (25 mM) and no sucrose. Formulation 30 comprises Trolox (2 mM), sodium ascorbate (50 mM) and sucrose (IM).

[0061] FIG. 39 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 31-34 comprise Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), sucrose (reduced from 1 M to 0.5 M), strontium acetate and glycerol. Formulation 31 comprises Trolox (2 mM) and sodium ascorbate (25 mM). Formulation 32 comprises Trolox quinone (2 mM) and sodium ascorbate (25 mM). Formulation 33 comprises Trolox (aged for 16 hours) (2 mM) and sodium ascorbate (25 mM). Formulation 34 comprises Trolox (aged for 3 hours) (2 mM) and sodium ascorbate (25 mM).

[0062] FIG. 40 is a series of box plots showing the effects of imaging reagents comprising different formulations (with varying concentrations of ethylene glycol) on signal intensity of labeled multivalent molecules. Labeled multivalent molecules: dG-CF57 (top) and dA-AF647 (bottom). The imaging reagents comprise Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 18 08 25 mM to 75 mM), Triton X-100 (RTM), strontium acetate, glycerol, varying concentrations of ethylene glycol, Trolox (2 mM), and sodium ascorbate (25 mM) (and no sucrose).

[0063] FIG. 41 is a series of box plots showing the effects of imaging reagents comprising different formulations (with varying concentrations of ethylene glycol) on signal intensity of labeled multivalent molecules. Labeled multivalent molecules: dU-CF532 (top) and dC-CF680 (bottom). The imaging reagents comprise Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), strontium acetate, glycerol, varying concentrations of ethylene glycol, Trolox (2 mM), and sodium ascorbate (25 mM) (and no sucrose).

[0064] FIG. 42 is a series of graphs showing the effects of imaging reagents comprising different formulations on average signal intensity with time. The different formulations contain different combinations of compounds that reduce photo damage. Dye 1 is AF647, dye 2 is CF532, dye 3 is CF570 and dye 4 is CD680. Formulations 35 and 36 comprise Tris-HCl (pH 7.2 - 7.5), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), strontium acetate, glycerol, ethylene glycol (30%), and no sucrose. Formulation 35 comprises Tris-HCl (pH 7.5), Trolox (aged) (2 mM) and sodium ascorbate (25 mM). Formulation 36 comprises Tris-HCl (pH 7.5), Trolox (non-aged) (2 mM) and sodium ascorbate (25 mM). Formulation 37 comprises Tris-HCl (pH 8), EDTA, NaCl (reduced from 100 mM to 75 mM), Triton X-100 (RTM), strontium acetate, glycerol, ethylene glycol (30%), no sucrose, Trolox (aged) (2 mM), and sodium ascorbate (25 mM). Formulation 38 comprises Tris-HCl (pH 8.8), EDTA, NaCl (100 mM), Triton X-100 (RTM), sucrose (1 M), strontium acetate, glycerol, Trolox (non-aged) (2 mM), and sodium ascorbate (25 mM).

[0065] FIG. 43 is a series of images of dye-labeled nucleotides where the nucleotides are joined to a dye via a cleavable moiety (CM). The images show no residual fluorescent signals after a 90 second exposure to high laser power. The imaging reagent comprises Tris-HCl (pH 8.8), EDTA, NaCl, Triton X-100 (RTM), sucrose, strontium acetate, glycerol, 1,3,5,7 cyclooctatetraene (COT) (2 mM), sodium ascorbate (50 mM) and Trolox (2 mM).

[0066] FIG. 44 is an amino acid sequence of wild type DNA polymerase from Candidatus altiarchaeales archaeon (SEQ ID NO:221).

[0067] FIG. 45 is an amino acid sequence of mutant DNA polymerase from Candidatus altiarchaeales archaeon (SEQ ID NO:222).

[0068] FIG. 46 is an amino acid sequence of mutant DNA polymerase from Candidatus altiarchaeales archaeon (SEQ ID NO:223). 18 08 25

[0069] FIG. 47 is an amino acid sequence of mutant DNA polymerase from Candidatus altiarchaeales archaeon (SEQ ID NO :224).

[0070] FIG. 48 is an amino acid sequence of mutant DNA polymerase from Candidatus altiarchaeales archaeon (SEQ ID NO:225).

[0071] FIG. 49 is an amino acid sequence of 9°N polymerase (SEQ ID NO:226).

[0072] FIG. 50 is an amino acid sequence of 9°N polymerase UniProtKB - Q56366 (DPOL THES9) (SEQ ID NO:227).

[0073] FIG. 51 is an amino acid sequence of therminator polymerase (SEQ ID NO:228).

[0074] FIG. 52 is an amino acid sequence of vent polymerase UniProtKB - P30317 (DPOL THELI) (SEQ ID NO:229).

[0075] FIG. 53 is an amino acid sequence deep vent polymerase UniProtKB - Q51334 (DPOL PYRSD) (SEQ ID NO:230).

[0076] FIG. 54 is an amino acid sequence of Pfu polymerase UniProtKB - P61875 (DPOLPYRFU) (SEQ ID NO:231).

[0077] FIG. 55 is an amino acid sequence of pyrococcus abyssi polymerase UniProtKB -P0CL77 (DPOL PYRAB) (SEQ ID NO:232).

[0078] FIG. 56 is an amino acid sequence of RB69 polymerase UniProtKB - Q38087 (DPOL BPR69) (SEQ ID NO:233). DETAILED DESCRIPTION

[0079] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0080] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0081] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0082] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: 18 08 25 “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0083] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0084] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0085] The term “cellular biological sample” refers to a single cell, a plurality of cells, a tissue, an organ, an organism, or section of any of these cellular biological samples. The cellular biological sample can be extracted (e.g., biopsied) from an organism, or obtained from a cell culture grown in liquid or in a culture dish. The cellular biological sample comprises a sample that is fresh, frozen, fresh frozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE). The cellular biological sample can be embedded in a wax, resin, epoxy or agar. The cellular biological sample can be fixed, for example in any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton (RTM) or glutaraldehyde. The cellular biological sample can be sectioned or nonsectioned. The cellular biological sample can be stained, de-stained or non-stained.

[0086] The nucleic acids of interest can be extracted from cells or cellular biological samples using any of a number of techniques known to those of skill in the art. For example, 18 08 25 a typical DNA extraction procedure comprises (i) collection of the cell sample or tissue sample from which DNA is to be extracted, (ii) disruption of cell membranes (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treatment of the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate out the precipitated proteins, lipids, and RNA, and (iv) purification of DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during the cell membrane lysis. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kits (for isolation of genomic DNA from human samples) and DNAeasy kits (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI).

[0087] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring intemucleosidic linkages, for example phosphdiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural intemucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0088] The term “template nucleic acid”, “template polynucleotide”, “target nucleic acid” “target polynucleotide”, “template strand” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the analysis methods describe herein (e.g., hybridization, amplifying and / or sequencing). The template nucleic acid can be singlestranded or double-stranded, or the template nucleic acid can have single-stranded or doublestranded portions. The template nucleic acid can be clonally amplified. The template nucleic acid can be a concatemer having tandem repeats of the nucleic acid sequence of interest operably joined to at least one adaptor sequence. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include 18 08 25 any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acids can include an insert portion having an insert sequence. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The insert portion can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert portion can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis. The template nucleic acid can be linear, concatemeric, circular, or other forms.

[0089] The term “primer” and related terms used herein refers to an oligonucleotide that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers comprise natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from 4-50 nucleotides. A typical primer comprises a 5’ end and 3’ end. The 3’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or can be immobilized to a support (e.g., a capture primer). Primers can be single-stranded along their entire length or have singlestranded and double-stranded portions

[0090] The term “universal sequence” and related terms refers to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, 18 08 25 an adaptor having a universal sequence can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Examples of universal adaptor sequences include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble or immobilized capture primers).

[0091] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked (appended) to a target polynucleotide, where the adaptor confers a function to the co-joined adaptor-target molecule. Adaptors comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5’ overhang and 3’ overhang ends. The 5’ end of a single-stranded adaptor, or one strand of a double-stranded adaptor, can have a 5’ phosphate group or lack a 5’ phosphate group. Adaptors can include a 5’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed. An adaptor can include a universal sequence. At least a portion of the adaptors comprise a known and pre-determined sequence. An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection. Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs or type IIB.

[0092] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtaposed components can be linked together 18 08 25 covalently. For example, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, linkage between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and / or translation initiation sequence, transcription and / or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and / or location of the transgene.

[0093] The terms “linked”, “joined”, “attached”, “appended” and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedure can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking (e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and / or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such linkage occurs intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998). 18 08 25

[0094] When used in reference to concentrations, the symbol “%” may refer to % by volume. When used in reference to concentrations, the symbol “%” may refer to % by mass. When used in reference to concentrations, the symbol “%” may refer to % by mol.

[0095] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0096] When used in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase.

[0097] In some embodiments, any of the amplification primer sequences, sequencing primer sequences, capture primer sequences (capture oligonucleotides), target capture sequences, circularization anchor sequences, sample barcode sequences, spatial barcode sequences, or anchor region sequences can be about 3-50 nucleotides in length, or about 5-40 nucleotides in length, or about 5-25 nucleotides in length.

[0098] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any 18 08 25 enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins or fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. A polymerase comprises DNA-directed DNA polymerase and RNA-directed DNA polymerase.

[0099] The term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a templatebased manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include mesophilic and thermophilic polymerases. Strand displacing polymerases include wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). 18 08 25

[00100] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety. The nucleotides can have 1-10 phosphate groups.

[00101] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[00102] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chern. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic &Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chern. 36: 2627-2638; Kim, et al., 1993 J. Med. Chern. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 18 08 25 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[00103] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[00104] The term “reporter moiety”, “reporter moieties” or related terms refers to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or support (e.g., surfaces).

[00105] A reporter moiety (or label) comprises a fluorescent label or a fluorophore. Exemplary fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives 18 08 25 such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes and others such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl] -3,3 -dimethyl -1,3 -dihy dro-2H-indol-2-ylidene } prop-1 -en-1 -yl)-3,3 -dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{ l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise 1 -(6-((2,5 -dioxopyrrolidin-1 -yl)oxy )-6-oxohexyl)-2-(( 1 E,3 E)-5 -((E)-1 -(6-((2,5 -dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium or l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and 18 08 25 Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3 H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

[00106] In some embodiments, the reporter moiety can be a FRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[00107] The term “support” as used herein refers to a substrate that is designed for deposition of biological molecules or biological samples for assays and / or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of biological samples include but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears and fluids from tissues or organs.

[00108] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example comprising a capillary or interior surface of a capillary.

[00109] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.

[00110] In some embodiments, the support comprises a bead having any shape, including spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like.

[00111] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated. 18 08 25

[00112] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid templates have the same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a different site on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a site on the support.

[00113] The term “array” refers to a support comprising a plurality of sites located at predetermined locations on the support to form an array of sites. The sites can be discrete and separated by interstitial regions. In some embodiments, the pre-determined sites on the support can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. In some embodiments, the plurality of pre-determined sites is arranged on the support in an organized fashion. In some embodiments, the plurality of pre-determined sites is arranged in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. The pitch between different pairs of sites can be that same or can vary. In some embodiments, the support comprises at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, at least 109 sites, at least 1010 sites, at least 1011 sites, at least 1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites, or more, where the sites are located at pre-determined locations on the support. In some embodiments, a plurality of pre-determined sites on the support (e.g., 102 -1015 sites or more) are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites, for example immobilized at 102 - 1015 sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of pre-determined sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[00114] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The location of the randomly located sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and / or 18 08 25 unpredictable fashion. In some embodiments, the support comprises at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, at least 109 sites, at least 1010 sites, at least 1011 sites, at least 1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites, or more, where the sites are randomly located on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 102 - 1015 sites or more) are immobilized with nucleic acid templates to form a support immobilized with nucleic acid templates. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites, for example immobilized at 102 - 1015 sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of randomly located sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[00115] When used in reference to support, the term “feature” refers to a region on a support. In some embodiments, the feature is a region on a coating which is layered on the support. In some embodiments, the feature is a region on a low non-specific binding coating which is layered on a support. A support or coating can have a plurality of regions (e.g., features) located at different pre-determined locations on the support or coating (FIG. 3, right). The different features on the support can be placed at non-overlapping positions or at overlapping positions on the support. The features can be configured to have any shape, for example circular, ovular, square, rectangular, or polygonal. The features can be arranged in a grid pattern having rows and columns, or can be arranged in a row or a column. In some embodiments, any given feature contains a plurality of capture oligonucleotides and / or a plurality of circularization oligonucleotides immobilized to the support or to the coating. The plurality of features includes at least a first and second feature.

[00116] In some embodiment, the plurality of immobilized surface capture primers on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and the like) onto the support so that the plurality of immobilized surface capture primers on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to conduct nucleic acid amplification 18 08 25 reactions (e.g., RCA, MD A, PCR and bridge amplification) essentially simultaneously on the plurality of immobilized surface capture primers.

[00117] In some embodiment, the plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, and the like) onto the support so that the plurality of immobilized nucleic acid clusters on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid clusters can be used to conduct nucleotide binding assays and / or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on the plurality of immobilized nucleic acid clusters, and optionally to conduct detection and imaging for massively parallel sequencing.

[00118] When used in reference to immobilized enzymes, the term “immobilized” and related terms refer to enzymes (e.g., polymerases) that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support.

[00119] When used in reference to immobilized nucleic acids, the term “immobilized” and related terms refer to nucleic acid molecules that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support, where the nucleic acid molecules include surface capture primers, nucleic acid template molecules and extension products of capture primers. Extension products of capture primers includes nucleic acid single copy molecules having one copy of insert sequences and at least one adaptor sequence, and includes concatemers having repeat tandem copies of insert and adaptor sequences. The nucleic acid molecules can be immobilized at pre-determined or random locations on the support. The nucleic acid molecules can be immobilized at pre-determined or random locations on or within a coating passivated on the support. In some embodiments, the term “immobilized” and related terms refer to enzymes (e.g., polymerases) that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support. The enzymes can be immobilized at pre-determined or random locations on the support. The enzymes can be immobilized at pre-determined or random locations on or within a coating passivated on the support.

[00120] In some embodiments, one or more nucleic acid templates are immobilized on the support, for example immobilized at the sites on the support. In some embodiments, the one 18 08 25 or more nucleic acid templates are clonally-amplified. In some embodiments, the one or more nucleic acid templates are clonally-amplified off the support (e.g., in-solution) and then deposited onto the support and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid templates is conducted on the support resulting in immobilization on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, including any one or any combination of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-stranded binding (SSB) protein-dependent amplification.

[00121] The term “surface capture primer”, “capture primer”, “capture oligonucleotide” and related terms refers to single-stranded oligonucleotides that are immobilized to a support and comprise a sequence that can hybridize to at least a portion of a nucleic acid template molecule. Surface capture primers can be used to immobilize template molecules to a support via hybridization. Surface capture primers can be immobilized to a support in a manner that resists primer removal during flowing, washing, aspirating, and changes in temperature, pH, salts, chemical and / or enzymatic conditions. Typically, but not necessarily, the 5’ end of a surface capture primer can be immobilized to a support. Alternatively, an interior portion or the 3’ end of a surface capture primer can be immobilized to a support.

[00122] The sequence of surface capture primers can be wholly or partially complementary along their length to at least a portion of the nucleic acid template molecule. A support can include a plurality of immobilized surface capture primers having the same sequence, or having two or more different sequences. Surface capture primers can be any length, for example 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longer lengths.

[00123] A surface capture primer can have a terminal 3’ nucleotide having a 3’ sugar moiety which is extendible for nucleotide polymerization (e.g., polymerase catalyzed polymerization). A surface capture primer can have a terminal 3’ nucleotide having the 3’ sugar position linked to a chain-terminating moiety that inhibits nucleotide polymerization. The 3’ chain-terminating moiety can be removed (e.g., de-blocked) to convert the 3’ end to an extendible 3’ OH end using a de-blocking agent. Examples of chain terminating moi eties 18 08 25 include alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. Azide type chain terminating moi eties including azide, azido and azidomethyl groups. Examples of deblocking agents include a phosphine compound, such as Tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfo triphenyl phosphine (BS-TPP), for chain-terminating groups azide, azido and azidomethyl groups. Examples of de-blocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ), for chain-terminating groups alkyl, alkenyl, alkynyl and allyl. Examples of a de-blocking agent includes Pd / C for chain-terminating groups aryl and benzyl. Examples of de-blocking agents include phosphine, beta-mercaptoethanol or dithiothritol (DTT), for chain-terminating groups amine, amide, keto, isocyanate, phosphate, thio and disulfide. Examples of de-blocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, and Zn in acetic acid (AcOH), for carbonate chainterminating groups. Examples of de-blocking agents include tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, and triethylamine trihydrofluoride, for chainterminating groups urea and silyl.

[00124] The term “branched polymer” and related terms refers to a polymer having a plurality of functional groups that help conjugate a biologically active molecule such as a nucleotide, and the functional group can be either on the side chain of the polymer or directly attaches to a central core or central backbone of the polymer. The branched polymer can have linear backbone with one or more functional groups coming off the backbone for conjugation. The branched polymer can also be a polymer having one or more sidechains, wherein the side chain has a site suitable for conjugation. Examples of the functional group include but are limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

[00125] The term “sequencing” and related terms refers to a method for obtaining nucleotide sequence information from a nucleic acid molecule, typically by determining the identity of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, the sequence information of a given region of a nucleic acid molecule includes identifying each and every nucleotide within a region that is sequenced. In some embodiments, sequencing information determines only some of the 18 08 25 nucleotides a region, while the identity of some nucleotides remains undetermined or incorrectly determined. Any suitable method of sequencing may be used. In an exemplary embodiment, sequencing can include label-free or ion based sequencing methods. In some embodiments, sequencing can include labeled or dye-containing nucleotide or fluorescent based nucleotide sequencing methods. In some embodiments, sequencing can include clusterbased sequencing or bridge sequencing methods. In some embodiments, the sequencing employs polymerases and multivalent molecules for generating at least one avidity complex, wherein individual multivalent molecules comprise a plurality of nucleotide units tethered to a core. In some embodiments, the sequencing employs polymerases and free nucleotides for performing sequencing-by-synthesis. In some embodiments, the sequencing employs a ligase enzyme and a plurality of sequence-specific oligonucleotides for performing sequence-by-ligation.

[00126] The term “persistence time” and related terms refers to the length of time that a binding complex, which is formed between the target nucleic acid, a polymerase, a conjugated or unconjugated nucleotide, remains stable without any binding component dissociates from the binding complex. The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary label is a fluorescent label.

[00127] When used in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, or is substantially identical to a sequence that is complementary to the template sequence.

[00128] The term “rolling circle” amplification generally refers to an amplification method that employs a circularized nucleic acid template molecule containing a target sequence of interest, an amplification primer binding sequence, and optionally one or more adaptor sequences such as a sequencing primer binding sequence and / or a barcode. The rolling circle amplification reaction can be conducted under isothermal amplification conditions, and 18 08 25 includes the circularized nucleic acid template molecule, an amplification primer, a stranddisplacing polymerase and a plurality of nucleotides, to generate a concatemer containing tandem repeat sequences of the circular template molecule and any adaptor sequences present in the original circularized nucleic acid template molecule. The concatemer can self-collapse to form a nucleic acid nanoball. The shape and size of the nanoball can be further compacted by including a pair of inverted repeat sequences in the circular template molecule, or by conducting the rolling circle amplification reaction with one or more compaction oligonucleotides. One of the advantages of using rolling circle amplification to generate clonal amplicons for a sequencing workflow, is that the repeat copies of the target sequence in the nanoball can be simultaneously sequenced to increase signal intensity. In some embodiments, the rolling circle amplification reaction can be conducted in the presence of a plurality of compaction oligonucleotides having at least four consecutive guanines. The rolling circle amplification reaction generates concatemers comprising repeat copies of the universal binding sequence for the compaction oligonucleotide. At least one compaction oligonucleotide can form a guanine tetrad and hybridize to the universal binding sequences for the compaction oligonucleotide, and the resulting concatemer can fold to form an intramolecular G-quadruplex structure. The concatemers can self-collapse to form compact nanoballs. Formation of the guanine tetrads and G-quadruplexes in the nanoballs may increase the stability of the nanoballs to retain their compact size and shape which can withstand repeated flows of reagents for conducting any of the sequencing workflows described herein.

