Methods and compositions for nucleic acid sequencing

Abstract

Embodiments of the present disclosure relate to methods, kits, and compositions for dual-channel nucleic acid sequencing using blue and violet light excitation (e.g., 450-460 nm and 400-405 nm lasers, respectively). In particular, nucleotides can be directly labeled with blue dye, violet dye, or both blue and violet dyes. Alternatively, one or more nucleotides for incorporation can be unlabeled, and affinity reagents containing blue dye, violet dye, or both blue and violet dyes can be used to specifically bind to each type of incorporated nucleotide.

Description

[Technical Field]

[0001] The present disclosure relates generally to methods, systems, kits, and compositions for nucleic acid sequencing applications. [Background technology]

[0002] For DNA sequencing, it is desirable to use multiple spectrally distinct fluorescent labels to achieve independent detection of multiple spatially overlapping analytes. Such multiplexing methods can reduce the number of reaction vessels, simplifying experimental protocols and facilitating the creation of application-specific reagent kits. For example, in multicolor automated DNA sequencing systems, multiplexed fluorescent detection allows the analysis of multiple nucleotide bases in a single electrophoretic lane, thereby increasing throughput compared to single-color methods and reducing uncertainties associated with lane-to-lane electrophoretic mobility variations.

[0003] However, multiplexed fluorescence detection can be problematic, and several important factors limit the selection of appropriate fluorescent labels. First, it can be difficult to find a dye compound with substantially resolved absorption and emission spectra for a given application. In addition, when several fluorescent dyes are used together, generating fluorescent signals in distinguishable spectral regions by simultaneous excitation can be complicated because the dye absorption bands are usually widely separated, making it difficult to achieve comparable fluorescence excitation efficiency even for two dyes. Many excitation methods use high-power light sources such as lasers, and therefore dyes must be sufficiently photostable to withstand such excitation. A final consideration, particularly important for molecular biology methods, is the degree to which fluorescent dyes must be compatible with reagent chemicals, such as DNA synthesis solvents and reagents, buffers, polymerase enzymes, and ligase enzymes.

[0004] Fluorescent dye molecules with improved fluorescence properties, such as favorable fluorescence intensity, shape, and wavelength maximum of the fluorescence band, can improve the speed and accuracy of nucleic acid sequencing. A strong fluorescent signal is particularly important when measurements are performed in aqueous biological buffers and at elevated temperatures, because the fluorescence intensity of most organic dyes is significantly lower under these conditions. Furthermore, the nature of the base to which the dye is attached also affects the fluorescence maximum, fluorescence intensity, and other spectral properties of the dye. The sequence-specific interaction between nucleic acid bases and fluorescent dyes can be tailored by specific design of the fluorescent dye. Optimizing the fluorescent dye structure can improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors, and reduce reagent usage in nucleic acid sequencing, thereby reducing the cost of nucleic acid sequencing.

[0005] Several optical and technological developments have already significantly improved image quality, but ultimately have been limited by insufficient optical resolution. Optical resolution is governed by Abbe's law. Generally, the optical resolution of optical microscopes is limited to the ability to distinguish objects separated by approximately half the wavelength of the light used. Practically, only objects very far apart (at least 200–350 nm) can be distinguished by optical microscopy. One way to improve image resolution and increase the number of distinguishable objects per unit surface area is to use shorter wavelength excitation light. For example, using the same optical system, shortening the wavelength by approximately 100 nm (Δλ) results in better resolution (approximately Δ50 nm / (approximately 15%)), recording images with less distortion, and increasing the density of objects on the recognizable area by approximately 35%.

[0006] However, broad-spectrum laser irradiation, especially in the shorter wavelength ranges of blue and violet, can bleach fluorescent dyes in solution / on the surface of the flow cell and damage nucleotide samples or those to which fluorescent dyes are conjugated. Exposure to such light can also cause damage to DNA samples. The type and extent of photobleaching and photodamage can vary depending on several physicochemical properties, such as the compound's chemical structure and redox potential, the excitation spectrum of a particular biolabel, the intensity of a particular light source's irradiation, and the exposure time in a particular measurement. Because lower wavelength light sources deliver higher energy photons, violet LEDs / lasers with shorter wavelengths are more likely to cause dye photobleaching and DNA damage. There are several chemical pathways by which nucleic acid damage can occur during irradiation in fluorescence detection. For example, it has been shown that exposure to ultraviolet (UV) radiation can cause DNA damage through the direct photochemical [2+2] photocycloaddition of thymine or cytosine to form cyclobutane-containing fused pyrimidine dimers such as TT, TC, and CC. Such direct photocycloaddition reactions can occur in the UV-B and UV-C regions, extending from about 100 nm to about 315 nm. In the UV-A region, which extends through a portion of the visible spectrum from about 315 nm to about 500 nm, a complex mix of indirect mechanisms can also cause DNA damage through photosensitization of other components. Such indirect mechanisms can result in oxidative DNA modification through interactions with different photoinduced reactive species, such as singlet oxygen, superoxide anion, and reactive oxygen species (ROS), such as hydroxyl radicals.