[00129] When used in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, and / or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, and / or is substantially identical to a sequence that is complementary to the template sequence.

[00130] The terms “resonance energy transfer” and “RET” and related terms used herein, refer to a radiationless transmission of excitation energy from a first moiety which is a donor moiety, to a second moiety which is an acceptor moiety. One type of RET includes Forster Resonance Energy Transfer (FRET), in which a donor fluorophore in an excited state transfers its energy to a proximal acceptor molecule by non-radiative dipole-dipole interaction. A description of FRET can be found in T. Forster, 1948, “Intermolecular Energy 18 08 25 Migration and Fluorescence”, Ann. Phys., 2:55-75; and J.R. Lakowicz, 1999, “Principles of Fluorescence Spectroscopy”, 2nd ed. Plenum, New York. 367-394. RET also includes luminescence resonance energy transfer, bioluminescence resonance energy transfer, chemiluminescence resonance energy transfer, and similar types of energy transfer that do not strictly follow the Forster's theory, such as nonoverlapping energy transfer occurring when nonoverlapping acceptors are utilized. See for example, Anal. Chern. 2005, 77: 1483-1487.

[00131] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically-synthesized forms. Polypeptides also include precursor molecules that have not yet been subjected to post-translation modification such as proteolytic cleavage, cleavage due to ribosomal skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation and / or disulfide bond formation. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins.

[00132] The present disclosure provides various reagents, and methods that employ the reagents for conducing massively parallel nucleic acid polony formation and sequencing. The various reagents can include at least one pH buffering agent. The full name of the pH buffering agents is listed herein.

[00133] The term “Tris” refers to a pH buffering agent Tris(hydroxymethyl)-aminomethane.

[00134] The term “Tris-HQ” refers to a pH buffering agent Tris(hydroxymethyl)-aminomethane hydrochloride. The term “Tris-acetate” refers to a pH buffering agent comprising an acetate salt of Tris (hydroxymethyl)-aminomethane.

[00135] The term “Tricine” refers to a pH buffering agent N-[tris(hydroxymethyl) methyl]glycine.

[00136] The term “Bicine” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)glycine.

[00137] The term “Bis-Tris propane” refers to a pH buffering agent 1,3 Bis[tris(hydroxymethyl)methylamino]propane

[00138] The term “HEPES” refers to a pH buffering agent 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 18 08 25

[00139] The term “MES” refers to a pH buffering agent 2-(Mmorpholino)ethanesulfonic acid).

[00140] The term “MOPS” refers to a pH buffering agent 3-(N-morpholino)propanesulfonic acid.

[00141] The term “MOPSO” refers to a pH buffering agent 3-(N-morpholino)-2-hydroxypropanesulfonic acid.

[00142] The term “BES” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

[00143] The term “TES” refers to a pH buffering agent 2-[(2-Hydroxy- 1,1 bi s(hydroxymethyl) ethyl)amino]ethanesulfonic acid).

[00144] The term “CAPS” refers to a pH buffering agent 3-(cyclohexylamino)-1-propanesuhinic acid.

[00145] The term “TAPS” refers to a pH buffering agent N-[Tris(hydroxymethyl)methyl]-3-amino propane sulfonic acid.

[00146] The term “TAPSO” refers to a pH buffering agent N-[Tris(hydroxymethyl)methyl]-3-amino-2-hyidroxypropansulfonic acid.

[00147] The term “ACES” refers to a pH buffering agent M(2-Acetamido)-2-aminoethanesulfonic acid.

[00148] The term “PIPES” refers to a pH buffering agent piperazine-l,4-bis(2-ethanesulfonic acid.

[00149] The term “Tris-acetate” refers to a pH buffering agent comprising an acetate salt of Tris (hydroxymethyl)-aminomethane.

[00150] The term “multivalent molecule” refers to a molecule comprising a plurality of binding motifs. In some embodiments, the plurality of binding motifs are each configured to bind to a nucleic acid base (e.g., A, C, T, G, or U). In some cases, the multivalent molecule comprises: (a) a core; and (b) a plurality of nucleotide arms which each comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit. FIG. 2B illustrates an example of a multivalent molecule, in accordance with some embodiments.

[00151] The term, “ternary complex” or “ternary binding complex” refers to a complex comprising three components. In some embodiments, the ternary complex comprises: (a) a multivalent molecule disclosed herein, (b) a primed nucleic acid sequence, and (c) a polymerizing enzyme. INTRODUCTION 18 08 25

[00152] The present disclosure provides compositions and methods that employ the compositions for conducting massively parallel nucleic acid sequencing workflows, where the workflows include, but are not limited to nucleic acid library preparation, library circularization, library molecule amplification, library immobilization to a support, polony formation of nucleic acid template molecules and sequencing the nucleic acid template molecules.

[00153] The present disclosure provides compositions comprising reagents used to conduct nucleic acid sequencing workflows, where the compositions include: a universal wash reagent; a nucleic acid hybridization reagent; a first and second amplification reagent; a wash-removal reagent; a trap reagent; a post-trap reagent; an imaging reagent; stepping reagent; and a cleaving reagent.

[00154] The present disclosure provides a nucleic acid sequencing workflow which employs the various reagents described above, where the sequencing workflow generally comprises polony formation and sequence-determining reactions.

[00155] In some embodiments, the polony-forming workflow generally comprises: hybridizing nucleic acid template molecules to amplification primers to form nucleic acid duplexes; amplifying the nucleic acid duplexes to form a plurality of concatemers where an individual concatemer in the plurality of concatemers is a polony.

[00156] In some embodiments, the sequence-determining reaction workflow generally comprises: forming ternary complexes by binding together the concatemer template molecules with sequencing primers, sequencing polymerases and multivalent molecules under conditions suitable for inhibiting polymerase-catalyzed incorporation of nucleotide units from the multivalent molecules; imaging signals from the bound multivalent molecules in the ternary complexes; dissociating the ternary complexes; forming ternary complexes by binding together the concatemer template molecules with sequencing primers, sequencing polymerases and nucleotides (e.g., chain terminator nucleotides) under conditions suitable for promoting polymerase-catalyzed incorporation of nucleotides thereby extending the sequencing primers with the nucleotides to form a nascent sequencing primer chain; and forming 3’ terminal extendible ends of the nascent sequencing primer chains. Nucleic Acid Fragmentation 18 08 25

[00157] The present disclosure provides reagents, kits and methods for preparing a population of fragmented nucleic acids. In some embodiments, individual fragmented nucleic acids will be covalently joined to at least one universal adaptor sequence for library preparation.

[00158] The insert region of a nucleic acid library molecule comprises a sequence of interest extracted from any source including a biological sample (e.g., fresh or live sample) such as a single cell, a plurality of cells or tissue. The insert region can be isolated from healthy or diseases cells or tissues. The insert region can be obtained from an archived sample such as a fresh frozen paraffin embedded (FFPE) sample, or from needle biopsies, circulating tumor cells, cell free circulating DNA (e.g., from tumor cells or a fetus). Cells or tissues are typically treated with a lysis buffer to release their DNA and RNA, and the desired nucleic acid is separated from non-desired macromolecules such as proteins.

[00159] The insert region of a nucleic acid library molecule can be isolated in any form, including chromosomal, genomic (e.g., whole genomic), organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned or amplified. The insert region of a nucleic acid library molecule can be methylated or non-methylated.

[00160] The insert region can be isolated from any organism including viruses, fungi, prokaryotes or eukaryotes. The insert region can be isolated from any organism including human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria. The insert region can be isolated from organisms borne in air, water, soil or food.

[00161] The insert region can be isolated from any biological fluid, including blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic samples, perspiration, semen, environmental samples or culture samples. The insert region can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[00162] The insert region can be prepared using recombinant nucleic acid technology including but not limited to any combination of vector cloning, transgenic host cell preparation, host cell culturing and / or PCR amplification.

[00163] The insert region can be in fragmented or un-fragmented form. Fragmented insert regions can be obtained by mechanical force, enzymatic or chemical fragmentation methods. The fragmented insert regions can be generated using procedures that yield a population of fragments having overlapping sequences or non-overlapping sequences. 18 08 25

[00164] Mechanical fragmentation typically generates randomly fragmented nucleic acid molecules. Mechanical fragmentation methods include mechanical shearing such as fluid shear, constant shear and pulsatile shear. Mechanical fragmentation methods also include mechanical stress including sonication, nebulization and acoustic cavitation. In some embodiments focused acoustic energy can be used to randomly fragment nucleic acid molecules. A commercially-available apparatus (e.g., Covaris) can be used to fragment nucleic acid molecules using focused acoustic energy.

[00165] Enzymatic fragmentation procedures can be conducted under conditions suitable to generate randomly or non-randomly fragmented nucleic acid molecules. For example, restriction endonuclease enzyme digestion can be conducted to completion to generate non-randomly fragmented nucleic acid molecule. Alternatively, partial or incomplete restriction enzyme digestion can be conducted to generate randomly-fragmented nucleic acid molecules. Enzymatic fragmentation using restriction endonuclease enzymes includes any one or any combination of two or more restriction enzymes selected from a group consisting of type I, type II, type Ils, type IIB, type III, or type IV restriction enzymes. Enzymatic fragmentation includes digestion of the nucleic acid with a rare-cutting restriction enzyme, comprising Not I, Asc I, Bae I, AspC I, Pac I, Fse I, Sap I, Sfi I or Psr I. Enzymatic fragmentation include use of any combination of a nicking restriction endonuclease, endonuclease and / or exonuclease. Enzymatic fragmentation can be achieved by conducting a nick translation reaction.

[00166] In some embodiments, enzymatic fragmentation can be achieved by reacting nucleic acids with an enzyme mixture, for example an enzyme that generates single-stranded nicks and another enzyme that catalyzes double-stranded cleavage. An exemplary enzyme mixture is FRAGMENTASE (e.g., from New England Biolabs).

[00167] Fragments of the insert region can be generated with PCR using sequence-specific primers that hybridize to target regions in genomic DNA samples to generate insert regions having known fragment lengths and sequences.

[00168] Targeted genome fragmentation methods using CRISPR / Cas9 can be used to generate fragmented insert regions.

[00169] Fragments of the insert portion can also be generated using a transposase-based tagmentation method using NEXTERA (from Epicentre).

[00170] The insert region can be single-stranded or double-stranded. The ends of the double-stranded insert region can be blunt-ended, or have a 5’ overhang or a 3’ overhang end, or any combination thereof. One or both ends of the insert region can be subjected to an enzymatic tailing reaction to generate a non-template poly-A tail by employing a terminal 18 08 25 transferase reaction. The ends of the insert region can be compatible for joining to at least one universal adaptor sequence.

[00171] The insert region can be any length, for example the insert region can be about 50-250, or about 250-500, or about 500-750, or about 750-1000 bases or base pairs in length.

[00172] The fragments containing the insert region can be subjected to a size selection process, or the fragments are not size selected. For example, the fragments can be size selected by gel electrophoresis and gel slice extraction. The fragments can be size selected using a solid phase adherence / immobilization method which typically employs micro paramagnetic beads coated with a chemical functional group that interacts with nucleic acids under certain ionic strength conditions with or without polyethylene glycol or polyalkylene glycol. Commercially-available solid phase adherence beads include SPRI (Solid Phase Reversible Immobilization) beads from Beckman Coulter (AMPUR XP paramagnetic beads, catalog No. B23318), MAGNA PURE magnetic glass particles (Roche Diagnostics, catalog No. 03003990001), MAGNASIL paramagnetic beads from Promega (catalog No. MD 1360), MAGTRATION paramagnetic beads and system from Precision System Science (catalog Nos. Al 120 and A1060), MAG-BIND from Omega Bio-Tek (catalog No. M1378-01), MAGPREP silica from Millapore (catalog No. 101193), SNARE DNA purification systems from Bangs Laboratories (catalog Nos. BP691, BP692 and BP693), and CHEMAGEN M-PVA beads from Perkin Elmer (catalog No. CMG-200).

[00173] In some embodiments, the fragmented nucleic acids can be subjected to enzymatic reactions for end-repair and / or A-tailing. The fragmented nucleic acids can be contacted with a plurality of enzymes under a condition suitable to generate nucleic acid fragments having blunt-ended 5’ phosphorylated ends. In some embodiments, the plurality of enzymes generates blunt-ended fragment having a non-template A-tail at their 3’ ends. The plurality of enzymes comprise two or more enzymes that can catalyze nucleic acid end-repair, phosphorylation and / or A-tailing. The end-repair enzymes include a DNA polymerase (e.g., T4 DNA polymerase) and KI enow fragment. The 5’ end phosphorylation enzyme comprises T4 polynucleotide kinase. The A-tailing enzyme includes a Taq polymerase (e.g., non-proofreading polymerase) and dATP. In some embodiments, the fragmenting, end-repair, phosphorylation and A-tailing can be conducted in a one-pot reaction using a mixture of enzymes. Appending Adaptors to Fragmented Nucleic Acids 18 08 25

[00174] The present disclosure provides reagents, kits and methods used to append one or more adaptor sequences to fragmented nucleic acids. In some embodiments, individual fragmented nucleic acids will be covalently joined to at least one universal adaptor sequence for library preparation. In general, a nucleic acid fragment is covalently joined at both ends to one or more universal adaptors to generate a linear library molecule having the arrangement left adaptor-insert-right adaptor. In some embodiments, at least one fragment in the population of fragmented nucleic acids comprises a sequence-of-interest. Individual library molecules in the population of library molecules can have an insert region that is the same or different as other library molecules in the population. In some embodiments, about 1-10 ng, or about 10-50 ng, or about 50-100 ng of input fragmented nucleic acids can be appended to one or more universal adaptors to generate a linear library.

[00175] Individual nucleic acid fragments can be appended on one or both ends to at least one universal adaptor sequence to form a recombinant nucleic acid linear library molecule having the general arrangement left adaptor-insert-right adaptor.

[00176] In some embodiments, the nucleic acid fragments can be appended with any one or any combination of two or more adaptor sequences comprising a left universal adaptor sequence having a binding sequence for a first surface primer, a right universal adaptor sequence having a binding sequence for a second surface primer, a left universal adaptor sequence having a binding sequence for a first sequencing primer, a right universal adaptor sequence having a binding sequence for a second sequencing primer, a left sample index sequence, a right sample index sequence, a left unique identification sequence, a right unique identification sequence and / or a universal adaptor sequence for binding a compaction oligonucleotide.

[00177] The universal adaptors can be prepared using chemical synthesis procedures using native nucleotides with or without nucleotide analogs or modified nucleotide linkages that confer certain properties, including resistance to enzymatic digestion, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease digestion include phosphorothioate, 2’-O-methyl RNA, inverted dT, and 2’ 3’ dideoxy-dT. Insert regions that include locked nucleic acids (LNA) have increased thermal stability.

[00178] The insert region can be joined at one or both ends to at least one universal adaptor sequence using a ligase enzyme and / or primer extension reaction to generate a linear library molecule. Covalent linkage between an insert region and the universal adaptor(s) can be achieved with a DNA or RNA ligase. Exemplary DNA ligases that can ligate double 18 08 25 stranded DNA molecules include T4 DNA ligase and T7 DNA ligase. A universal adaptor sequence can be appended to an insert sequence by PCR using a tailed primer having 5’ region carrying a universal adaptor sequence and a 3’ region that is complementary to a portion of the insert sequence. A universal adaptor sequence can be appended to an insert sequence which is flanked one side or both sides with first and second universal adaptor sequences by PCR using a tailed primer having 5’ region carrying a third universal adaptor sequence and a 3’ region that is complementary to a portion of the first or second adaptor sequence. Nucleic Acid Hybridization Reagents and Methods of Use

[00179] The present disclosure provides one or more nucleic acid hybridization reagents, and methods that employ the nucleic acid hybridization reagents where the methods comprise hybridizing nucleic acid template molecules to amplification primers to form a plurality of nucleic acid duplexes (e.g., step (a) of the methods described herein). The hybridization reagents can promote specific hybridization between template molecules of interest with amplification primers. The nucleic acid hybridization reagents can reduce background signals when determining the sequences of the amplified template molecules in a downstream step.

[00180] In some embodiments, the nucleic acid hybridization reagents can be used to hybridize library molecules to amplification primers that are immobilized to a support. The support can be a planar support or at least one bead. The nucleic acid hybridization reagents can be used for the massively parallel sequencing workflow step (a) as described below.

[00181] In some embodiments, the nucleic acid hybridization reagents comprise at least one solvent, a pH buffering agent, and at least one monovalent cation. The hybridization reagents further comprise any one or any combination of two or more of a detergent, a reducing agent, a chaotropic agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and / or a crowding agent.

[00182] In some embodiments, the nucleic acid hybridization reagents further comprise at least one nucleic acid template molecule which comprises DNA or RNA, or a mixture of RNA and DNA. In some embodiments, the nucleic acid template molecules comprise linear or circularized molecules, or a mixture of linear and circular molecules. In some embodiments, the nucleic acid template molecules comprise single-stranded molecules, double-stranded molecules or nucleic acid molecules having single- and double-stranded portions. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor, where the adaptor includes a capture primer binding sequence, 18 08 25 an amplification primer binding sequence and / or a sequencing primer binding sequence. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor having a sample barcode sequence or a unique molecular tag sequence. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor having a sequence that binds at least a portion of a condenser oligonucleotide.

[00183] In some embodiments, the nucleic acid hybridization reagents further comprise at least one nucleic acid amplification duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. The amplification primer can hybridize to at least a portion of the nucleic acid template molecule. The amplification primer comprises a 3’ extendible end or a 3’ non-extendible end.

[00184] In some embodiments, the amplification primer comprises soluble oligonucleotide primers (e.g., in-solution), or the amplification primer is immobilized to a support or immobilized to a coating (e.g., polymer coating) on the support.

[00185] In some embodiments, the nucleic acid hybridization reagents further comprise at least one nucleic acid duplex immobilized to a support, where the nucleic acid duplex comprise a nucleic acid template molecule hybridized to an amplification primer, and where the template molecule and / or the amplification primer is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, a plurality of amplification primers are immobilized to one or more layers of the coatings on the support where the density of the immobilized amplification primers is about 100-100,000 amplification primers per mm2.

[00186] In some embodiments, the plurality of immobilized nucleic acid amplification duplexes on the support are in fluid communication with each other to permit flowing a solution of the nucleic acid hybridization reagent onto the support so that the plurality of immobilized amplification duplexes on the support can be essentially simultaneously reacted with the nucleic acid hybridization reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized amplification duplexes can be used to conduct nucleic acid hybridization reactions in a massively parallel manner on the support.

[00187] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 8.8), EDTA (0.5 mM), NaCl (50 mM), and Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation HR-A. 18 08 25

[00188] In some embodiments, a nucleic acid hybridization reagent may comprise MES (100 mM, pH 8.8), EDTA (0.25 mM), MgCh (75 mM), ethylene glycol (5%) and Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation HR-B.

[00189] In some embodiments, a nucleic acid hybridization reagent may comprise MES (200 mM, pH 8.3), EDTA (0.5 mM), NaCl (100 mM), SDS (0.5 M), glycerol (5%), and Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation HR-C

[00190] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 7.7), EDTA (5 mM), NaCl (200 mM), SDS (0.25 M), and Triton-X (RTM) (0.03%). In some cases, this formulation may be referred to as Formulation HR-D.

[00191] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 8.2), EDTA (50 mM), MgCh (1 M), and Tween-20 (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation HR-E.

[00192] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 7.6), EDTA (0.5 mM), NaCl (100 mM), guanidium chloride (1 M), and Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation HR-F.

[00193] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), guanidium chloride (1 M), and Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation HR-G.

[00194] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 8), EDTA (5 mM), NaCl (100 mM), methanol (1 M), and Triton-X (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation HR-H.

[00195] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 7.9), EDTA (2 mM), NaCl (200 mM), phenol (2 M), and Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation HR-I.