[0007] To increase sequencing efficiency and reduce the cost per genome, it is essential to increase the pitch density so that more clusters can be packed into the same required surface area while maintaining good optical resolution, necessitating the use of light with shorter wavelengths (such as violet and blue lasers). Challenges remain in selecting a suitable set of dyes in the highly dense region of wavelengths (blue to violet region) for nucleic acid sequencing applications and mitigating DNA damage caused by shorter wavelength excitation. Summary of the Invention

[0008] The present disclosure relates to methods, kits, and compositions for dual-channel nucleic acid sequencing applications using blue and violet light excitation (e.g., 450-460 nm and 400-405 nm lasers).

[0009] Some aspects of the present disclosure relate to methods for determining the sequence of a target polynucleotide, the methods comprising: (a) contacting a primer polynucleotide with a mixture comprising one or more of a first type of nucleotides, a second type of nucleotides, a third type of nucleotides, and a fourth type of nucleotides, wherein the primer polynucleotide is complementary to at least a portion of a target polynucleotide; (b) incorporating one type of nucleotide from the mixture into a primer polynucleotide to generate an extended primer polynucleotide; (c) performing a first imaging event using a first excitation light source and collecting a first emission signal from the extended primer polynucleotide with a first emission filter; (d) performing a second imaging event using a second excitation light source and collecting a second emission signal from the extended primer polynucleotide with a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first and second emission filters has a detection wavelength of about 415 nm to about 450 nm, and the other of the first and second emission filters has a detection wavelength of about 480 nm to about 525 nm. In some embodiments, each of the first, second, and third type nucleotides is labeled with a detectable label. In other embodiments, one or more of the first, second, and third type nucleotides are unlabeled, and the method utilizes a second labeling step that includes the use of one or more affinity reagents that specifically bind to the unlabeled nucleotides incorporated into the primer polynucleotide / target polynucleotide complex. In a further embodiment, the fourth type nucleotide is unlabeled and does not emit any signal during the first and second imaging events.

[0010] Some aspects of the present disclosure relate to kits for sequencing applications, the kits comprising: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with a first detectable label; and a third type of nucleotide labeled with a second detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label being excitable by a first light source and detectable by a first emission filter, and the second detectable label being excitable by a second light source and detectable by a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first and second emission filters has a detection wavelength of approximately 415 nm to approximately 450 nm, and the other of the first and second emission filters has a detection wavelength of approximately 480 nm to approximately 525 nm.

[0011] Some aspects of the present disclosure relate to kits for sequencing applications, the kits comprising: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with a third detectable label; a third type of nucleotide labeled with a fourth detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label being excitable by a first light source and detectable by a first emission filter, and the second detectable label being excitable by a second light source and detectable by a second emission filter; the third detectable label and the fourth detectable label are spectrally distinguishable from one another, the third detectable label being excitable by a first light source and detectable by a first emission filter, and the fourth detectable label being excitable by a second light source and detectable by a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first and second emission filters has a detection wavelength of approximately 415 nm to approximately 450 nm, and the other of the first and second emission filters has a detection wavelength of approximately 480 nm to approximately 525 nm.

[0012] Some other aspects of the present disclosure relate to kits for sequencing applications, the kits comprising: a first type of unlabeled nucleotide; a second type of unlabeled nucleotide; and a third type of unlabeled nucleotide; and A set of affinity reagents, a first affinity reagent that specifically binds to a first type of unlabeled nucleotide; and a second affinity reagent that specifically binds to a second type of unlabeled nucleotide; a set of affinity reagents, wherein the first affinity reagent comprises one or more first detectable labels excitable by a first excitation light source and detectable by a first emission filter, and the second affinity reagent comprises one or more second detectable labels excitable by a second excitation light source and detectable by a second emission filter, and the first detectable labels are spectrally distinguishable from the second detectable labels; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm. In some embodiments, both the first affinity reagent and the second affinity reagent specifically bind to a third type of unlabeled nucleotide. In other embodiments, the set of affinity reagents further includes a third affinity reagent that specifically binds to a third type of nucleotide, the third affinity reagent including one or more third detectable labels excitable by the first excitation light source and detectable by the first emission filter, and one or more fourth detectable labels excitable by the second excitation light source and detectable by the second emission filter.

[0013] Some other aspects of the present disclosure relate to kits for sequencing applications, the kits comprising: a first type of nucleotide, either unlabeled or labeled with a first detectable label; a first type of nucleotide and a second type of nucleotide, which are either unlabeled or labeled with a second detectable label, wherein one of the first type of nucleotide and the second type of nucleotide is unlabeled; a third type of unlabeled nucleotide, and a third type of nucleotide labeled with the same detectable label as either the first or second type of nucleotide, wherein the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label is excitable by a first light source and detectable by a first emission filter, and the second detectable label is excitable by a second light source and detectable by a second emission filter; an affinity reagent comprising either unlabeled nucleotides of a third type and a first affinity reagent that specifically binds to nucleotides of the first type when the nucleotides of the first type are unlabeled, or unlabeled nucleotides of the third type and a second affinity reagent that specifically binds to nucleotides of the second type when the nucleotides of the second type are unlabeled, wherein the first affinity reagent comprises one or more first detectable labels and the second affinity reagent comprises one or more second detectable labels; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first and second emission filters has a detection wavelength of approximately 415 nm to approximately 450 nm, and the other of the first and second emission filters has a detection wavelength of approximately 480 nm to approximately 525 nm. [Brief explanation of the drawings]

[0014] [Figure 1]1 is a line graph showing DNA photodamage caused by violet light exposure as a function of time. [Figure 2] Scatter plot obtained from secondary labeled sequencing by synthesis method described in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure relates to methods, systems, kits, and compositions for nucleic acid sequencing applications, specifically sequencing-by-synthesis using blue and violet light excitation (e.g., 450-460 nm and 400-405 nm lasers) and dual-channel detection using filter bands of approximately 415-450 nm and approximately 480-525 nm. The methods, kits, and compositions described herein utilize a dye set containing blue and violet dyes (i.e., dyes with maximum absorption in the blue and violet light regions). The methods further utilize affinity reagents to reduce DNA damage and photobleaching caused by blue and violet excitation. The sequencing methods described herein, with shorter wavelength light sources, can increase the pitch or cluster density in patterned arrays or flow cells compared to current dual-channel sequencing used in Illumina's MiniSeq®, NextSeq®, and NovaSeq® systems, which use red / green or green / blue light source excitation. As used herein, the term "pitch" refers to the distance between two nanopatterns on a patterned solid support (e.g., the distance between two nanowells in a patterned flow cell). A detailed description of Illumina two-channel sequencing using red / green light source excitation is disclosed in U.S. Patent Publication No. 2013 / 0079232, which is incorporated herein by reference in its entirety. For example, in systems using green / red or blue / green excitation, the optical resolution is limited by the red or green fluorescent dye emission, respectively (e.g., approximately 715 nm for the green / red system and approximately 590 nm for the blue / green system). By using violet / blue light excitation, the optical resolution is limited by the blue dye emission (i.e., 480-525 nm). Thus, the disclosed methods and systems can provide up to a 50% increase in pitch density.

[0016] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms, such as "include," "includes," and "included," is not limiting. Furthermore, the use of the term "having" and other forms, such as "have," "has," and "had," is not limiting. As used herein, in transitional phrases or claim text, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrases "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0017] As used herein, common organic abbreviations are defined as follows: ℃ Celsius temperature dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine triphosphate ddNTP Dideoxynucleotide Triphosphate ffA fully functionalized A nucleotide ffC fully functionalized C nucleotide ffG fully functionalized G nucleotide ffN fully functionalized nucleotides ffT fully functionalized T nucleotide LED Light Emitting Diode SBS sequencing by synthesis

[0018] As used herein, the term "array" refers to a collection of different probe molecules bound to one or more substrates such that the different probe molecules can be distinguished from one another according to their relative positions. An array can include probe molecules each located at a different addressable location on the substrate. Alternatively, or additionally, an array can include separate substrates each carrying a different probe molecule, and the different probe molecules can be identified according to the position of the substrate on the surface to which the substrate is bound or according to the position of the substrate in a liquid. Exemplary arrays in which separate substrates are located on a surface include, but are not limited to, those containing beads in wells, as described, for example, in U.S. Pat. No. 6,355,431 B1, US 2002 / 0102578, and PCT Publication No. WO 00 / 63437. An exemplary format that can be used in the present invention to distinguish beads in a liquid array uses a microfluidic device, such as a fluorescence-activated cell sorter (FACS), as described, for example, in U.S. Pat. No. 6,524,793. Further examples of arrays that can be used in the present invention include those described in U.S. Patent Nos. 5,429,807, 5,436,327, 5,561,071, 5,583,211, 5,658,734, 5,837,858, 5,874,219, 5,919,523, 6,136,269, 6,287,768, These include Patent Nos. 6,287,776, 6,288,220, 6,297,006, 6,291,193, 6,346,413, 6,416,949, 6,482,591, 6,514,751, and 6,610,482, as well as WO93 / 17126, WO95 / 11995, WO95 / 35505, EP0 742 287, and EP0 799 897.