[00196] In some embodiments, a nucleic acid hybridization reagent may comprise MES (50 mM, pH 8), EDTA (0.5 mM), NaCl (10 mM), guanidium chloride (1 M), and Tween-20 (RTM) (1%). In some cases, this formulation may be referred to as Formulation HR-J. Nucleic Acid Amplification Reagents and Methods of Use

[00197] The present disclosure provides one or more nucleic acid amplification reagents, and methods that employ the nucleic acid amplification reagents where the methods comprise forming a plurality of complexed amplification polymerases each comprising an amplification polymerase and a nucleotide bound to a nucleic acid amplification duplex (e.g., 18 08 25 a nucleic acid template molecule hybridized to an amplification primer). In some embodiments, the nucleic acid amplification reactions can be conducted as a two-stage amplification reaction that employs first and second amplification reagents, respectively. The first amplification reagents can seed a plurality of nucleic acid duplexes with amplification polymerases under a condition that inhibits amplification. The second amplification reagents can promote amplification of the plurality of nucleic acid duplexes so that amplification coincides on the plurality of duplexes at the same time. In some embodiments, the two-stage amplification reaction can be used to generate clonally amplified template molecules which are copied from library molecules, where the clonally amplified template molecules are immobilized to a support.

[00198] In some embodiments, the nucleic acid amplification reagents comprises a first amplification reagent, and methods that employ the first amplification reagent where the methods comprise contacting a plurality of nucleic acid duplexes with the first amplification reagent under a condition suitable for forming a plurality of complexed amplification polymerases each comprising an amplification polymerase and a nucleotide bound to a nucleic acid duplex but amplification is inhibited. The first amplification reagent can be used for the massively parallel sequencing workflow of step (c) as described below. In some embodiments, the first amplification reagent comprises at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions, a plurality of nucleotides and an amplification polymerase enzyme. In some embodiments, the first amplification reagent further comprises any one or any combination of two or more of a detergent, a reducing agent, a viscosity agent. In some embodiments, the pH of the first amplification reagent is suitable for binding the amplification polymerase to a nucleic acid duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. In some embodiments, the pH of the first amplification reagent can reduce / inhibit activity of the amplification polymerase (e.g., polymerase-catalyzed nucleotide incorporation activity). For example, the pH of the first amplification reagent can be about pH 8 or lower (e.g., pH 7-8).

[00199] In some embodiments, the amplification polymerase has strand displacement activity. The amplification polymerase comprises a wild type or mutant amino acid sequence.

[00200] In some embodiments, the plurality of nucleotides comprise a mixture of two or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of four types of nucleotides including dATP, dGTP, dCTP and dTTP. 18 08 25

[00201] In some embodiments, the first amplification reagents further comprise at least one nucleic acid template molecule which comprises DNA or RNA, or a mixture of RNA and DNA. In some embodiments, the nucleic acid template molecules comprise linear or circularized molecules, or a mixture of linear and circular molecules. In some embodiments, the nucleic acid template molecules comprise single-stranded molecules, double-stranded molecules or nucleic acid molecules having single- and double-stranded portions. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor, where the adaptor includes a capture primer binding sequence, an amplification primer binding sequence and / or a sequencing primer binding sequence. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor having a sample barcode sequence or a unique molecular tag sequence. . In some embodiments, the amount of circular or linear library molecules that can be reacted with the first amplification reagent is about 1-10 frnol, or about 10-25 frnol, or about 25-50 frnol, or about 50-100 frnol, or about 100-200 frnol. In some embodiments, the amount of circular or linear library molecules that can be reacted with the first amplification reagent is about 1-10 pmol, or about 10-25 pmol, or about 25-50 pmol, or about 50-100 pmol, or about 100-200 pmol.

[00202] In some embodiments, the first amplification reagents further comprise at least one nucleic acid amplification duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. The amplification primer can hybridize to at least a portion of the nucleic acid template molecule. The amplification primer comprises a 3’ extendible end or a 3’ non-extendible end.

[00203] In some embodiments, the amplification primer comprises soluble oligonucleotide primers (e.g., in-solution), or the amplification primer is immobilized to a support or immobilized to a coating (e.g., polymer coating) on the support.

[00204] In some embodiments, the first amplification reagents further comprise at least one nucleic acid duplex immobilized to a support, where the nucleic acid duplex comprise a nucleic acid template molecule hybridized to an amplification primer, and where the template molecule and / or the amplification primer is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, a plurality of amplification primers are immobilized to one or more layers of 18 08 25 the coatings on the support where the density of the immobilized amplification primers is about 100-100,000 amplification primers per mm2.

[00205] In some embodiments, the plurality of immobilized nucleic acid amplification duplexes on the support are in fluid communication with each other to permit flowing a solution of the first amplification reagent onto the support so that the plurality of immobilized amplification duplexes on the support can be essentially simultaneously reacted with the first amplification reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized amplification duplexes can be used to conduct nucleic acid amplification reactions in a massively parallel manner on the support.

[00206] In some embodiments, the nucleic acid amplification reagents comprises a second amplification reagent, and methods that employ the nucleic acid amplification reagents where the methods comprise contacting a plurality of the complexed amplification polymerases with a second amplification reagent under a condition that is suitable for retaining the complexed amplification polymerases and promoting amplification to generate amplicons having sequences that are complementary to their respective library molecule. The second amplification reagents can be used for the massively parallel sequencing workflow of step (d) described below).

[00207] In some embodiments, the amplification reaction can generate concatemer template molecules containing tandem repeat sequences of the circular library molecule including any insert and adaptor sequences present in the original circularized nucleic acid library molecule. In some embodiments, the amplification reaction can generate linear template molecules having one copy of the linear library molecule including any insert and adaptor sequences present in the original linear nucleic acid library molecule. In some embodiments, the second amplification reagent comprises at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions and a plurality of nucleotides. In some embodiments, the second amplification reagent lacks an amplification polymerase enzyme. In some embodiments, the second amplification reagent further comprise any one or any combination of two or more of a detergent, a reducing agent, a viscosity agenT. In some embodiments, the first and second amplification reagents have a different pH. In some embodiments, the pH of the second amplification reagent is suitable for retaining a complex having the amplification polymerase (e.g., from the first amplification reagent) bound to a nucleic acid duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. In some embodiments, the pH of the second amplification reagent can be suitable for promoting activity of the amplification polymerase 18 08 25 (e.g., polymerase-catalyzed nucleotide incorporation activity). For example, the pH of the second amplification reagent can be about pH 8.5 or higher (e.g., pH 8.5-8.8).

[00208] In some embodiments, the plurality of nucleotides comprise a mixture of two or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of four types of nucleotides including dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of five types of nucleotides including dATP, dGTP, dCTP, dTTP and dUTP.

[00209] In some embodiments, the second amplification reagents further comprise at least one nucleic acid amplification duplex which comprises a nucleic acid template molecule (e.g., linear or circular template molecules) hybridized to an amplification oligonucleotide primer. The amplification primer can hybridize to at least a portion of the nucleic acid template molecule. The amplification primer comprises a 3’ extendible end or a 3’ nonextendible end.

[00210] In some embodiments, the amplification primer comprises soluble oligonucleotide primers (e.g., in-solution), or the amplification primer is immobilized to a support or immobilized to a coating (e.g., polymer coating) on the support.

[00211] In some embodiments, the second amplification reagents further comprise at least one nucleic acid duplex immobilized to a support, where the nucleic acid duplex comprise a nucleic acid template molecule (e.g., linear or circular template molecule) hybridized to an amplification primer, and where the template molecule and / or the amplification primer is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, a plurality of amplification primers are immobilized to one or more layers of the coatings on the support where the density of the immobilized amplification primers is about 100-100,000 amplification primers 2 per mm .

[00212] In some embodiments, the plurality of immobilized nucleic acid amplification duplexes on the support are in fluid communication with each other to permit flowing a solution of the second amplification reagent onto the support so that the plurality of immobilized amplification duplexes on the support can be essentially simultaneously reacted with the second amplification reagent in a massively parallel manner. In some embodiments, 18 08 25 the fluid communication of the plurality of immobilized amplification duplexes can be used to conduct nucleic acid amplification reactions in a massively parallel manner on the support.

[00213] In some embodiments, the second amplification reagents further comprise at least one concatemer immobilized to a support, and where the concatemer is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, a plurality of concatemers are immobilized to one or more layers of the coatings on the support where the density of the immobilized concatemers is about 100-100,000 amplification primers per mm2.

[00214] In some embodiments, the plurality of immobilized concatemers on the support are in fluid communication with each other to permit flowing a solution of the second amplification reagent onto the support so that the plurality of immobilized concatemers on the support can be essentially simultaneously reacted with the second amplification reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized concatemers can be used to conduct nucleic acid amplification reactions in a massively parallel manner on the support.

[00215] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 8), MgSO4 (20 mM), KC1 (50mM), (NH4)SO4 (30 mM), Tween-80 (RTM) (0.5%), DTT (1 mM), Betaine (0.8 M), sucrose (0.3 M), dATP (2 mM), dGTP (2 mM), dCTP (2 mM), dTTP (2 mM), dUTP (0.01 mM), and an amplification polymerase (120 nM). In some cases, this formulation may be referred to as Formulation AR1-A.

[00216] In some embodiments, a first amplification reagent may comprise Tris (10 mM, pH 8), MgSO4 (30 mM), KC1 (20mM), Triton-X (RTM) (0.2%), DTT (2 mM), Betaine (0.8 M), sucrose (0.1 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.02 mM), and an amplification polymerase (240 nM). In some cases, this formulation may be referred to as Formulation AR1-B.

[00217] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 8), MgSO4 (5 mM), KC1 (50 mM), NaCl (40 mM), (NH4)SO4 (5 mM), Tween-80 (RTM) (0.5%), DTT (2 mM), Betaine (0.1 M), sucrose (0.1 M), dATP (2 mM), dGTP (2 mM), dCTP (2 mM), dTTP (0.99 mM), dUTP (0.02 mM), and an amplification polymerase (120 nM). In some cases, this formulation may be referred to as Formulation AR1-C.

[00218] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 7), MgSO4 (10 mM), KC1 (90mM), (NH4)SO4 (10 mM), Tween-80 (RTM) (0.1%), DTT 18 08 25 (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.01 mM), and an amplification polymerase (240 nM). In some cases, this formulation may be referred to as Formulation AR1-D.

[00219] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 7), MgSO4 (70 mM), KC1 (30mM), NaCl (40 mM), (NH4)SO4 (20 mM), Tween-80 (RTM) (0.5%), DTT (5 mM), Betaine (0.4 M), sucrose (1 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.01 mM), and an amplification polymerase (500 nM). In some cases, this formulation may be referred to as Formulation AR1-E.

[00220] In some embodiments, a first amplification reagent may comprise Tris (25 mM, pH 7), MgSO4 (10 mM), KC1 (90mM), (NH4)SO4 (20 mM), Triton-X (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.01 mM), and an amplification polymerase (500 nM). In some cases, this formulation may be referred to as Formulation AR1-F.

[00221] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 7.5), MgSO4 (10 mM), KC1 (lOmM), (NH4)SO4 (30 mM), Tween-80 (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.01 mM), and an amplification polymerase (120 nM). In some cases, this formulation may be referred to as Formulation AR1-G.

[00222] In some embodiments, a first amplification reagent may comprise Tris (50 mM, pH 7.5), KC1 (90mM), (NH4)SO4 (100 mM), Triton-X (RTM) (0.1%), DTT (10 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), dUTP (0.01 mM), and an amplification polymerase (240 nM). In some cases, this formulation may be referred to as Formulation AR1-H.

[00223] In some embodiments, a second amplification reagent may comprise Tris (75 mM, pH 8.5), MgSO4 (10 mM), KC1 (200 mM), (NH4)SO4 (30 mM), Tween-80 (RTM) (0.3%), DTT (10 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-A.

[00224] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (10 mM), KC1 (90mM), (NH4)SO4 (10 mM), Tween-80 (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-B. 18 08 25

[00225] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (20 mM), KC1 (150mM), (NH4)SO4 (10 mM), Tween-80 (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-C.

[00226] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (10 mM), KC1 (90 mM), (NH4)SO4 (10 mM), Triton-X (RTM) (0.5%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-D.

[00227] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (50 mM), NaCl (150 mM), (NH4)SO4 (10 mM), Triton-X (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-E.

[00228] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (10 mM), NaCl (200 mM), (NH4)SO4 (30 mM), Tween-80 (RTM) (0.3%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-F.

[00229] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (5 mM), NaCl (90 mM), (NH4)SO4 (10 mM), SDS (0.5%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-G.

[00230] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), MgSO4 (20 mM), KC1 (15 mM), (NH4)SO4 (10 mM), SDS (0.3%), DTT (5 mM), Betaine (0.4 M), sucrose (0.4 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 mM), and dUTP (0.01 mM). In some cases, this formulation may be referred to as Formulation AR2-H.

[00231] In some embodiments, a second amplification reagent may comprise Tris (50 mM, pH 8.5), NaCl (45 mM), (NH4)SO4 (50 mM), Tween-80 (RTM) (0.1%), DTT (5 mM), Betaine (0.4 M), sucrose (0.8 M), dATP (1 mM), dGTP (1 mM), dCTP (1 mM), dTTP (0.99 18 08 25 mM), and dUTP (0.05 mM). In some cases, this formulation may be referred to as Formulation AR2-I. Universal Wash Reagents and Methods of Use

[00232] The present disclosure provides one or more universal wash reagents, and methods that employ the universal wash reagents. The universal wash reagents can be used to wash away unreacted components after any of the steps described herein. In some embodiments, the universal wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation and a detergent.

[00233] The universal wash reagents can be used for the massively parallel sequencing workflow prior to step (a), at step (b), at step (f), at step (1), at step (o) and / or at step (q), as described below.

[00234] In some embodiments, methods using the universal wash reagents include: conducting a step pre-(a) which is conducted prior to conducting the nucleic acid hybridization of step (a), the step pre(a) comprises: washing the immobilized amplification primers with the wash reagent.

[00235] In some embodiments, methods using the universal wash reagents include: conducting a washing step (b) which comprises washing the plurality of immobilized nucleic acid duplexes with a universal wash reagent.

[00236] In some embodiments, methods using the universal wash reagents include: conducting a washing step (f) which comprises washing the plurality of concatemers with the universal wash reagent.

[00237] In some embodiments, methods using the universal wash reagents include: conducting a washing step (1) which comprises washing the plurality of immobilized nucleic acid duplexes with the universal wash reagent.

[00238] In some embodiments, methods using the universal wash reagents include: conducting a washing step (o) which comprises washing the plurality of immobilized nucleic acid duplexes having extended sequencing primers with the universal wash reagent.

[00239] In some embodiments, methods using the universal wash reagents include: conducting a washing step (q) which comprises washing the plurality of immobilized nucleic acid duplexes having extended sequencing primers with the universal wash reagent.

[00240] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-A. 18 08 25

[00241] In some embodiments, a universal wash reagent may comprise Tris (20 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-B.

[00242] In some embodiments, a universal wash reagent may comprise Tris (30 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-C.

[00243] In some embodiments, a universal wash reagent may comprise Tris (40 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-D.

[00244] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.2), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-E.

[00245] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.4), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-F.

[00246] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.6), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-G.

[00247] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-H.

[00248] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (1 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-I.

[00249] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (1.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-J.

[00250] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (2 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-K.

[00251] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (2.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-L. 18 08 25

[00252] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (0.5%). In some cases, this formulation may be referred to as Formulation UWR-M.

[00253] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (1%). In some cases, this formulation may be referred to as Formulation UWR-N.

[00254] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (2%). In some cases, this formulation may be referred to as Formulation UWR-O.

[00255] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8), EDTA (0.5 mM), NaCl (100 mM), Tween-20 (RTM) (3%). In some cases, this formulation may be referred to as Formulation UWR-P.

[00256] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.2), EDTA (0.1 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-Q.

[00257] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.6), EDTA (0.2 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-R.

[00258] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.4), EDTA (0.3 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-S.

[00259] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.0), EDTA (0.4 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-T.

[00260] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (10 mM), Triton-X (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-U.

[00261] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (25 mM), Triton-X (RTM) (1%). In some cases, this formulation may be referred to as Formulation UWR-V.

[00262] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (50 mM), Triton-X (RTM) (0.2%). In some cases, this formulation may be referred to as Formulation UWR-W. 18 08 25

[00263] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (75 mM), Triton-X (RTM) (2%). In some cases, this formulation may be referred to as Formulation UWR-X.

[00264] In some embodiments, a universal wash reagent may comprise Tris (40 mM, pH 8.8), EDTA (0.4 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-Y.

[00265] In some embodiments, a universal wash reagent may comprise Tris (30 mM, pH 8.8), EDTA (0.1 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-Z.

[00266] In some embodiments, a universal wash reagent may comprise Tris (20 mM, pH 8.8), EDTA (0.3 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AA.

[00267] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.0), EDTA (0.1 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AB.

[00268] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.2), EDTA (0.2 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AC.

[00269] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.4), EDTA (0.5 mM), NaCl (100 mM), Triton-X (RTM) (0.2%). In some cases, this formulation may be referred to as Formulation UWR-AD.

[00270] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.6), EDTA (1 mM), NaCl (100 mM), Triton-X (RTM) (0.4%). In some cases, this formulation may be referred to as Formulation UWR-AE.

[00271] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.4), EDTA (0.3 mM), NaCl (100 mM), Triton-X (RTM) (0.2%). In some cases, this formulation may be referred to as Formulation UWR-AF.

[00272] In some embodiments, a universal wash reagent may comprise Tris (10 mM, pH 8.8), EDTA (0.5 mM), NaCl (100 mM), Triton-X (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AG.

[00273] In some embodiments, a universal wash reagent may comprise Tris-HCl (5 mM, pH 8.8), EDTA (0.1 mM), NaCl (50 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AH. 18 08 25

[00274] In some embodiments, a universal wash reagent may comprise Tris-HCl (25 mM, pH 8.5), EDTA (0.5 mM), NaCl (60 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AI.

[00275] In some embodiments, a universal wash reagent may comprise Tris-HCl (10 mM, pH 8.2), EDTA (0.2 mM), NaCl (70 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AJ.

[00276] In some embodiments, a universal wash reagent may comprise Tris-HCl (25 mM, pH 8.7), EDTA (0.7 mM), NaCl (80 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AK.

[00277] In some embodiments, a universal wash reagent may comprise Tris-HCl (50 mM, pH 8.4), EDTA (0.4 mM), NaCl (90 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AL.

[00278] In some embodiments, a universal wash reagent may comprise Tris-HCl (25 mM, pH 8.8), EDTA (0.1 mM), NaCl (100 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AM.

[00279] In some embodiments, a universal wash reagent may comprise Tris-HCl (5 mM, pH 8.5), EDTA (0.4 mM), NaCl (75 mM), Triton-X-lOOk (RTM) (0.4%). In some cases, this formulation may be referred to as Formulation UWR-AN.

[00280] In some embodiments, a universal wash reagent may comprise Tris-HCl (20 mM, pH 8.2), EDTA (0.2 mM), NaCl (75 mM), Triton-X-lOOk (RTM) (0.7%). In some cases, this formulation may be referred to as Formulation UWR-AO.

[00281] In some embodiments, a universal wash reagent may comprise Tris-HCl (40 mM, pH 8.4), EDTA (0.7 mM), NaCl (200 mM), Triton-X-lOOk (RTM) (0.3%). In some cases, this formulation may be referred to as Formulation UWR-AP.

[00282] In some embodiments, a universal wash reagent may comprise Tris-HCl (100 mM, pH 8.6), EDTA (0.3 mM), NaCl (50 mM), Triton-X-lOOk (RTM) (2%). In some cases, this formulation may be referred to as Formulation UWR-AQ.

[00283] In some embodiments, a universal wash reagent may comprise Tris-HCl (30 mM, pH 8.1), EDTA (0.2 mM), NaCl (20 mM), Triton-X-lOOk (RTM) (0.1%). In some cases, this formulation may be referred to as Formulation UWR-AR.

[00284] In some embodiments, a universal wash reagent may comprise Tris-HCl (50 mM, pH 8), EDTA (0.1 mM, pH 7.5), NaCl (750 mM), Tween-20 (RTM) (0.01%). In some cases, this formulation may be referred to as Formulation UWR-AS. 18 08 25

[00285] In some embodiments, a universal wash reagent may comprise Tris-HCl (60 mM, pH 8), EDTA (0.1 mM, pH 7.8), NaCl (50 mM), Tween-20 (RTM) (0.05%). In some cases, this formulation may be referred to as Formulation UWR-AT.

[00286] In some embodiments, a universal wash reagent may comprise Tris-HCl (70 mM, pH 8), EDTA (0.2 mM, pH 7.8), NaCl (250 mM), Tween-20 (RTM) (0.07%). In some cases, this formulation may be referred to as Formulation UWR-AU.

[00287] In some embodiments, a universal wash reagent may comprise Tris-HCl (70 mM, pH 8), EDTA (0.2 mM, pH 7.7), NaCl (100 mM), Tween-20 (RTM) (0.04%). In some cases, this formulation may be referred to as Formulation UWR-AV.

[00288] In some embodiments, a universal wash reagent may comprise Tris-HCl (50 mM, pH 8), EDTA (0.3 mM, pH 7.6), NaCl (300 mM), Tween-20 (RTM) (0.02%). In some cases, this formulation may be referred to as Formulation UWR-AW.

[00289] In some embodiments, a universal wash reagent may comprise Tris-HCl (60 mM, pH 8), EDTA (0.3 mM, pH 7.5), NaCl (20 mM), Tween-20 (RTM) (0.01%). In some cases, this formulation may be referred to as Formulation UWR-AX.

[00290] In some embodiments, a universal wash reagent may comprise Tris-HCl (70 mM, pH 8), EDTA (0.4 mM, pH 7.7), NaCl (10 mM), Tween-20 (RTM) (0.05%). In some cases, this formulation may be referred to as Formulation UWR-AY.

[00291] In some embodiments, a universal wash reagent may comprise Tris-HCl (80 mM, pH 8), EDTA (0.4 mM, pH 7.5), NaCl (100 mM), Tween-20 (RTM) (0.07%). In some cases, this formulation may be referred to as Formulation UWR-AZ.

[00292] In some embodiments, a universal wash reagent may comprise Tris-HCl (30 mM, pH 8), EDTA (0.5 mM, pH 7.6), NaCl (50 mM), Tween-20 (RTM) (0.04%). In some cases, this formulation may be referred to as Formulation UWR-BA.

[00293] In some embodiments, a universal wash reagent may comprise Tris-HCl (40 mM, pH 8), EDTA (0.5 mM, pH 7.7), NaCl (750 mM), Tween-20 (RTM) (0.02%). In some cases, this formulation may be referred to as Formulation UWR-BB.

[00294] In some embodiments, a universal wash reagent may comprise Tris-HCl (50 mM, pH 8), EDTA (0.6 mM, pH 7.6), NaCl (1 M), Tween-20 (RTM) (0.01%). In some cases, this formulation may be referred to as Formulation UWR-BC. Wash-Removal Reagents and Methods of Use

[00295] The present disclosure provides one or more wash-removal reagents, and methods that employ the wash-removal reagents. 18 08 25

[00296] In some embodiments, methods that employ the wash-removal reagents include contacting the plurality of concatemers with the wash-removal reagent to remove amplification polymerases and unreacted nucleotides after using the first and second amplification reagents. The wash-removal reagents can be used for the massively parallel sequencing workflow at step (e) as described helow.

[00297] In some embodiments, methods using the wash-removal reagents include contacting the plurality of immobilized fluorescently-labeled ternary complexes with a washremoval reagent to remove unbound sequencing polymerases and unbound multivalent molecules. The wash-removal reagent can be used for the massively parallel sequencing workflow at step (k) as described below.

[00298] In some embodiments, the wash-removal reagent comprises at least one solvent, a pH buffering agent, a chelating agent, a detergent and a chaotropic agent.

[00299] In some embodiments, a wash-removal reagent comprises Tris (50 mM, pH 8.4), EDTA (10 mM, pH 7.2), SDS (0.1%), Tween-20 (RTM) (0.5%), and KC1 (300 mM). In some cases, this formulation may be referred to as Formulation WRR-A.

[00300] In some embodiments, a wash-removal reagent comprises Tris (40 mM, pH 8.2), EDTA (50 mM, pH 7.3), SDS (0.5%), Tween-20 (RTM) (0.5%), and LiCl (250 mM). In some cases, this formulation may be referred to as Formulation WRR-B.

[00301] In some embodiments, a wash-removal reagent comprises Tris (30 mM, pH 8.6), EDTA (100 mM, pH 7.4), SDS (1%), Tween-20 (RTM) (0.4%), and KC1 (350 mM). In some cases, this formulation may be referred to as Formulation WRR-C.

[00302] In some embodiments, a wash-removal reagent comprises Tris (20 mM, pH 8.4), EDTA (100 mM, pH 7.5), SDS (2%), Tween-20 (RTM) (0.2%), and LiCl (200 mM). In some cases, this formulation may be referred to as Formulation WRR-D.

[00303] In some embodiments, a wash-removal reagent comprises Tris (10 mM, pH 8.2), EDTA (50 mM, pH 7.6), SDS (0.3%), Tween-20 (RTM) (0.1%), and KC1 (300 mM). In some cases, this formulation may be referred to as Formulation WRR-E.

[00304] In some embodiments, a wash-removal reagent comprises Tris (50 mM, pH 8.6), EDTA (20 mM, pH 7.3), SDS (1%), Triton X-100 (RTM) (0.5%), and KC1 (250 mM). In some cases, this formulation may be referred to as Formulation WRR-F.

[00305] In some embodiments, a wash-removal reagent comprises Tris (40 mM, pH 8.4), EDTA (10 mM, pH 7.4), Triton X-100 (RTM) (2%), and LiCl (350 mM). In some cases, this formulation may be referred to as Formulation WRR-G. 18 08 25

[00306] In some embodiments, a wash-removal reagent comprises Tris (30 mM, pH 8.2), EDTA (50 mM, pH 7.5), SDS (0.3%), Triton X-100 (RTM) (1%), and KC1 (200 mM). In some cases, this formulation may be referred to as Formulation WRR-H.

[00307] In some embodiments, a wash-removal reagent comprises Tris (20 mM, pH 8.4), EDTA (20 mM, pH 7.5), SDS (0.5%), Triton X-100 (RTM) (0.1%), and LiCl (300 mM). In some cases, this formulation may be referred to as Formulation WRR-I.

[00308] In some embodiments, a wash-removal reagent comprises Tris (10 mM, pH 8.2), EDTA (200 mM, pH 7.6), SDS (0.3%), Triton X-100 (RTM) (0.5%), and KC1 (350 mM). In some cases, this formulation may be referred to as Formulation WRR-J. Trap Reagents and Methods of Use

[00309] The present disclosure provides one or more trap reagents, and methods that employ the trap reagents where the methods comprise forming a plurality of ternary complexes by contacting the plurality of template molecules (e.g., concatemers or single copy template molecules) with a plurality of sequencing primers that hybridize to a sequencing primer binding site on the template molecules, and the trap reagent. The trap reagent comprises a plurality of first sequencing polymerases and nucleotide reagents (e.g., multivalent molecules) and compounds that promote formation of ternary complexes where polymerase-catalyzed incorporation of nucleotide units is inhibited. The template molecules and sequencing primers form ternary complexes in the presence of the trap reagent. The trap reagents can be used for the massively parallel sequencing workflow at step (g) as described below.

[00310] In some embodiments, the trap reagents comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent, a plurality of multivalent molecules and a first sequencing polymerase enzyme. The first sequencing polymerases can be labeled with a detectable moiety (e.g., a fluorophore) or can be unlabeled. In some embodiments, the trap reagent further comprises at least one viscosity agent. In some embodiments, the trap reagent further comprises a plurality of sequencing primers which hybridize to at least a portion of a nucleic acid template molecule. The sequencing primer comprises a 3’ extendible end or a 3’ nonextendible end. In some embodiments, the sequencing primer comprises soluble oligonucleotide primers (e.g., in-solution).

[00311] In some embodiments, die trap reagents comprise a plurality of nucleotide reagents which comprises a plurality of multivalent molecules. A multivalent molecule 18 08 25 generally comprises: (1) a core, and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit. See FIGS. 2A to 2D.

[00312] In some embodiments, the multivalent molecule comprises a core which is attached to the plurality of nucleotide arms. In some embodiments, the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, sugar and at least one phosphate group. In some embodiments, the linker is attached to the nucleotide unit through the base. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits and optionally the linker includes an aromatic moiety (FIGS. 2A to 2D). In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, and wherein the multiple nucleotide arms have the same type of nucleotide unit which is selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of multivalent molecules comprises a mixture of different types of multivalent molecules having two or more different types of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. For example, the mixture comprises a plurality of a first type of multivalent molecules each having one type of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The mixture also comprises a plurality of a second type of multivalent molecules each having a different type of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, which differ from the first type of nucleotide units in the first plurality.

[00313] In some embodiments, at least one of the multivalent molecules in the plurality is fluorescently-labeled, where the fluorophore is attached to the core or attached to at least one base on a nucleotide unit. In some embodiments, the fluorophore which is attached to the multivalent molecule corresponds to the base of the nucleotide unit to permit distinguishing nucleotide base units of the different fluorescently-labeled multivalent molecules.

[00314] In some embodiments, at least one of the multivalent molecules comprises at least one nucleotide arm having a cleavable moiety. In some embodiments, a multivalent molecule comprises 1, 2, 3, 4 or more nucleotide arms where each nucleotide arm includes a cleavable moiety. The cleavable moiety in the nucleotide arm can be cleaved with a cleavable agent to separate the nucleotide arm from the core.

[00315] In some embodiments, at least one of the multivalent molecules in the plurality includes a chain terminating moiety which inhibits polymerase-catalyzed incorporation of the 18 08 25 nucleotide unit. The chain terminating moiety can be attached to the 2’ or 3’ sugar position of the nucleotide unit. The chain terminating moiety can be removable from the nucleotide unit by contacting the multivalent molecule with a compound that cleaves / removes the chain terminating moiety to form a nucleotide unit with a 2’ or 3’ extendible group.

[00316] In some embodiments, the trap reagents are formulated to promote formation of a stable ternary complex which comprises a polymerase (e.g., the first sequencing polymerase) bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, in the ternary complex, the nucleotide unit is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand. In some embodiments, the trap reagents are formulated to promote formation of a ternary complex without incorporation of the complementary nucleotide unit into the 3’ end of the sequencing primer (e.g., no polymerase-catalyzed nucleotide incorporation). In some embodiments, the trap reagent comprises a non-catalytic divalent cation that promotes formation of the ternary complex without incorporation of the complementary nucleotide unit. In some embodiments, the non-catalytic divalent cation comprises strontium ions and / or barium ions. In some embodiments, the trap reagent lacks a catalytic divalent cation that promotes polymerase-catalyzed incorporation of the complementary nucleotide unit. Exemplary catalytic divalent cations include magnesium and / or manganese.

[00317] In some embodiments, the trap reagent can also include a monovalent salt which can promote formation of the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations such as NaCl, KC1, LiCl, CsCl, AgCl, (NEL^SCh or potassium glutamate or other such monovalent salt solutions. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. In some embodiments, the trap reagent can include a monovalent salt at a concentration of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.

[00318] The multivalent molecules used during the sequencing reaction offer many advantages that are not provided by free nucleotides. The multivalent molecules comprise a core attached to multiple arms with each arm tethered to a nucleotide unit. The multivalent molecules increase the local concentration of the nucleotide units in proximity to a polymerase / template binding site which favors formation of the ternary complex. Formation of the ternary complex is conducted under a condition that does not promote incorporation of 18 08 25 the nucleotide unit into the sequencing primer. The multivalent molecules also increase the persistence time in forming and preserving a stable ternary complex. When a sequencing reaction is conducted with a multivalent molecule labeled with a detectable moiety (e.g., fluorescent moiety) the longer persistence time of the ternary complex provides shorter imaging time and increases signal intensity during a sequencing reaction. The high signal intensity from the ternary complex remains for the entire binding and imaging step. Strong binding between the polymerase, the primed template strand, and the nucleotide unit also provides a stable ternary complex that remains intact during a subsequent washing step. The high intensity signal is retained when unreacted or non-complementary nucleotide units are removed during a washing step. After the signal imaging step (e.g., see the imaging reagent description below), the ternary complex can be destabilized and the sequencing primer can be extended by one nucleotide base in a subsequent incorporation / extension reaction (e.g., see the nucleotide incorporation reagent description below). After the incorporation / extension reaction, the binding and imaging steps can be repeated again using another cycle of binding with multivalent molecules to determine the identity of the next base in the template molecule.

[00319] The persistence time refers to the length of time that a stable complex (e.g., ternary complex) remains intact (e.g., without dissociation), where the complex comprises a nucleic acid template molecule hybridized to a primer, a polymerase, and a free nucleotide or nucleotide unit from a multivalent molecule. The persistence time is indicative of the stability of the complex and strength of the binding interactions between the nucleic acid template molecule hybridized to a primer, the polymerase, and the free nucleotide or nucleotide unit. Persistence time can be measured by observing the onset and / or duration of the complex, for example by observing a signal from a labeled component of the complex. For example, a labeled nucleotide or a labeled multivalent molecule may be present in the complex, thus allowing the signal from the label to be detected during the persistence time of the complex. An exemplary label is a fluorescent label. The stable ternary complex can exhibit a persistence time of more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 second, or longer than 1 second.

[00320] The multivalent molecules bind to a polymerase / duplex complex to form a ternary complex with a rate that is time-dependent, though substantially slower than the rate of association known for free nucleotides in solution. The on-rate (Kon) of a multivalent molecule is substantially slower than the on rate for free nucleotides. Importantly, the off rate (Koff) of the multivalent molecule is substantially slower than that observed for free 18 08 25 nucleotides. Therefore, the multivalent molecules provide a surprising and unexpected beneficial improvement of the persistence time of ternary complexes compared to ternary complexes formed using free nucleotides. Thus, multivalent molecules form stable ternary complexes which do not readily dissociate thereby improving imaging quality of the ternary complex during a sequencing workflow. The ternary complex is stable until exposed to a dissociation condition.

[00321] In some embodiments, the first sequencing polymerase can bind a complementary nucleotide unit of a multivalent molecule and nucleic acid duplex to form a ternary complex. The first sequencing polymerase comprises a recombinant wild-type or mutant polymerase comprising an amino acid sequence that is at least 80% identical to a backbone sequence of a polymerase from Candidatus altiarchaeales archaeon (e.g., any of SEQ ID NOS: 2, 3, 4, OR 5.)

[00322] In some embodiments, the first sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. The first sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the first sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some embodiments, the first sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, or 5.

[00323] In some embodiments, the first sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7, or 8. In some embodiments, the first sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7, or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567.

[00324] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In 18 08 25 some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00325] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00326] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00327] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12. 18 08 25

[00328] In some embodiments, the trap reagents further comprise at least one nucleic acid template molecule which comprises DNA or RNA, or a mixture of RNA and DNA. In some embodiments, the nucleic acid template molecules comprise linear or circularized molecules, or a mixture of linear and circular molecules. In some embodiments, the template molecules comprise a concatemer or a single-copy template molecule. In some embodiments, the nucleic acid template molecules comprise single-stranded molecules, double-stranded molecules or nucleic acid molecules having single- and double-stranded portions. In some embodiments, the nucleic acid template molecules are soluble or immobilized to a support or immobilized to a coating on the support. In some embodiments, the nucleic acid template molecules comprise concatemers each comprising tandem repeat sequences of a sequence of interest and any adaptor sequences operably joined to the sequence of interest. In some embodiments, the nucleic acid template molecules comprise amplified molecules (e.g., clonally amplified molecules).

[00329] In some embodiments, the nucleic acid template molecules comprise single copy template molecules (e.g., linear molecules) each comprising one copy of a sequence of interest and any adaptor sequences are operably linked or joined to the sequence of interest. In some embodiments, the nucleic acid template molecules comprise amplified molecules (e.g., clonally amplified molecules).

[00330] In some embodiments, the trap reagents further comprise a plurality of sequencing primers that hybridize to a universal sequencing primer binding site on the template molecules.

[00331] In some embodiments, the individual nucleic acid template molecules are joined to at least one adaptor, where the adaptor includes a capture primer binding sequence, an amplification primer binding sequence and / or a sequencing primer binding sequence. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor having a sample barcode sequence or a unique molecular tag sequence. In some embodiments, the nucleic acid template molecules are soluble or immobilized to a support or immobilized to a coating on the support.

[00332] In some embodiments, the nucleic acid template molecules comprise concatemers each comprising tandem repeat sequences of a sequence of interest and any adaptor sequences operably joined to the sequence of interest. In some embodiments, the nucleic acid template molecules comprise amplified molecules (e.g., clonally amplified molecules).

[00333] In some embodiments, the trap reagent further comprises a plurality of ternary complexes each comprising a first sequencing polymerase bound to a nucleic acid duplex 18 08 25 which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, the multivalent molecules are labeled with a fluorophore, where the multivalent molecules are part of the ternary complexes.

[00334] In some embodiments, the trap reagents further comprise a plurality of ternary complexes immobilized to a support, where the template molecule (e.g., concatemer) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2.

[00335] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the trap reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the trap reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct nucleic acid sequencing reactions (e.g., trapping reactions) in a massively parallel manner on the support.

[00336] In some embodiments, the trap reagent comprises HEPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), Gd-HCl (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and a sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation TR-A.

[00337] In some embodiments, the trap reagent comprises Bicine (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), Tween-20 (RTM) (0.02%), Gd-HCl (0.06 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (100 nM). In some cases, this formulation may be referred to as Formulation TR-B.

[00338] In some embodiments, the trap reagent comprises MOPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), Gd-HCl (0.04 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (500 nM). In some cases, this formulation may be referred to as Formulation TR-C. 18 08 25

[00339] In some embodiments, the trap reagent comprises BES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), Tween-20 (RTM) (0.02%), Gd-HCl (0.02 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (300 nM). In some cases, this formulation may be referred to as Formulation TR-D.

[00340] In some embodiments, the trap reagent comprises TES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), Gd-HCl (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (100 nM). In some cases, this formulation may be referred to as Formulation TR-E.

[00341] In some embodiments, the trap reagent comprises CAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), Triton-X (RTM) (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation TR-F.

[00342] In some embodiments, the trap reagent comprises TAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), Triton-X (RTM) (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (500 nM). In some cases, this formulation may be referred to as Formulation TR-G.

[00343] In some embodiments, the trap reagent comprises ACES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), Triton-X (RTM) (0.02%), urea (0.06 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (400 nM). In some cases, this formulation may be referred to as Formulation TR-H.

[00344] In some embodiments, the trap reagent comprises PIPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), Triton-X (RTM) (0.02%), urea (0.04 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (500 nM). In some cases, this formulation may be referred to as Formulation TR-I.

[00345] In some embodiments, the trap reagent comprises Tris (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), Triton-X (RTM) (0.02%), urea (0.02 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 18 08 25 pM), and sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation TR-J. Post-Trap Reagents and Methods of Use

[00346] The present disclosure provides one or more post-trap reagents, and methods that employ the post-trap reagents where the methods comprise preserving the ternary complexes without polymerase-catalyzed incorporation of the nucleotide units by contacting the plurality of ternary complexes with the post-trap reagent (e.g., step (h) of the methods described herein).

[00347] In some embodiments, the post-trap reagents comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and first sequencing polymerase. The post-trap reagents lack a plurality of multivalent molecules.

[00348] In some embodiments, the post-trap reagent further comprises at least one viscosity agent. In some embodiments, the post- trap reagent optionally comprises a plurality of sequencing primers which hybridize to at least a portion of a nucleic acid template molecule. The sequencing primer comprises a 3’ extendible end or a 3’ non-extendible end. In some embodiments, the sequencing primer comprises soluble oligonucleotide primers (e.g., in-solution).

[00349] In some embodiments, although the post-trap reagent lacks a plurality of multivalent molecules, the post-trap reagents are formulated to preserve the stable ternary complex which forms when using the trap reagent described above.

[00350] In some embodiments, the post-trap reagents are formulated to preserve a ternary complex without incorporation of the complementary nucleotide unit into the 3’ end of the sequencing primer (e.g., no polymerase-catalyzed incorporation of the nucleotide unit). In some embodiments, the post-trap reagent comprises a non-catalytic divalent cation that promotes formation of the ternary complex without incorporation of the complementary nucleotide unit. In some embodiments, the non-catalytic divalent cation comprises strontium ions and / or barium ions. In some embodiments, the post-trap reagent lacks a catalytic divalent cation that promotes polymerase-catalyzed incorporation of the complementary nucleotide unit. Exemplary catalytic divalent cations include magnesium and / or manganese.

[00351] In some embodiments, the post-trap reagent can also include a monovalent salt which can preserve the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent 18 08 25 cations and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NH^hSCU or potassium glutamate or other such monovalent salt solutions. The post-trap reagent can include a monovalent salt at a concentration of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.

[00352] In some embodiments, the post-trap reagents contain a first sequencing polymerase which can be the same type or a different type of first sequencing polymerase contained in the trap reagent.

[00353] In some embodiments, the first sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. The first sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the first sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some embodiments, the first sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, 5. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 1. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 2, 3, 4, or 5.

[00354] In some embodiments, the first sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7, or 8. In some embodiments, the first sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7, or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. . In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 6, 7, or 8.

[00355] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent 18 08 25 polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00356] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00357] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00358] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12. 18 08 25

[00359] In some embodiments, the post-trap reagent further comprises a plurality of ternary complexes each comprising a first sequencing polymerase bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, the multivalent molecules are labeled with a fluorophore, where the multivalent molecules are part of the ternary complexes.

[00360] In some embodiments, the post-trap reagents further comprise a plurality of ternary complexes immobilized to a support, where the template molecule (e.g., concatemer) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2.

[00361] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the post-trap reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the post-trap reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct nucleic acid sequencing reactions in a massively parallel manner on the support.

[00362] In some embodiments, the stable ternary complex comprises a first sequencing polymerase bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule wherein the multivalent molecule is fluorescently-labeled to permit detection of the stable ternary complex. The stable ternary complex which is preserved by the post-trap reagent is stable enough to be imaged by contacting the stable ternary complex (e.g., fluorescently-labeled) with an imaging reagent (see the imaging reagents described below). The stable ternary complex does not dissociate until contacted with the wash-removal reagent which contains a chaotropic agent (e.g., see the wash-removal reagent described above).

[00363] In some embodiments, a post-trap reagent comprises TAPSO (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton-X (RTM) (0.01%), 18 08 25 sucrose (0.2 M), and a sequencing polymerase (100 nM). In some cases, this formulation may be referred to as Formulation PTR-A.

[00364] In some embodiments, a post-trap reagent comprises MES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (100 mM), Ba-acetate (5 mM), Triton-X (RTM) (0.02%), sucrose (0.2 M), and sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation PTR-B.

[00365] In some embodiments, a post-trap reagent comprises MOPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (100 mM), Sr-acetate (5 mM), Triton-X (RTM) (0.04%), sucrose (0.2 M), and sequencing polymerase (50 nM). In some cases, this formulation may be referred to as Formulation PTR-C.

[00366] In some embodiments, a post-trap reagent comprises MOPSO (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (100 mM), Ba-acetate (5 mM), Triton-X (RTM) (0.08%), sucrose (0.2 M), and sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation PTR-D.

[00367] In some embodiments, a post-trap reagent comprises Tricine (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (100 mM), Sr-acetate (5 mM), Triton-X (RTM) (0.2%), sucrose (0.2 M), and sequencing polymerase (50 nM).. In some cases, this formulation may be referred to as Formulation PTR-E.

[00368] In some embodiments, a post-trap reagent comprises Bicine (10 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (5 mM), Tween-20 (RTM) (0.01%), sucrose (0.1 M), and a sequencing polymerase (100 nM). In some cases, this formulation may be referred to as Formulation PTR-F.

[00369] In some embodiments, a post-trap reagent comprises HEPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), sucrose (0.2 M), and a sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation PTR-G.

[00370] In some embodiments, a post-trap reagent comprises TES (50 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (200 mM), Ba-acetate (5 mM), Tween-20 (RTM) (0.04%), sucrose (0.4 M), and sequencing polymerase (50 nM). In some cases, this formulation may be referred to as Formulation PTR-H.

[00371] In some embodiments, a post-trap reagent comprises ACES (75 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (300 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.08%), sucrose (0.8 M), and sequencing polymerase (100 nM). In some cases, this formulation may be referred to as Formulation PTR-I. 18 08 25

[00372] In some embodiments, a post-trap reagent comprises PIPES (100 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (400 mM), Ba-acetate (5 mM), Tween-20 (RTM) (0.2%), sucrose (2 M), and sequencing polymerase (200 nM). In some cases, this formulation may be referred to as Formulation PTR-J. Imaging Reagents and Methods of Use

[00373] The present disclosure provides one or more imaging reagents, and methods that employ the imaging reagents. In some embodiments, the imaging reagent can be employed after formation of ternary complexes, for example after a trapping step and / or after a stepping step. Formation of ternary complexes can include binding a polymerase, a nucleic acid duplex which comprises a nucleic acid template molecule hybridized to a sequencing primer, and a nucleotide reagent (e.g., a nucleotide unit of a multivalent molecule or a nonconjugated nucleotide), to form a ternary complex. In some embodiments, in a ternary complex, the nucleotide unit or the nucleotide is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand. The imaging reagents can be formulated to preserve (e.g., stabilize) the ternary complexes that were previously formed during a trapping and / or stepping step.

[00374] The trapping and / or stepping reagents can be formulated to promote formation of ternary complexes but these reagents may, or may not, be formulated to reduce photo-damage of the ternary complex caused by exposing the ternary complex to excitation illumination during a subsequent imaging / detecting step. Thus, imaging reagent formulations can differ from that of the trapping and stepping reagents. The imaging reagents can be formulated to reduce photo-damage of any of the components of the ternary complexes where the damage may be caused by exposing the ternary complex to excitation illumination during an imaging / detecting step. The imaging reagents can also be formulated to preserve the ternary complex that formed during trapping and / or stepping by reducing dissociation of the components of the ternary complex that would otherwise occur in the absence of the imaging reagent. The imaging reagents preserve a ternary complex thereby increasing the persistence time of an assembled ternary complex. The stable ternary complex can exhibit a persistence time of more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 second, or longer than 1 second. By reducing dissociation of the ternary complex the imaging reagent increases the time period for imaging detectably labeled ternary complexes. The imaging reagents can also be formulated to preserve a ternary complex while inhibiting a polymerase-catalyzed nucleotide incorporation reaction. 18 08 25

[00375] The imaging reagent increases the persistence time of an assembled ternary complex, and decreases photo-damage of the template molecule, which effectively reduces the decay in fluorescent signal intensities of ternary complexes formed in a first sequencing cycle and ternary complexes formed in subsequent sequencing cycles, compared to signal intensities detected in subsequent sequencing cycles in the absence of the imaging reagent.

[00376] In some embodiments, the imaging reagents can be formulated to preserve (e.g., stabilize) a ternary complex that formed in the presence of any of the trap and / or post-trap reagents described herein. For example, the ternary complex comprises a polymerase (e.g., the first sequencing polymerase) bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer or single copy template molecule) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, in the ternary complex, the nucleotide unit is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand. In some embodiments, the multivalent molecule is detectably labeled with a fluorophore. In some embodiments, the imaging reagent is employed to image the signals (e.g., fluorescent signals) emitted by a plurality of ternary complexes.

[00377] In some embodiments, the imaging reagents can be formulated to preserve (e.g., stabilize) a ternary complex that formed in the presence of any of the stepping reagents described herein. For example, the ternary complex comprises a polymerase (e.g., the second sequencing polymerase) bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer or single copy template molecule) hybridized to a sequencing primer, and a complementary nucleotide (e.g., a non-conjugated free nucleotide). In some embodiments, in the ternary complex, the nucleotide is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand. In some embodiments, the nucleotide is detectably labeled with a fluorophore, or the nucleotide is non-labeled. In some embodiments, the nucleotide comprises a chain terminating moiety attached to the 3’ sugar position, or the nucleotide lacks a chain terminating moiety. In some embodiments, the imaging reagent is employed to image the signals (e.g., fluorescent signals) emitted by a plurality of ternary complexes.

[00378] The present disclosure provides one or more imaging reagents, and methods that employ the imaging reagents where the methods comprise (1) contacting the plurality of stable ternary complexes (e.g., plurality of immobilized fluorescently-labeled ternary complexes) with an imaging reagent under a condition suitable to preserve the ternary complexes; and (2) detecting the presence of at least one of the plurality of ternary complexes 18 08 25 by exposing the plurality of ternary complexes to an excitation illumination and detecting a fluorescent signal emitted from the immobilized fluorescently-labeled ternary complexes in response to the excitation illumination (e.g., step (i) of the methods described herein). In some embodiments, the methods further comprise identifying the nucleotide unit of a multivalent molecule that is bound to the sequencing primer, as part of the fluorescently-labeled ternary complex (e.g., step (j) of the methods described herein). The imaging reagents can be used for the massively parallel sequencing workflow at step (i) as described below. In some embodiments, the plurality of ternary complexes comprises a non-conjugated nucleotide that is bound to the sequencing primer, as part of a ternary complex. The imaging reagents can be used for the massively parallel sequencing workflow at step (m) as described below.

[00379] In some embodiments, the ternary complexes can be formed in the presence of a trapping or stepping reagent, and the trapping or stepping reagent can be removed from ternary complexes, and the removed reagent can be replaced with an imaging reagent that is formulated to preserve the ternary complexes.

[00380] In some embodiments, the ternary complexes can be formed in the presence of a trapping or stepping reagent, and the trapping or stepping reagent is mixed with an imaging reagent, where the imaging reagent is formulated to preserve the ternary complexes.

[00381] The imaging reagents can be formulated to include at least one compound for reducing photo-damage caused by exposing the ternary complexes to excitation illumination. The excitation illumination can induce formation of reactive oxygen species which can damage any component of the ternary complexes including the nucleic acid template molecule, the sequencing primer, the sequencing polymerase, the nucleotide unit, the nonconjugated nucleotide, the fluorophore attached to the multivalent molecule and / or the fluorophore attached to a non-conjugated nucleotide.

[00382] The inclusion of one or more photo-damage reducing compound in the imaging reagents can reduce formation of the excited triplet state and counteract the damaging effect of reactive oxygen species thereby improving photostability of the fluorophores, reducing photo-bleaching and / or retaining fluorescence intensity. The inclusion of one or more photodamage reducing compound in the imaging reagents may also reduce non-specific binding of the fluorescent dyes to other biomolecules (e.g., polymerases, nucleic acid template molecules and / or sequencing primer oligonucleotides). In some embodiments, the imaging reagents comprise any one or any combination of two or more photo-damage reducing compounds comprising ascorbic acid (or derivatives thereof), ascorbyl palmitate, D- 18 08 25 isoascorbic acid (erythorbic acid), sodium ascorbate, citric acid, coumaric acid, ferulic acid, caffeic acid, chi orogenic acid, sinapic acid, ellagic acid, gallic acid, gentisic acid, salicylic acid, vanillic acid, butylated hydroxytoluene (BTH), butylated hydroxy toluene (BHT), polyphenol antioxidants, polyvinyl alcohols, butylated hydroxy anisol (BHA) and / or Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). In some embodiments, the Trolox comprises a vitamin E analog including nitrated and nitroalkene Trolox derivatives. In some embodiments, the Trolox comprises Trolox quinone or Trolox hydroquinone. In some embodiments, the imaging reagents comprise Trolox that has been subjected to an aging process. In some embodiments, the imaging reagents comprise 1,3,5,7 cyclo-octatetraene (COT) and / or methyl viologen. In some embodiments, the gallic acid comprises sulfonated forms having 1, 2 or 3 sulfonate groups.

[00383] In some embodiments, the imaging reagents can be formulated to include at least one reducing compound which can stabilize any of the photo-damage reducing compound(s) that is / are present in the imaging reagents. For example, an imaging reagent that includes ascorbic acid (e.g., as a photo-damage reducing compound) can further include at least one reducing compound to reduce or inhibit oxidation of ascorbic acid. Oxidized ascorbic acid can form dehydroascorbic acid which can turn color from a clear to yellow. Oxidized ascorbic acid can form crystals. Ascorbic acid can oxidize after exposure to elevated temperatures of 25-65 °C. In some embodiments, imaging reagents that contain ascorbic acid can also include at least one reducing compound to inhibit oxidation, inhibit crystallization and / or inhibit oxidation at elevated temperatures. Exemplary reducing compounds include DTT (dithiothreitol), 2-beta mercaptoethanol, TCEP, (tris(2- carboxyethyl)phosphine), formamide, DMSO (dimethylsulfoxide), sodium dithionite (^282()4), glutathione, methionine, betaine, Tris(3-hydroxypropyl)phosphine (THPP) andN-acetyl cysteine.

[00384] In some embodiments, the imaging reagents can be formulated to include at least one compound that stabilizes ascorbic acid. In some embodiments, an imaging reagent that includes ascorbic acid can further include at least one stabilizing compound to reduce or inhibit degradation of ascorbic acid upon exposure to light. Exemplary stabilizer compounds include boric acid, tartaric acid, citric acid and Tiron (4,5-Dihydroxy-l,3-benzenedisulfonic acid; e.g., disodium salt). The imaging reagents can include a stabilizing compound at a concentration of about 1-20 mM, or about 20-40 mM, or about 40-60 mM, or about 60-100 mM.

[00385] In some embodiments, the imaging reagents comprise at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent 18 08 25 cation, a detergent and at least one compound for reducing photo-damage. In some embodiments, the imaging reagents include two, three, four or five photo-damage reducing compounds. In some embodiments, the imaging reagents further comprise, or lack, at least one viscosity agent. In some embodiments, a high viscosity imaging reagent includes one, two, or more viscosity agents. In some embodiments, a low viscosity imaging reagent lacks a viscosity agent.

[00386] In some embodiments, the imaging reagents are formulated to preserve the stable ternary complex which forms when using the trap and post-trap reagents described above.

[00387] In some embodiments, the imaging reagents are formulated to preserve a ternary complex without incorporation of the complementary nucleotide unit into the 3’ end of the sequencing primer (e.g., no polymerase-catalyzed incorporation of the nucleotide unit). In some embodiments, the imaging reagent comprises a non-catalytic divalent cation that promotes formation of the ternary complex without incorporation of the complementary nucleotide unit. In some embodiments, the non-catalytic divalent cation comprises strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin, and / or terbium ions. In some embodiments, the imaging reagent lacks a catalytic divalent cation that promotes polymerase-catalyzed incorporation of the complementary nucleotide unit. Exemplary catalytic divalent cations include magnesium and / or manganese.

[00388] In some embodiments, the imaging reagent can also include a monovalent salt which can preserve the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NHThSCU or potassium glutamate or other such monovalent salt solutions. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. In some embodiments, the imaging reagent can include a monovalent salt at a concentration of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.

[00389] In some embodiments, the imaging reagents comprise at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and at least one compound for reducing photo-damage. In some embodiments, the imaging reagents further comprise at least one reducing agent. In some embodiments, the imaging reagents further comprise at least one stabilizing compound for stabilizing ascorbic acid. In some embodiments, the imaging reagents further comprise one or more compounds for increasing viscosity of the imaging reagents. 18 08 25

[00390] In some embodiments, the imaging reagent comprises a pH buffering agent at a pH of about 6.5 - 9, or a pH of about 7 - 8.5, or a pH of about 7.5 - 8. In some embodiments, the imaging reagent comprises a pH buffering agent at a pH of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or a pH of about 8.

[00391] In some embodiments, the imaging reagent comprises a chelating agent, for example EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylene triamine pentaacetic acid) or NTA (N,N-bis(carboxymethyl)glycine).

[00392] In some embodiments, the imaging reagent comprises a monovalent cation comprising sodium, for example as sodium chloride, sodium fluoride, sodium bromide, sodium iodide, sodium sulfate, sodium bicarbonate, sodium carbonate or sodium amide. In some embodiments, the imaging reagent comprises sodium in an amount that does not promote crystal formation in the imaging reagent. In some embodiments, the imaging reagent lacks a monovalent cation comprising sodium.

[00393] In some embodiments, the imaging reagent comprises a non-catalytic divalent cation such as for example strontium, barium, calcium and / or tin. In some embodiments, the non-catalytic divalent cation comprises strontium chloride, strontium acetate, barium acetate or nickel chloride.

[00394] In some embodiments, the imaging reagent comprises a detergent, for example a non-ionic detergent such as Triton X-100 (RTM), Tween 20 (RTM), Tween 80 (RTM) or Nonidet P-40 (RTM).

[00395] In some embodiments, the imaging reagent comprises a reducing agent, for example DTT (dithiothreitol), TCEP, (tris(2- carboxyethyl)phosphine), or Tris(3-hydroxypropyl)phosphine (THPP). In some embodiments, the imaging reagent lacks a reducing agent.

[00396] In some embodiments, the imaging reagents further comprise, or lack, at least one viscosity agent. In some embodiments, a high viscosity imaging reagent includes one, two, or more viscosity agents. In some embodiments, a low viscosity imaging reagent lacks a viscosity agent. In some embodiments, the imaging reagents comprise at least one viscosity agent comprising any one or any combination or two or more of sucrose, ethylene glycol and / or glycerol. In some embodiments, the concentration of the sucrose in the imaging reagents can be about 0.1-0.3 M, or about 0.3-0.6 M, or about 0.6-0.9 M, or about 0.9-1 M, or about 1-2 M. In some embodiments, the concentration of the ethylene glycol in the imaging reagents can be about 1-5%, or about 5-10%, or about 10-20%, or about 20-40%, or about 40- 18 08 25 60%, or about 60-80%. In some embodiments, the concentration of the glycerol in the imaging reagents can be about 1-2%, or about 2-4%, or about 4-6%, or about 6-8%, or about 8-10%.

[00397] In some embodiments, the imaging reagents include two, three, four or five photodamage reducing compounds. The photo-damage reducing compounds can be water soluble, for example at a pH of about 6.5 - 9, or a pH of about 7 - 8.5, or a pH of about 7.5 - 8. The imaging reagent includes at least photo-damage reducing compound at a concentration that reduces crystal formation in the imaging reagent.

[00398] In some embodiments, the imaging reagents comprise a photo-damage reducing compound including ascorbic acid in any form. Ascorbic acid includes both L-isomer and D-isomer and mixtures of L- and D-isomers, and racemic mixtures. In some embodiments, the ascorbic acid comprises a salt for example sodium L-ascorbate. In some embodiments, the ascorbic acid comprises dehydroascorbic acid (DHA). In some embodiments, includes ascorbate and analogs and derivatives thereof. In some embodiments, derivatives include ascorbate having an esterified 5-hydroxy and / or 6-hydroxy group. In some embodiments, derivatives include ascorbate in which the 5- and / or 6-hydroxy group is replaced with a halo or amino group. In some embodiments, derivatives include ascorbate in which the 5- and / or 6-hydroxy group lacks a hydroxy group such as for example a hydrogen atom replaces the hydroxyl group. In some embodiments, ascorbate derivatives include 5-deoxy-L-ascorbate, 6-bromo-6-deoxy-L-ascorbate, 6-amino-6-deoxy-L-ascorbate, L-ascorbic acid 6-carb oxy late, 6-O-tosyl-L-ascorbate, and 6-O-ascorbyl alkanoates such as 6-ascorbyl palmitate (palmitoyl ascorbate). The concentration of the ascorbate in the imaging reagents can be about 1-10 mM, or about 10-20 mM, or about 20-30 mM, or about 30-40 mM, or about 40-50 mM, or about 50-60 mM, or about 60-70 mM, or about 70-80 mM, or about 80-90 mM, or about 90-100 mM.

[00399] In some embodiments, the imaging reagents comprise a photo-damage reducing compounds including Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or other vitamin E analogs including nitrated and nitroalkene Trolox derivatives. In some embodiments, the Trolox comprises Trolox quinone or Trolox hydroquinone. In some embodiments, the Trolox that has been subjected to an aging process. The concentration of the Trolox or the Trolox derivative in the imaging reagent can be about 0.1-1 mM, or about 1-2 mM, or about 2-3 mM, or about 3-4 mM, or about 4-5 mM, or about 5-10 mM, or about 10-15 mM. 18 08 25

[00400] In some embodiments, the imaging reagents comprise a Trolox compound with or without an ascorbate. In some embodiments, the imaging reagents comprise a combination of photo-damage reducing compounds including an ascorbate (e.g., sodium ascorbate) and a Trolox compound (e.g., Trolox or Trolox quinone or Trolox hydroquinone or aged Trolox). In some embodiments, Trolox can be exposed to UV light for minutes or hours to produce aged Trolox which is hydroxylated and can exhibit increased water solubility. For example the Trolox can be exposed to UV light for 15-30 minutes, or 30-45 minutes, or 45-60 minutes. Trolox can be exposed to UV light for 1-4 hours, or 4-8 hours, or 8-12 hours, or 12-16 hours, or 16-20 hours, or up to 60 hours. Conducting a sequencing workflow using imaging reagents that include Trolox (non-aged), Trolox quinone, Trolox hydroquinone or aged Trolox, can reduce sequencing phasing and pre-phasing rates compared to sequencing workflows that do not include these Trolox compounds. The phasing and / or pre-phasing rates can be reduced by about 1-3%, or about 3-6%, or about 6-9%, or more than 9%.

[00401] In some embodiments, the imaging reagents comprise an ascorbate (e.g., sodium ascorbate) and a Trolox (or Trolox quinone or Trolox hydroquinone or aged Trolox), and ethylene glycol.

[00402] FIGS. 17-18 and FIGS. 35-42 show data of the effects of various imaging reagents on signal intensity of fluorescent dyes with time. The different formulations contain either no compounds that reduce photo damage or different combinations of compounds that reduce photo damage.

[00403] In some embodiments, the imaging reagent further comprises a plurality of ternary complexes each comprising a first sequencing polymerase bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, the multivalent molecules are labeled with a fluorophore, where the multivalent molecules are part of the ternary complexes. In the ternary complex, the nucleotide unit of the multivalent molecule is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand.

[00404] In some embodiments, the imaging reagent further comprises a plurality of ternary complexes each comprising a sequencing polymerase (e.g., a second sequencing polymerase) bound to a nucleic acid duplex which includes a nucleic acid template molecule hybridized to a sequencing primer, and a nucleotide reagent (e.g., a complementary non-conjugated nucleotide). In some embodiments, the non-conjugated nucleotide is unlabeled or is labeled with a fluorophore. In some embodiments, in the ternary complex, the non-conjugated 18 08 25 nucleotide is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand.

[00405] The imaging reagents are formulated to include at least one compound for reducing photo-damage caused by exposing the ternary complexes to excitation illumination. The excitation illumination can induce formation of reactive oxygen species which can damage any component of the ternary complexes including the nucleic acid template molecule, the sequencing primer, the sequencing polymerase and / or the fluorophore attached to the multivalent molecule.

[00406] The inclusion of one or more photo-damage reducing compound in the imaging reagents can reduce formation of the excited triplet state and counteract the damaging effect of reactive oxygen species thereby improving photostability of the fluorophores, reducing photo-bleaching and / or retaining fluorescence intensity (see FIGS. 17 and 18). The inclusion of one or more photo-damage reducing compound in the imaging reagents may also reduce non-specific binding of the fluorescent dyes to other biomolecules (e.g., polymerases, nucleic acid template molecules and / or sequencing primer oligonucleotides).

[00407] In some embodiments, the imaging reagents further comprise a plurality of ternary complexes immobilized to a support, where the template molecule (e.g., concatemer or single copy template molecule) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of about 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2. In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the imaging reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the imaging reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct imaging reactions in a massively parallel manner on the support.

[00408] The inclusion of one or more photo-damage reducing compounds in the imaging reagents can also mitigate / reduce residual signal from a fluorescently-labeled multivalent molecule during an imaging step in one or more subsequent nucleic acid sequencing cycles (e.g., repeated sequencing cycles), or can mitigate / reduce residual signal from a 18 08 25 fluorescently-labeled non-conjugated nucleotide during an imaging step in one or more subsequent nucleic acid sequencing cycles (e.g., repeated sequencing cycles). For example, in a first cycle, the imaging step includes detecting the fluorescent signal emitted from the immobilized fluorescently-labeled ternary complexes in the presence of an imaging reagent. In a subsequent step, the fluorescently-labeled ternary complexes are washed to remove the fluorescently-labeled multivalent molecules and sequencing polymerases using a washremoval reagent that dissociates ternary complexes to generate nucleic acid duplexes of templates (e.g., concatemers) hybridized to sequencing primers. In yet another subsequent step, a plurality of free nucleotides and a second sequencing polymerase are bound to the nucleic acid duplexes under conditions suitable for incorporating a complementary nucleotide into the sequencing primer, thereby advancing the polymerase to the next base position. In a second cycle, the sequencing workflow can be repeated by binding sequencing polymerases and fluorescently-labeled multivalent molecules onto the nucleic acid duplexes using the pretrap, trap and post-trap reagents. Another imaging step follows in which fluorescent signals from residual fluorescently-labeled multivalent molecules remaining from the first cycle using could increase background signals in the second cycle and interfere with accurate signal detection in the current imaging cycle (See FIGS. 19-21). Thus, the imaging reagents described herein can mitigate residual signals from a fluorescently-labeled multivalent molecule during an imaging step in a subsequent nucleic acid sequencing cycle.

[00409] The present disclosure provides methods for inhibiting photo-damage of a ternary complex during a detecting step of a nucleic acid sequencing reaction comprising the steps of: (a) providing a ternary complex comprising a polymerase, a nucleic acid duplex having a nucleic acid template molecule hybridized to a primer, and a nucleotide reagent that is complementary to a next base of the primer-hybridized template molecule, wherein (i) the complementary nucleotide reagent and / or the polymerase is labeled with a fluorescent dye, and (ii) the ternary complex is a stable ternary complex that maintains a persistence time of longer than 1 second without incorporation of the nucleotide reagent; (b) illuminating the ternary complex with light in the presence of an imaging reagent comprising ascorbic acid or a salt thereof; and (c) determining the identity of the complementary nucleotide reagent in the ternary complex.

[00410] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the fluorescently labelled nucleotide reagent comprises a multivalent molecule which comprises a core attached to a plurality of nucleotide arms where each nucleotide arm comprises (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit. 18 08 25

[00411] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the fluorescently labelled nucleotide reagent comprises a nucleotide polyphosphate having 3-10 phosphate groups.

[00412] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the fluorescently labelled nucleotide reagent comprises a nucleotide triphosphate having a removable chain terminating moiety.

[00413] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, (i) the nucleotide reagent is fluorescently labeled and the polymerase lacks a fluorescent label, (ii) the nucleotide reagent lacks a fluorescent label and the polymerase is fluorescently labeled, or (iii) the nucleotide reagent is fluorescently labeled and the polymerase is fluorescently labeled.

[00414] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the ternary complex is immobilized to a support. In some embodiments, the support is coated with a hydrophilic layer having a water contact angle of less than 50 degrees.

[00415] In some embodiments, the methods for inhibiting photo-damage of a ternary complex further comprises providing a plurality of ternary complexes, each complex comprising a polymerase, a nucleic acid duplex having a nucleic acid template molecule hybridized to a primer, and a nucleotide reagent that is complementary to a next base of the primer-hybridized template molecule, wherein the plurality of ternary complexes are immobilized to a support. In some embodiments, the plurality of ternary complexes are immobilized to the support at pre-determined locations on the support. In some embodiments, the plurality of ternary complexes are immobilized to the support at random locations on the support.

[00416] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the nucleic acid duplex comprises a nucleic acid concatemer template molecule hybridized to a plurality of primers.

[00417] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the nucleic acid duplex further comprises a cluster of nucleic acid duplexes each duplex in the cluster comprising a single copy template molecule having one copy of an insert sequence and at least one universal adaptor sequence wherein the single copy template molecules are each hybridized to a primer.

[00418] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the nucleic acid duplex further comprises a plurality of clonally amplified nucleic 18 08 25 acid template molecules immobilized to a bead, and wherein each template molecule is hybridized to a primer.

[00419] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the imaging reagent comprises ascorbic acid or salt thereof at a concentration of at least 10 mM. In some embodiments, the imaging reagent comprises ascorbic acid or salt thereof at a concentration of at least 20 mM. In some embodiments, the imaging reagent comprises ascorbic acid or salt thereof at a concentration of up to 100 mM.

[00420] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the ascorbic acid comprises sodium ascorbate.

[00421] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the imaging reagent comprises ascorbic acid or salt thereof and further comprises Trolox and / or Trolox quinone. In some embodiments, the imaging reagent further comprises ethylene glycol.

[00422] In some embodiments, in the methods for inhibiting photo-damage of a ternary complex, the imaging reagent comprises a non-catalytic divalent cation that inhibits incorporation of the complementary nucleotide reagent wherein the non-catalytic divalent cation comprises strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium ions.

[00423] In some embodiments, the imaging reagent is employed to image the signals (e.g., fluorescent signals) emitted by a plurality of ternary complexes, each ternary complex comprising a first sequencing polymerase bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, the multivalent molecules are labeled with a fluorophore, where the multivalent molecules are part of the ternary complexes. In the ternary complex, the nucleotide unit of the multivalent molecule is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand.

[00424] In some embodiments, a multivalent molecule generally comprises: (1) a core, and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit. See FIGS. 2A and B.

[00425] In some embodiments, the multivalent molecule comprises a core which is attached to the plurality of nucleotide arms. In some embodiments, the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the 18 08 25 nucleotide unit comprises a base, sugar and at least one phosphate group. In some embodiments, the linker is attached to the nucleotide unit through the base. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits and optionally the linker includes an aromatic moiety (FIGS. 2A and 2B). In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, and wherein the multiple nucleotide arms have the same type of nucleotide unit which is selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of multivalent molecules comprises a mixture of different types of multivalent molecules having two or more different types of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. For example, the mixture comprises a plurality of a first type of multivalent molecules each having one type of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The mixture also comprises a plurality of a second type of multivalent molecules each having a different type of nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, which differ from the first type of nucleotide units in the first plurality.

[00426] In some embodiments, at least one of the multivalent molecules in the plurality is fluorescently-labeled, where the fluorophore is attached to the core or attached to at least one base on a nucleotide unit. In some embodiments, the fluorophore which is attached to the multivalent molecule corresponds to the base of the nucleotide unit to permit distinguishing nucleotide base units of the different fluorescently-labeled multivalent molecules.

[00427] In some embodiments, at least one of the multivalent molecules comprises at least one nucleotide arm having a cleavable moiety. In some embodiments, a multivalent molecule comprises 1, 2, 3, 4 or more nucleotide arms where each nucleotide arm includes a cleavable moiety. The cleavable moiety in the nucleotide arm can be cleaved with a cleavable agent to separate the nucleotide arm from the core.

[00428] In some embodiments, at least one of the multivalent molecules in the plurality includes a chain terminating moiety which inhibits polymerase-catalyzed incorporation of the nucleotide unit. The chain terminating moiety can be attached to the 2’ or 3’ sugar position of the nucleotide unit. The chain terminating moiety can be removable from the nucleotide unit by contacting the multivalent molecule with a compound that cleaves / removes the chain terminating moiety to form a nucleotide unit with a 2’ or 3’ extendible group.

[00429] In some embodiments, the first sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some 18 08 25 embodiments, the first sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. The first sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the first sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some embodiments, the first sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, or 5. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 1. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 2, 3, 4, or 5.

[00430] In some embodiments, the first sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7, or 8. In some embodiments, the first sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7, or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 6, 7, or 8.

[00431] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00432] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant 18 08 25 Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00433] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00434] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12.

[00435] In some embodiments, the nucleic acid template molecules comprise concatemers each comprising tandem repeat sequences of a sequence of interest and one or more adaptor sequences operably joined to a sequence of interest. In some embodiments, the sequence of interest is operably joined to any one or any combination of two or more adaptor sequence(s) including a surface capture primer binding sequence, an amplification primer binding sequence, a sequencing primer binding sequence, a sample barcode sequence, unique molecular tag sequence. In some embodiments, the nucleic acid template molecules comprise amplified molecules (e.g., clonally amplified molecules).

[00436] In some embodiments, the imaging reagent further comprises a plurality of ternary complexes each comprising a first sequencing polymerase bound to a nucleic acid duplex 18 08 25 which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide unit of a multivalent molecule. In some embodiments, the multivalent molecules are labeled with a fluorophore, where the multivalent molecules are part of the ternary complexes.

[00437] In some embodiments, the imaging reagents further comprise a plurality of ternary complexes immobilized to a support, where the template molecule (e.g., concatemer) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2.

[00438] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the imaging reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the imaging reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct imaging reactions in a massively parallel manner on the support.

[00439] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.8), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), sucrose (1 M), glycerol (5%), Trolox (2 mM), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-A.

[00440] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.8), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), sucrose (1 M), glycerol (5%), Trolox (2 mM), ascorbic acid (50 mM), and methyl viologen (2 mM). In some cases, this formulation may be referred to as Formulation IR-B.

[00441] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.8), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), sucrose (1 M), glycerol (5%), Trolox (2 mM), ascorbic acid (50 mM), and 1,3,5,7 cyclo-octatetraene (2 mM). In some cases, this formulation may be referred to as Formulation IR-C.

[00442] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) 18 08 25 (0.1%), sucrose (1 M), glycerol (5%), Trolox (2 mM), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-D.

[00443] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), glycerol (5%), Trolox (8 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-E.

[00444] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), glycerol (5%), Trolox (5 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-F.

[00445] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), glycerol (5%), Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-G.

[00446] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), Trolox (8 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-H.

[00447] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), Trolox (5 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-I.

[00448] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (100 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-J.

[00449] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), sucrose (0.5 M), glycerol (5%), aged Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-K.

[00450] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), glycerol (5%), aged Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-L. 18 08 25

[00451] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), sucrose (0.5 M), glycerol (5%), Trolox quinone (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation M.

[00452] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), glycerol (5%), Trolox quinone (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-N.

[00453] In some embodiments, an imaging reagent may comprise Tris-HCl (20 mM; pH 7.2-7.5), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), ethylene glycol (30%), glycerol (5%), Trolox (2 mM), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-O.

[00454] In some embodiments, an imaging reagent may comprise Tris-HCl (20 mM; pH 7.2), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), ethylene glycol (30%), glycerol (5%), Trolox (2 mM), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-P.

[00455] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), ethylene glycol (30%), glycerol (5%), Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-Q.

[00456] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), ethylene glycol (30%), glycerol (5%), aged Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-R.

[00457] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 8.0), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), ethylene glycol (30%), glycerol (5%), Trolox quinone (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-S.

[00458] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 7.2-7.5), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), ethylene glycol (30%), glycerol (5%), Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-T.

[00459] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 7.2-7.5), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) 18 08 25 (0.1%), ethylene glycol (30%), glycerol (5%), aged Trolox (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-U.

[00460] In some embodiments, an imaging reagent may comprise Tris-HCl (10 mM; pH 7.2-7.5), EDTA (0.5 mM; pH 7.5), NaCl (75 mM), Sr-acetate (5 mM), Triton X-100 (RTM) (0.1%), ethylene glycol (30%), glycerol (5%), Trolox quinone (2 mM), and ascorbic acid (25 mM). In some cases, this formulation may be referred to as Formulation IR-V.

[00461] In some embodiments, an imaging reagent may comprise Tris-HCl (20 mM; pH 7.2), EDTA (0.5 mM; pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), ethylene glycol (30%), glycerol (5%), Trolox (2 mM, fresh Trolox), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-W.

[00462] In some embodiments, an imaging reagent may comprise Tris-HCl (20 mM; pH 7.2), EDTA (0.5 mM; pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), TCEP (0.5 mM), ethylene glycol (30%), glycerol (5%), Trolox (2 mM, fresh Trolox), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-X.

[00463] In some embodiments, an imaging reagent may comprise Tris-HCl (20 mM; pH 7.2), EDTA (0.5 mM; pH 7.5), Sr-acetate (5 mM), Tween-20 (RTM) (0.02%), TCEP (0.5 mM), ethylene glycol (30%), glycerol (5%), Trolox (2 mM, fresh Trolox), and ascorbic acid (50 mM). In some cases, this formulation may be referred to as Formulation IR-Y. Stepping Reagents and Methods of Use

[00464] The present disclosure provides one or more stepping reagents, and methods that employ the stepping reagents where the methods comprise contacting the plurality of immobilized nucleic acid duplexes with the stepping reagent (e.g., step (m) of the methods described herein) under a condition suitable for promoting polymerase-catalyzed nucleotide incorporation. The stepping reagents can be used for the massively parallel sequencing workflow at step (m) as described below.

[00465] In some embodiments, the stepping reagents comprise: at least one solvent, at least one pH buffering agent, at least one monovalent cation, a catalytic divalent cation, a detergent, a second sequencing polymerase enzyme and a plurality of nucleotides (e.g., free nucleotides). In some embodiments, the second sequencing polymerases can be labeled with a detectable moiety (e.g., a fluorophore) or can be unlabeled. In some embodiments, the catalytic divalent cation comprises magnesium and / or manganese. In some embodiments, the stepping reagent lacks a non-catalytic divalent cation. Exemplary non-catalytic divalent 18 08 25 cations include strontium and / or barium. In some embodiments, the monovalent salt which can promote formation of the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations as, and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NH4)2SO4 or potassium glutamate or other such monovalent salt solutions. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. In some embodiments, the catalytic divalent cation comprises magnesium and / or manganese. In some embodiments, the stepping reagent lacks a non-catalytic divalent cation. Exemplary non-catalytic divalent cations include strontium and / or barium. In some embodiments, the monovalent salt which can promote formation of the ternary complex. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate.

[00466] In some embodiments, the stepping reagent further comprises at least one viscosity agent.

[00467] In some embodiments, the stepping reagent can optionally include a plurality of sequencing primers which hybridize to at least a portion of a nucleic acid template molecule. The plurality of sequencing primers comprise a 3’ extendible end or a 3’ non-extendible end. In some embodiments, the plurality of sequencing primers comprise soluble oligonucleotide primers (e.g., in-solution).

[00468] In some embodiments, individual nucleotides in the plurality of nucleotides comprise an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. In some embodiments, the nucleotides comprise a polyphosphate chain having 1-10 phosphate groups.

[00469] In some embodiments, the plurality of nucleotide comprises at least one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of nucleotide comprises a mixture of two or more, in any combination of nucleotides, selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

[00470] In some embodiments, at least one of the nucleotides in the plurality is fluorescently-labeled. The fluorophore can be attached to the base of the nucleotide. In some embodiments, the fluorophore corresponds to the base of the nucleotide to permit distinguishing nucleotide base of the different fluorescently-labeled nucleotides. In some embodiments, the labeled nucleotide comprises a fluorophore is attached to the base of the nucleotide via a cleavable linker that is cleavable with a compound that cleaves / removes the cleavable linker thereby removing the fluorophore label from the nucleotide. 18 08 25

[00471] In some embodiments, at least one of the nucleotides in the plurality of nucleotides includes a chain terminating moiety which inhibits nucleotide incorporation in a subsequent polymerase-catalyzed nucleotide incorporation reaction. The chain terminating moiety can be attached to the 2’ or 3’ sugar position of the nucleotide. The chain terminating moiety can be removable from the nucleotide by contacting the chain terminating nucleotide with a compound that cleaves / removes the chain terminating moiety to form a nucleotide with a 2’ or 3’ extendible group. For example, a chain terminating nucleotide comprising a chain terminating moiety attached to its 3’ sugar position, can be converted to a nucleotide having an extendible 3’OH sugar group by contacting the chain terminating nucleotide with a compound that cleaves / removes the chain terminating moiety.

[00472] In some embodiments, the stepping reagents are formulated to promote formation of a stable ternary complex which comprises a polymerase (e.g., the second sequencing polymerase) bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide. In some embodiments, in the ternary complex, the nucleotide is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand. In some embodiments, the stepping reagents are formulated to promote incorporation of the complementary nucleotide unit into the 3’ end of the sequencing primer (e.g., polymerase-catalyzed nucleotide incorporation) to generate a plurality of nucleic acid duplexes each having a template molecule (e.g., concatemer) hybridized to a nascent strand (e.g., extended sequencing primer) where the nascent strand is extended by one nucleotide. In some embodiments, the catalytic divalent cation comprises magnesium and / or manganese.

[00473] In some embodiments, the stepping reagent can also include a monovalent salt which can promote formation of the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations as are known in the art, and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NH4)2SO4 or potassium glutamate or other such monovalent salt solutions as are known in the art. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. The stepping reagent can include a monovalent salt at a concentration of about 5-200 mM, or about 25-100 mM.

[00474] In some embodiments, the second sequencing polymerase can bind a complementary nucleotide and nucleic acid duplex to form a ternary complex. The second sequencing polymerase comprises a recombinant wild-type or mutant polymerase comprising an amino acid sequence that is at least 80% identical to a backbone sequence of a polymerase 18 08 25 from Candidatus altiarchaeales archaeon (e.g., any of SEQ ID NOS:221-225), or from 9°N (e.g., SEQ ID NOS:226 or 227), or from Therminator (e.g., SEQ ID NO:228), or from Vent (e.g., SEQ ID NO:229), or from Deep Vent (e.g., SEQ ID NO:230), or from Pfu (e.g., SEQ ID NO:231), or from Pyrococcus abyssi (e.g., SEQ ID NO:232), or from RB69 (SEQ ID NO:233). FIG 16A and 16B list exemplary positionally equivalent mutations for various polymerases.

[00475] In some embodiments, the stepping reagents further comprise at least one nucleic acid template molecule which comprises DNA or RNA, or a mixture of RNA and DNA. In some embodiments, the nucleic acid template molecules comprise concatemer or single-copy template molecules, or a mixture of concatemer and single-copy template molecules. In some embodiments, the nucleic acid template molecules comprise single-stranded molecules, double-stranded molecules or nucleic acid molecules having single- and double-stranded portions. In some embodiments, individual nucleic acid template molecules include at least one adaptor, where the adaptor includes a capture primer binding sequence, an amplification primer binding sequence and / or a sequencing primer binding sequence. In some embodiments, individual nucleic acid template molecules are operably linked to at least one adaptor having a sample barcode sequence or a unique molecular tag sequence. In some embodiments, the nucleic acid template molecules are soluble or immobilized to a support or immobilized to a coating on the support.

[00476] In some embodiments, the nucleic acid template molecules comprise concatemers each comprising tandem repeat sequences of a sequence of interest and any adaptor sequences operably joined to the sequence of interest. In some embodiments, the nucleic acid template molecules comprise amplified molecules (e.g., clonally amplified molecules).

[00477] In some embodiments, the second sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some embodiments, the second sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. The second sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the second sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some 18 08 25 embodiments, the second sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, or 5. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 1. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 2, 3, 4, or 5.

[00478] In some embodiments, the second sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7 or 8. In some embodiments, the second sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7 or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 6, 7, or 8.

[00479] In some embodiments, the second sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00480] In some embodiments, the second sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00481] In some embodiments, the second sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu 18 08 25 polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00482] In some embodiments, the second sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12.

[00483] In some embodiments, the stepping reagent further comprises a plurality of ternary complexes each comprising a second sequencing polymerase bound to a nucleic acid duplex which includes a nucleic acid template molecule (e.g., a concatemer) hybridized to a sequencing primer, and a complementary nucleotide. In some embodiments, the nucleotide is labeled with a fluorophore, where the nucleotide is part of the ternary complex. In the ternary complex, the nucleotide is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand.

[00484] In some embodiments, the stepping reagents further comprise a plurality of ternary complexes immobilized to a support, where the template molecule (e.g., concatemer) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2.

[00485] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the stepping reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the stepping reagent in a massively parallel manner. 18 08 25 In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct stepping reactions in a massively parallel manner on the support.

[00486] In some embodiments, a stepping reagent comprises Bis-Tris propane (25 mM, pH 8.8), KC1 (40 mM), NH4SO4 (10 mM), MgSO4 (10 mM), Tween-20 (RTM) (0.2%), Betaine (250 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (0.5%), and a sequencing polymerase (0.24 pM). In some cases, this formulation may be referred to as Formulation SR-A.

[00487] In some embodiments, a stepping reagent comprises HEPES (25 mM, pH 8.6), KC1 (40 mM), NH4SO4 (10 mM), MgSO4 (20 mM), Triton-X (RTM) (0.5%), glutathione (250 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (0.5%), and a sequencing polymerase (0.37 pM). In some cases, this formulation may be referred to as Formulation SR-B.

[00488] In some embodiments, a stepping reagent comprises MES (25 mM, pH 8.4), KC1 (40 mM), NH4SO4 (40 mM), MgSO4 (30 mM), Tween-20 (RTM) (1%), tris(2-carboxyethyl)phosphine (250 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (0.5%), and a sequencing polymerase (0.48 pM). In some cases, this formulation may be referred to as Formulation SR-C.

[00489] In some embodiments, a stepping reagent comprises MOPS (25 mM, pH 8.2), KC1 (40 mM), NH4SO4 (10 mM), MgSO4 (10 mM), Triton-X (RTM) (1%), TCEP (250 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (0.5%), and a sequencing polymerase (0.2 pM). In some cases, this formulation may be referred to as Formulation SR-D.

[00490] In some embodiments, a stepping reagent comprises MOPSO (25 mM, pH 8.0), KC1 (40 mM), NH4SO4 (40 mM), MgSO4 (20 mM), Triton-X (RTM) (1%), sodium dithionite (250 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (0.5%), and a sequencing polymerase (1.20 pM). In some cases, this formulation may be referred to as Formulation SR-E.

[00491] In some embodiments, a stepping reagent comprises Bis-Tris propane (25 mM, pH 8.8), NaCl (40 mM), NH4SO4 (10 mM), MgSO4 (10 mM), Tween-20 (RTM) (0.2%), 2-beta mercaptoethanol (50 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (2%), and a sequencing polymerase (0.88 pM). In some cases, this formulation may be referred to as Formulation SR-F. 18 08 25

[00492] In some embodiments, a stepping reagent comprises BES (25 mM, pH 8.6), NaCl (40 mM), NH4SO4 (40 mM), MgSO4 (20 mM), Triton-X (RTM) (0.5%), DTT (50 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (2%), and a sequencing polymerase (0.44 pM). In some cases, this formulation may be referred to as Formulation SR-G.

[00493] In some embodiments, a stepping reagent comprises TES (25 mM, pH 8.4), NaCl (40 mM), NH4SO4 (10 mM), MgSO4 (30 mM), Tween-20 (RTM) (1%), formamide (50 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), and a sequencing polymerase (0.42 pM). In some cases, this formulation may be referred to as Formulation SR-H.

[00494] In some embodiments, a stepping reagent comprises Bis-Tris propane (25 mM, pH 8.2), NaCl (40 mM), NH4SO4 (40 mM), MgSO4 (10 mM), Triton-X (RTM) (1%), glutathione (50 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), and a sequencing polymerase (0.52 pM). In some cases, this formulation may be referred to as Formulation SR-I.

[00495] In some embodiments, a stepping reagent comprises CAPS (25 mM, pH 8.0), NaCl (40 mM), NH4SO4 (10 mM), MgSO4 (20 mM), Triton-X (RTM) (1%), Betaine (50 mM), sucrose (200 mM), N3-dATP (2 pM), N3-dGTP (2 pM), N3-dCTP (2 pM), N3-dTTP (2 pM), DMSO (2%), and a sequencing polymerase (0.88 pM). In some cases, this formulation may be referred to as Formulation SR-J. Cleaving Reagents and Methods of Use

[00496] The present disclosure provides one or more cleaving reagents, and methods that employ the cleaving reagents where the methods comprise cleaving a chain terminating moiety from the terminal nucleotide on a nascent strand (e.g., extended sequencing primer) thereby generating an extended sequencing primer with a 3’ extendible end (e.g., step (p) of the methods described herein).

[00497] In some embodiments, the cleaving reagent comprises: at least one solvent, a pH buffering agent, at least one monovalent cation, a detergent, a cleaving agent and a cleaving catalyst.

[00498] The cleaving reagents are formulated to retain the plurality of nucleic acid duplexes each having a template molecule (e.g., concatemer) hybridized to a nascent strand (e.g., extended sequencing primer). 18 08 25

[00499] In some embodiments, when the cleaving reagent includes at least one nucleotide having a chain terminating moiety at the 2’ or 3’ sugar position, then the cleaving reagents are formulated to include a cleaving agent that cleaves the chain terminating moiety from the terminal nucleotide on the nascent strand (e.g., on the extended sequencing primer) to generate an extended sequencing primer with a 3’ extendible end.

[00500] In some embodiments, when the stepping reagent includes at least one nucleotide having a fluorophore attached to its base via a cleavable linker, then the cleaving reagents are formulated to include a cleaving agent that cleaves the cleavable linker to generate a nucleotide that lacks a fluorophore.

[00501] In some embodiments, the cleaving reagent further comprise a plurality of nucleic acid duplexes each having a template molecule (e.g., concatemer) hybridized to a nascent strand (e.g., extended sequencing primer) where the nascent strand is extended by one nucleotide. In some embodiments, the one nucleotide is a terminal nucleotide on the nascent strand and the nucleotide comprises a chain terminating moiety. In some embodiments, the one nucleotide is a terminal nucleotide on the nascent strand and the nucleotide comprises an extendible 3’ OH group. In some embodiments, the nucleic acid duplexes are each bound to a polymerase (e.g., a second sequencing polymerase).

[00502] In some embodiments, the cleaving reagents further comprise a plurality of nucleic acid duplexes immobilized to a support, where the template molecule (e.g., concatemer) within the nucleic acid duplex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of nucleic acid duplexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized nucleic acid duplexes is about 100-100,000 per mm2.

[00503] In some embodiments, the plurality of immobilized nucleic acid duplexes are in fluid communication with each other to permit flowing a solution of the cleaving reagent onto the support so that the plurality of immobilized nucleic acid duplexes on the support can be essentially simultaneously reacted with the cleaving reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid duplexes can be used to conduct cleaving reactions in a massively parallel manner on the support. 18 08 25

[00504] In some embodiments, the cleaving reagent comprises HEPES (50 mM, pH 9.2), MgCh (4 mM), NaCl (500 mM), Triton-X (RTM) (0.1%), Trolox (2 mM), ascorbic acid (50 mM), TCEP (50 mM). In some cases, this formulation may be referred to as Formulation CR-A

[00505] In some embodiments, the cleaving reagent comprises MES (50 mM, pH 9.2), MgCh (4 mM), KC1 (500 mM), CHAPS (0.1%), Trolox (2 mM), ascorbyl palmitate (50 mM), THPP (50 mM). In some cases, this formulation may be referred to as Formulation CR-B

[00506] In some embodiments, the cleaving reagent comprises Bis Tris propane (50 mM, pH 9.2), MgCh (4 mM), NaCl (500 mM), 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) (0.1%), Trolox (2 mM), ascorbic acid (50 mM), THPP (50 mM). In some cases, this formulation may be referred to as Formulation CR-C.

[00507] In some embodiments, the cleaving reagent comprises MOPS (50 mM, pH 9.2), MgCh (4 mM), KC1 (500 mM), CHAPS (0.1%), Trolox (2 mM), citric acid (50 mM), betaine (50 mM). In some cases, this formulation may be referred to as Formulation CR-D.

[00508] In some embodiments, the cleaving reagent comprises MOPSO (50 mM, pH 9.2), MgCh (4 mM), NaCl (500 mM), CHAPS (0.1%), Trolox (2 mM), butylated hydroxytoluene (50 mM), 2-beta mercaptoethanol (50 mM). In some cases, this formulation may be referred to as Formulation CR-E.

[00509] In some embodiments, the cleaving reagent comprises BES (50 mM, pH 9.2), MgCh (4 mM), KC1 (500 mM), Tween-20 (RTM) (0.1%), Trolox (2 mM), Trolox (50 mM), N-acetyl cysteine (50 mM). In some cases, this formulation may be referred to as Formulation CR-F.

[00510] In some embodiments, the cleaving reagent comprises TES (50 mM, pH 9.2), MgCh (4 mM), NaCl (500 mM), Tween-20 (RTM) (0.1%), Trolox (2 mM), butylated hydroxy toluene (50 mM), glutathione (50 mM). In some cases, this formulation may be referred to as Formulation CR-G.

[00511] In some embodiments, the cleaving reagent comprises CAPS (50 mM, pH 9.2), MgCh (4 mM), KC1 (500 mM), Tween-20 (RTM) (0.1%), Trolox (2 mM), butylated hydroxy anisol (50 mM), THPP (50 mM). In some cases, this formulation may be referred to as Formulation CR-H.

[00512] In some embodiments, the cleaving reagent comprises TAPS (50 mM, pH 9.2), MgCh (4 mM), NaCl (500 mM), Triton-X (RTM) (0.1%), Trolox (2 mM), coumaric acid (50 18 08 25 mM), betaine (50 mM). In some cases, this formulation may be referred to as Formulation CR-I

[00513] In some embodiments, the cleaving reagent comprises TAPSO (50 mM, pH 9.2), MgCl2 (4 mM), KC1 (500 mM), Triton-X (RTM) (0.1%), Trolox (2 mM), caffeic acid (50 mM), THPP (50 mM). In some cases, this formulation may be referred to as Formulation CR-J METHODS

[00514] The present disclosure provides methods for conducting a nucleic acid sequencing workflow. In some embodiments, a method may comprise contacting a plurality of nucleic acid template molecules with a plurality of amplification primers in the presence of a nucleic acid hybridization reagent under a condition suitable to form a plurality of nucleic acid duplexes each duplex comprising a template molecule hybridized to an amplification primer. In some embodiments, the amplification primers are soluble primers or are immobilized to a support. In some embodiments, the plurality of nucleic acid duplexes comprises at least a first nucleic acid template molecule hybridized to a first amplification primer to form at least a first nucleic acid duplex, and at least a second nucleic acid template molecule hybridized to a second amplification primer to form a second nucleic acid duplex.

[00515] In some embodiments, the nucleic acid hybridization reagent comprises at least one solvent, a pH buffering agent, and at least one monovalent cation. In some embodiments, the hybridization reagent further comprises any one or any combination of two or more of a detergent, a reducing agent, a chaotropic agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and / or a crowding agent.

[00516] In some embodiments, the contacting is conducted at a temperature of about 20-25 °C, or about 25-35 °C, or about 35-45 °C, or about 45-55 °C, or about 55-65 °C, or about 65-75 °C, or higher temperatures.

[00517] In some embodiments, the plurality of nucleic acid template molecules comprises DNA, RNA, or a mixture of RNA and DNA. In some embodiments, the plurality of nucleic acid template molecules comprises linear or circularized molecules, or a mixture of linear and circular molecules. In some embodiments, the plurality of nucleic acid template molecules comprises single-stranded molecules, double-stranded molecules or nucleic acid molecules having single- and double-stranded portions. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid template molecules are operably linked to at least one adaptor sequence, where the adaptor sequence includes a capture primer binding 18 08 25 sequence, an amplification primer binding sequence and / or a sequencing primer binding sequence. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid template molecules are operably linked to at least one adaptor sequence having a sample barcode sequence or a unique molecular tag sequence.

[00518] In some embodiments, the amplification primers can hybridize to at least a portion of the nucleic acid template molecules. The amplification primer comprises a 3’ extendible end or the 3’ end comprises a chain terminating moiety.

[00519] In some embodiments, the amplification primers comprise soluble oligonucleotide primers (e.g., in-solution), or the amplification primers are immobilized to a support or immobilized to a coating on the support. In some embodiments, the density of the immobilized amplification primers on the support is about 104 - 1012 per mm2.

[00520] In some embodiments, the plurality of nucleic acid duplexes are immobilized to a support, where the template molecules (e.g., circularized template molecules) and / or the amplification primers are immobilized to a support or are immobilized to a coating on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiment, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, a plurality of amplification primers are immobilized to one or more layers of the coating on the support where the density of the immobilized amplification primers is about 100-100,000 amplification primers per 2 mm.

[00521] In some embodiment, the plurality of immobilized amplification primers on the support are in fluid communication with each other to permit flowing a solution of the reagents (e.g., hybridization reagent) onto the support so that the plurality of immobilized amplification primers on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized amplification primers can be used to conduct nucleic acid hybridization reactions in a massively parallel manner on the support.

[00522] In some embodiments, when the amplification primers are immobilized to a support or immobilized to a coating on the support, prior to forming the plurality of nucleic acid duplexes, the methods for conducting a nucleic acid sequencing workflow may comprise (i) providing a plurality of amplification primers that are immobilized to a support or immobilized to a coating on the support, and (ii) washing the immobilized amplification primers with a wash reagent. In some embodiments, the wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, and a 18 08 25 detergent. In some embodiments, the wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, or a detergent. In some embodiments, the washing is conducted at a temperature of about 20-45 °C, or at a temperature of about 20-30 °C.

[00523] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: washing the plurality of immobilized nucleic acid duplexes with a universal wash reagent. In some embodiments, the wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, and a detergent. In some embodiments, the wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, or a detergent. In some embodiments, the washing is conducted at a temperature of about 20-45 °C, or at a temperature of about 20-30 °C.

[00524] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise a two-stage amplification reaction employing a first and second amplification reagent where the pH of the first amplification reagent is formulated to inhibit nucleic acid amplification and the pH of the second amplification reagent is formulated to promote nucleic acid amplification.

[00525] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: contacting the plurality of nucleic acid duplexes with a first amplification reagent which comprises at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions, a plurality of nucleotides, and / or an amplification polymerase enzyme. In some embodiments, the first amplification reagent further comprises any one or any combination of two or more of a detergent, a reducing agent, and / or a viscosity agent.. In some embodiments, the pH of the first amplification reagent is suitable for binding the amplification polymerase to a nucleic acid duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. In some embodiments, the pH of the first amplification reagent can reduce / inhibit activity of the amplification polymerase. In some embodiments, the pH of the first amplification reagent can be about pH 8 or lower (e.g., pH 7-8).

[00526] In some embodiments, the contacting is conducted under a condition suitable for forming a plurality of complexed amplification polymerases each comprising an amplification polymerase and a nucleotide bound to a nucleic acid duplex. In some embodiments, the condition is not suitable for conducting nucleic acid amplification reactions. 18 08 25

[00527] In some embodiments, the plurality of nucleic acid duplexes are immobilized to the support, where the template molecules comprise circularized template molecules that are hybridizes to the amplification primers. In some embodiments, the amplification primers are immobilized to the support or are immobilized to a coating on the support.

[00528] In some embodiments, the amplification polymerase has strand displacement activity and comprise phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bea (exo-) DNA polymerase, KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[00529] In some embodiments, the plurality of nucleotides comprise a mixture of two or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of three or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of four types of nucleotides including dATP, dGTP, dCTP and dTTP.

[00530] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: contacting the plurality of complexed amplification polymerases with a second amplification reagent which comprises at least one solvent, a pH buffering agent, at least one monovalent cation, ammonium ions, and / or a plurality of nucleotides. In some embodiments, the second amplification reagent lacks an amplification polymerase enzyme. In some embodiments, the second amplification reagent further comprises any one or any combination of two or more of a detergent, a reducing agent, and / or a viscosity agent. In some embodiments, the first amplification reagent and the second amplification reagent have a different pH. In some embodiments, the pH of the second amplification reagent is suitable for retaining a complex having the amplification polymerase (e.g., from the first amplification reagent) bound to a nucleic acid duplex which comprises a nucleic acid template molecule hybridized to an amplification oligonucleotide primer. In some embodiments, the pH of the second amplification reagent can be suitable for promoting activity of the amplification polymerase. For example, the pH of the second amplification reagent can be about pH 8.5 or higher (e.g., pH 8.5-8.8). 18 08 25

[00531] In some embodiments, the contacting is conducted under a condition suitable for retaining the plurality of complexed amplification polymerases from each comprising an amplification polymerase and a nucleotide bound to a nucleic acid duplex, and the condition is suitable for conducting a plurality of nucleic acid amplification reactions to form a plurality of clonally amplified template molecules (e.g., concatemers or single copy template molecules).

[00532] In some embodiments, the plurality of concatemers comprises at least a first concatemer and a second concatemer. In some embodiments, the first concatemer is immobilized to a first location on the support (or a first location on the coating on the support). In some embodiments, the second concatemer is immobilized to a second location on the support (or a second location on the coating on the support) which differs from the location of the first immobilized concatemer.

[00533] In some embodiments, the second amplification reaction comprises a rolling circle amplification reaction to generate a plurality of concatemers each containing tandem repeat sequences of the circular template molecule and any adaptor sequences present in the original circularized nucleic acid template molecule. In some embodiments, the rolling circle amplification reaction can be conducted under isothermal amplification conditions at a constant temperature such as, for example about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about40°C, about42°C, about50°C, about60°C, about65°C, about70°C, about 75°C or at a higher temperature, or within a temperature range defined by any two of the foregoing temperatures.

[00534] In some embodiments, the plurality of nucleotides comprise a mixture of two or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of three or more nucleotides selected from a group consisting of dATP, dGTP, dCTP and dTTP. In some embodiments, the plurality of nucleotides comprise a mixture of four types of nucleotides including dATP, dGTP, dCTP and dTTP.

[00535] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: contacting the plurality of concatemers with a wash-removal reagent under a condition suitable to retain the immobilized concatemers, and the condition is suitable to remove (e.g., wash away) components of a first reaction (e.g., a first amplification reaction) and / or the second reaction (e.g., a second amplification reaction), including removing the amplification polymerase and the nucleotides. In some embodiments, the washremoval reagent comprises at least one solvent, a pH buffering agent, a chelating agent, a 18 08 25 detergent, and / or a chaotropic agent. In some embodiments, the contacting is conducted at a temperature of about 20-60 °C, or at a temperature of about 25-45 °C.

[00536] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: washing the plurality of concatemers with a universal wash reagent. In some embodiments, the washing can remove the chaotropic agent from the wash-removal reagent. In some embodiments, the universal wash reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, and / or a detergent. In some embodiments, the washing is conducted at a temperature of about 20-65 °C, or at a temperature of about 25-45 °C.

[00537] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: contacting the plurality of concatemers (e.g., immobilized concatemers) with a trap reagent, wherein the trap reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent, a plurality of multivalent molecules, a plurality of sequencing primers, and / or a first sequencing polymerase enzyme. In some embodiments, the trap reagent further comprises at least one viscosity agent. In some embodiments, the non-catalytic divalent cations comprise strontium and / or barium.

[00538] In some embodiments, the contacting is conducted under a condition suitable for forming a plurality of immobilized ternary complexes by binding the immobilized concatemers to sequencing primers, the first sequencing polymerases and a plurality of multivalent molecules, thereby forming a plurality of immobilized ternary complexes.

[00539] In some embodiments, the immobilized template molecule comprises an immobilized concatemer molecule comprising tandem repeat sequences of a sequence-of-interest and any adaptor sequences present in the original circularized nucleic acid template molecule. The contacting of step (g) can generate multiple ternary complexes along the same concatemer molecule, wherein individual ternary complexes comprise a sequencing primer, a first sequencing polymerase and a nucleotide unit of a multivalent molecule.

[00540] In some embodiments, the trap reagent further comprises a monovalent salt which can promote formation of the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations as are known in the art, and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NH4)2SO4 or potassium glutamate or other such monovalent salt solutions as are known in the art. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4, or potassium glutamate. 18 08 25

[00541] In some embodiments, the trap reagent further comprises a plurality of sequencing primers which hybridize to at least a portion of a nucleic acid template molecule. In some embodiments, the sequencing primer comprises a 3’ extendible end or a 3’ non-extendible end. In some embodiments, the sequencing primer comprises soluble oligonucleotide primers (e.g., in-solution).

[00542] In some embodiments, the contacting is conducted at a temperature of about 20-65 °C, or at a temperature of about 25-45 °C.

[00543] In some embodiments, the plurality of immobilized concatemers are contacted with the trap reagent and the plurality of sequencing primers under a condition suitable for forming at least a first immobilized ternary complex by binding the first immobilized concatemer (e.g., first immobilized template) to a sequencing primer, at least one of the first sequencing polymerases and at least one of the multivalent molecules, thereby forming the first immobilized ternary complex.

[00544] In some embodiments, the plurality of immobilized concatemers are contacted with the trap reagent and the plurality of sequencing primers under a condition suitable for forming at least a second immobilized ternary complex by binding the second immobilized concatemer (e.g., second immobilized template) to a sequencing primer, at least one of the first sequencing polymerases and at least one of the multivalent molecules, thereby forming the second immobilized ternary complex.

[00545] In some embodiments, in an individual ternary complex in the plurality of ternary complexes, the nucleotide unit from the multivalent molecule is bound to the 3’ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template strand.

[00546] In some embodiments, the plurality of immobilized concatemers are contacted with the trap reagent under a condition suitable for forming a plurality of ternary complexes, (e.g., at least a first and second ternary complex), and in the plurality of ternary complexes the suitable condition inhibits incorporation of a nucleotide unit from a multivalent molecule into a sequencing primer.

[00547] In some embodiments, the plurality of immobilized concatemers are contacted with the trap reagent under a condition suitable for forming a plurality of stable ternary complexes, (e.g., at least a first and second stable ternary complex) wherein the plurality of stable ternary complexes have a longer persistence time (e.g., little or no dissociation) compared to a plurality of ternary complexes formed by binding template molecules to the sequencing primer, the sequencing polymerase and a free nucleotide. 18 08 25

[00548] In some embodiments, individual multivalent molecule in the plurality comprise (a) a core, and (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit. See FIGS. 2A to 2D.

[00549] In some embodiments, the multivalent molecule comprises a core which is attached to the plurality of nucleotide arms. In some embodiments, the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, sugar and at least one phosphate group. In some embodiments, the linker is attached to the nucleotide unit through the base. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits and optionally the linker includes an aromatic moiety

[00550] In some embodiments, the multivalent molecule comprises a core which is attached to the plurality of nucleotide arms. In some embodiments, the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, sugar and at least one phosphate group. In some embodiments, the linker is attached to the nucleotide unit through the base. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits and optionally the linker includes an aromatic moiety (FIGS. 2A to 2D). In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, and wherein the multiple nucleotide arms have the same type of nucleotide unit which is selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of multivalent molecules comprises a mixture of multivalent molecules having two or more different types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, at least one of the multivalent molecules in the plurality is fluorescently-labeled, where the fluorophore is attached to the core or attached to at least one base on a nucleotide unit. In some embodiments, the fluorophore corresponds to the base of the nucleotide unit to permit distinguishing nucleotide base units of the different fluorescently-labeled multivalent molecules.

[00551] In some embodiments, the first sequencing polymerase can bind a complementary nucleotide unit of a multivalent molecule and nucleic acid duplex to form a ternary complex. The first sequencing polymerase comprises a recombinant wild-type or mutant polymerase comprising an amino acid sequence that is at least 80% identical to a backbone sequence of a 18 08 25 polymerase from Candidatus altiarchaeales archaeon (e.g., any of SEQ ID NOS:221-225), or from 9°N (e.g., SEQ ID NOS:226 or 227), or from Therminator (e.g., SEQ ID NO:228), or from Vent (e.g., SEQ ID NO:229), or from Deep Vent (e.g., SEQ ID NO:230), or from Pfu (e.g., SEQ ID NO:231), or from Pyrococcus abyssi (e.g., SEQ ID NO:232), or from RB69 (SEQ ID NO:233).

[00552] In some embodiments, the first sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the first sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some embodiments, the first sequencing polymerase comprises the amino acid sequence of any one of SEQ ID NOS: 2, 3, 4, or 5. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 1. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 2, 3, 4, or 5.

[00553] In some embodiments, the first sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7 or 8. In some embodiments, the first sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7 or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 6, 7, or 8.

[00554] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ 18 08 25 ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00555] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00556] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00557] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12.

[00558] In some embodiments, the plurality of ternary complexes are immobilized to a support. In some embodiments, the template molecule (e.g., concatemer) within the ternary 18 08 25 complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiments, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2. In some embodiments, at least one of the plurality of immobilized ternary complexes is a fluorescently-labeled ternary complex which comprises a nucleic acid duplex having a template molecule (e.g., a concatemer) hybridized to a sequencing primer, where the duplex is bound to a first sequencing polymerase and a fluorescently-labeled multivalent molecule.

[00559] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the trap reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the trap reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct nucleic acid sequencing reactions in a massively parallel manner on the support.

[00560] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: contacting the plurality of stable ternary complexes (e.g., immobilized ternary complexes) with a post-trap reagent, where the post-trap reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent and a first sequencing polymerase. In some embodiments, the post-trap reagents lack a plurality of multivalent molecules. In some embodiments, the post-trap reagent further comprises at least one viscosity agent. In some embodiments, the non-catalytic divalent cations comprise strontium and / or barium.

[00561] In some embodiments, the post-trap reagent further comprises a monovalent salt which can promote formation of the ternary complex. The monovalent salt may comprise, for example, one or more of Sodium, Potassium, Lithium, Rubidium, Cesium, Silver, or other monovalent cations as are known in the art, and may be supplied as NaCl, KC1, LiCl, CsCl, AgCl, (NH4)2SO4 or potassium glutamate or other such monovalent salt solutions as are known in the art. In some embodiments, the monovalent salt comprises NaCl, KC1, (NH4)2SO4 or potassium glutamate. 18 08 25

[00562] In some embodiments, the post-trap reagent optionally comprises a plurality of sequencing primers which hybridize to at least a portion of a nucleic acid template molecule. In some embodiments, the sequencing primer comprises a 3’ extendible end or a 3’ nonextendible end. In some embodiments, the sequencing primer comprises soluble oligonucleotide primers (e.g., in-solution).

[00563] In some embodiments, the plurality of immobilized ternary complexes (e.g., at least the first and second ternary complexes) that formed during step (g) are contacted with the post-trap reagent of step (h) under a condition suitable for preserving the ternary complexes without polymerase-catalyzed incorporation of the nucleotide units. In some embodiments, the post-trap reagent lacks a plurality of multivalent molecules. In some embodiments, the post-trap reagent reduces or eliminates dissociation of the existing polymerase from the multivalent molecules and nucleic acid duplexes so that the ternary complexes remain intact.

[00564] In some embodiments, the plurality of stable ternary complexes have a longer persistence time (e.g., little or no dissociation) compared to a plurality of ternary complexes formed by binding template molecules to the sequencing primer, the sequencing polymerase and a free nucleotide.

[00565] In some embodiments, the contacting is conducted at a temperature of about 20-65 °C, or at a temperature of about 25-45 °C.

[00566] In some embodiments, the post-trap reagents contain a first sequencing polymerase which can be the same type or a different type of first sequencing polymerase contained in the trap reagent.

[00567] In some embodiments, the first sequencing polymerase comprises a recombinant wild-type or mutant polymerase from Candidatus altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises a DNA polymerase having an amino acid sequence backbone of a DNA polymerase from a Candidatus Altiarchaeales archaeon. In some embodiments, the first sequencing polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, where the mutant DNA polymerase comprises an amino acid substitution at one or more positions selected from a group consisting of Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the first sequencing polymerase comprises (i) an amino acid substitution selected from a group consisting of Leu416Ser, Leu416Phe and Leu416Tyr; and (ii) the amino acid substitutions of Tyr417Ala, Pro418Gly, Ala493Ser, Ile529His, Arg515Leu and Asn567Asp. In some embodiments, the positions of the mutations described herein are with 18 08 25 reference to SEQ ID NO: 1. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NOS: 2, 3, 4, or 5.

[00568] In some embodiments, the first sequencing polymerase comprises a DNA polymerase from 9°N which comprises the amino acid sequence of SEQ ID NOS: 6, 7, or 8. In some embodiments, the first sequencing polymerase comprises a mutant 9°N polymerase having a backbone amino acid sequence of a polymerase from 9°N (e.g., SEQ ID NOS: 6, 7, or 8) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to any one of SEQ ID NO: 6, 7, or 8.

[00569] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 9 (Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 9) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 9.

[00570] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 10 (Deep Vent polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Deep Vent polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 10) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 10.

[00571] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 11 (Pfu polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pfu polymerase having a backbone amino acid sequence of a polymerase from Pfu (e.g., SEQ ID NO: 11) with mutations at positions that are equivalent to amino acid positions in a mutant 18 08 25 polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 11.

[00572] In some embodiments, the first sequencing polymerase comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 12 (Pyrococcus abyssi polymerase). In some embodiments, the first and / or second sequencing polymerase comprises a mutant Pyrococcus abyssi polymerase having a backbone amino acid sequence of a polymerase from Vent (e.g., SEQ ID NO: 12) with mutations at positions that are equivalent to amino acid positions in a mutant polymerase from a Candidatus Altiarchaeales archaeon, such as, for example, one or more mutations at positions Leu416, Tyr417, Pro418, Ala493, Arg515, Ile529 and Asn567. In some embodiments, the positions of the mutations described herein are with reference to SEQ ID NO: 12.

[00573] In some embodiments, the plurality of ternary complexes are immobilized to a support. In some embodiments, the template molecule (e.g., concatemer) within the ternary complex is immobilized to a support or is immobilized to a coating (e.g., polymer coating) on the support. In some embodiments, the support can be coated with at least one hydrophilic polymer coating. In some embodiments, the hydrophilic polymer coating has a water contact angle of no more than 45-50 degrees. In some embodiments, the plurality of ternary complexes are immobilized to one or more layers of the coatings on the support where the density of the immobilized ternary complexes is about 100-100,000 per mm2. In some embodiments, at least one of the plurality of immobilized ternary complexes is a fluorescently-labeled ternary complex which comprises a nucleic acid duplex having a template molecule (e.g., a concatemer) hybridized to a sequencing primer, where the duplex is bound to a first sequencing polymerase and a fluorescently-labeled multivalent molecule.

[00574] In some embodiments, the plurality of immobilized ternary complexes are in fluid communication with each other to permit flowing a solution of the post-trap reagent onto the support so that the plurality of immobilized ternary complexes on the support can be essentially simultaneously reacted with the post-trap reagent in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized ternary complexes can be used to conduct nucleic acid sequencing reactions in a massively parallel manner on the support.

[00575] In some embodiments, the methods for conducting a nucleic acid sequencing workflow may comprise: (i) contacting the plurality of stable ternary complexes (e.g., 18 08 25 plurality of immobilized fluorescently-labeled ternary complexes) with an imaging reagent, and (ii) detecting the presence of at least one of the plurality of ternary complexes by exposing the plurality of ternary complexes to an excitation illumination and detecting a fluorescent signal emitted from the immobilized fluorescently-labeled ternary complexes in response to the excitation illumination.

[00576] In some embodiments, the imaging reagent comprises at least one solvent, a pH buffering agent, a chelating agent, at least one monovalent cation, a non-catalytic divalent cation, a detergent, and / or at least one compound for reducing photo-damage. In some embodiments, the imaging reagents further comprise at least one viscosity agent.

[00577] In some embodiments, the imaging reagent preserves (e.g., stabilizes) the ternary complexes that were formed in the presence of a trap reagent. In some embodiments, the imaging reagent inhibits polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the imaging reagent reduces or inhibits photo-damage of the ternary complex by exposure to excitation illumination.

[00578] In some embodiments, the fluorescent signal emitted from the immobilized fluorescently-labeled ternary complexes can be detected by employing a system comprising a excitation illumi...

Claims

18 08 25What is claimed:

1. A method for sequencing a template nucleic acid, comprising:a) providing a fluorescently-labeled multivalent molecule comprising a core attached to a plurality of nucleotide-arms wherein individual nucleotide-arms comprise (i) a core attachment moiety, and (ii) a nucleotide moiety;b) forming a binding complex by contacting the template nucleic acid with (i) a polymerizing enzyme, (ii) a sequencing primer comprising a 3’ extendible end, and (iii) the multivalent molecule, wherein one of the nucleotide moieties of the multivalent molecule is bound to the 3' end of the sequencing primer at a position that is opposite a complementary nucleotide in the template nucleic acid;c) contacting the binding complex with a trap reagent comprising a non-catalytic divalent cation that promotes formation of the binding complex and inhibits polymerase-catalyzed incorporation of the nucleotide moiety into the 3’ extendible end of the sequencing primer; andd) contacting the binding complex with an imaging reagent, wherein the imaging reagent comprises a photobleaching reducing agent.

2. The method of claim 1, wherein the non-catalytic divalent cation is a strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium cation.

3. The method of claim 1, wherein the trap reagent further comprises a pH buffering agent.

4. The method of claim 3, wherein the pH buffering agent comprises Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (MEA), a citrate compound, a citrate mixture, NaOH, or KOH, or a mixture thereof.

5. The method of claim 1, wherein the trap reagent further comprises a chelating agent.18 08 256. The method of claim 5, wherein the chelating agent is selected from the group consisting of EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylene triamine pentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate, magnesium citrate, or a mixture thereof.

7. The method of claim 1, wherein the trap reagent further comprises a monovalentcation.

8. The method of claim 7, wherein the monovalent cation is sodium, potassium, or a mixture thereof.

9. The method of claim 1, wherein the trap reagent further comprises a detergent.

10. The method of claim 9, wherein the detergent comprises SDS (sodium dodecylsulfate), t-Octylphenoxypolyethoxyethanol, polysorbate 20, polysorbate 80, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate) or A-Dodecyl-AA-dimethyl-S-amonio-l-propanesulfate (DetX), LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate, or a mixture thereof.

11. The method of claim 1, wherein the trap reagent further comprises a fluorescently-labeled nucleotide conjugate.

12. The method of claim 11, wherein the fluorescently-labeled nucleotide conjugate is dATP, dGTP, dCTP, dTTP and dUTP, or a mixture thereof, wherein one of more of the dATP, dGTP, dCTP, dTTP and dUTP is labeled with a detectable reporter moiety.18 08 2513. The method of claim 1, wherein the trap reagent further comprises a first sequencingpolymerase enzyme.

14. The method of claim 13, wherein the first sequencing polymerase enzyme comprisesthe amino acid sequence of any one of SEQ ID NOs 1 to 12.

15. The method of claim 1, wherein the trap reagent further comprises a viscosity agent.

16. The method of claim 15, wherein the viscosity agent comprises trehalose, sucrose,cellulose, xylitol, mannitol, sorbitol or inositol, glycerol, ethylene glycol or propylene glycol, or a mixture thereof.

17. The method of claim 1, wherein the trap reagent comprises:(i) HEPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), polysorbate-20 (0.02%), Gd-HCl (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and a sequencing polymerase (200 nM);(ii) Bicine (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), polysorbate-20 (0.02%), Gd-HCl (0.06 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (100 nM);(iii) MOPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), polysorbate-20 (0.02%), Gd-HCl (0.04 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (500 nM);(iv) BES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), polysorbate-20 (0.02%), Gd-HCl (0.02 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (300 nM);(v) TES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), polysorbate-20 (0.02%), Gd-HCl (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (100 nM);(vi) CAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), t-Octylphenoxypolyethoxyethanol (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (200 nM);18 08 25(vii) TAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), t-Octylphenoxypolyethoxyethanol (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (500 nM)(viii) ACES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), t-Octylphenoxypolyethoxyethanol (0.02%), urea (0.06 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (400 nM);PIPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (25 mM), Sr-acetate (5 mM), t-Octylphenoxypolyethoxyethanol (0.02%), urea (0.04 M), ethylene glycol (10%), dATP (0.02 pM), dGTP (0.02 pM), dCTP (0.02 pM), dUTP (0.02 pM), and sequencing polymerase (500 nM); orTris (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KC1 (50 mM), Ba-acetate (10 mM), t-Octylphenoxypolyethoxyethanol (0.02%), urea (0.02 M), ethylene glycol (10%), dATP (0.04 pM), dGTP (0.04 pM), dCTP (0.04 pM), dUTP (0.04 pM), and sequencing polymerase (200 nM).

18. The method of claim 1, wherein the trap reagent does not comprise manganese or magnesium ions.

19. The method of claim 1, further comprising: detecting a fluorescence signal emitted from the binding complex in response to an excitation illumination.

20. The method of claim 1, wherein the photobleaching reagent comprises ascorbate.

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