[0019] As used herein, the terms "covalently attached" or "covalently bonded" refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, as compared to attaching to the surface by other means, such as adhesion or electrostatic interactions. It will be understood that a polymer covalently attached to a surface can be attached by means in addition to covalent bonds.

[0020] In each instance where a single mesomeric form of a compound described herein is shown, the alternative mesomeric form is contemplated as well.

[0021] As used herein, a "nucleotide" comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. The nitrogen-containing heterocyclic base can be a purine, deazapurine, or pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof, such as 7-deazaadenine or 7-deazaguanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of deoxyribose is linked to the N-1 atom of the pyrimidine or the N-9 atom of the purine. In some instances, the term "nucleotide" may also encompass a nucleotide conjugate, which is a nucleotide labeled with a fluorescent moiety, optionally via a linker, as described herein.

[0022] As used herein, "unlabeled nucleotide" refers to a nucleotide that does not contain a fluorescent moiety. In some examples, an unlabeled nucleotide may contain a cleavable linker and / or a functional moiety (e.g., a hapten) that allows it to be attached to an affinity reagent described herein. In other examples, an unlabeled nucleotide does not have a cleavable linker or functional moiety that allows it to be attached to an affinity reagent described herein.

[0023] As used herein, a "nucleoside" is structurally similar to a nucleotide but lacks a phosphate moiety. An example of a nucleoside analog is one in which a label is linked to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, ribonucleosides containing a ribose moiety and deoxyribonucleosides containing a deoxyribose moiety. Modified pentose moieties are those in which an oxygen atom is replaced with a carbon and / or a carbon is replaced with a sulfur or oxygen atom. A "nucleoside" is a monomer that may have a substituted base and / or sugar moiety. Furthermore, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0024] The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, deazapurine, 7-deazaadenine, 7-deazaguanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0025] As used herein, when an oligonucleotide or polynucleotide is described as "comprising" a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as being part of an oligonucleotide or polynucleotide, such as being "incorporated" into an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between the 3' hydroxy group of the oligonucleotide or polynucleotide and the 5' phosphate group of a nucleotide described herein, or as a phosphodiester bond between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

[0026] As used herein, the term "cleavable linker" does not imply that the entire linker must be removed. The cleavage site can be located on the linker at a position that ensures that a portion of the linker remains attached to the detectable label and / or nucleoside or nucleotide moiety after cleavage.

[0027] As used herein, "derivative" or "analog" refers to a synthetic nucleotide or nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also include modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, and phosphoramidate linkages. As used herein, "derivative," "analog," and "modified" can be used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein.

[0028] As used herein, the term "phosphate" is used in its ordinary sense as understood by one of ordinary skill in the art and includes its protonated form (e.g.,

[0029] [ka] As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used in their ordinary sense as understood by those of skill in the art and include protonated forms.

[0030] As will be appreciated by those skilled in the art, compounds such as nucleotides described herein may be prepared using, for example, -CO2 - , -SO3 - , or -O - The compounds may exist in ionized forms, including: When the compounds contain positively or negatively charged substituents, e.g., S, they may also contain negatively or positively charged counterions, such that the compounds are overall neutral. In other embodiments, the compounds may exist in salt form, with the counterion being provided by a conjugate acid or base.

[0031] As used herein, the term "phasing" refers to a phenomenon in SBS caused by incomplete removal of 3' terminators and fluorophores and / or incomplete incorporation of some DNA strands within a cluster by the polymerase in a given sequencing cycle. Prephasing is caused by the incorporation of a nucleotide without an effective 3' terminator, and the incorporation event advances one cycle due to the incomplete termination. Phasing and prephasing cause the signal intensity measured in a particular cycle to consist of signal from the current cycle and noise from the previous and next cycles. As the number of cycles increases, the proportion of sequences per cluster affected by phasing and prephasing increases, preventing the identification of the correct base. Prephasing can be caused by the presence of traces of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides can be generated during the manufacturing process or, in some cases, during storage and reagent handling processes.

[0032] As used herein, the term "spectrally distinguishable fluorescent dye" refers to a fluorescent dye that emits fluorescent energy at wavelengths that can be distinguished by a fluorescence detection instrument when two or more such dyes are present in a sample.

[0033] Blue / purple dual channel sequencing Some aspects of the present disclosure relate to methods for determining the sequence of a target polynucleotide (e.g., a single-stranded target polynucleotide), the methods comprising: (a) contacting the primer polynucleotide / target polynucleotide complex with a mixture comprising one or more of a first type of nucleotides, a second type of nucleotides, a third type of nucleotides, and a fourth type of nucleotides, wherein the primer polynucleotide is complementary to at least a portion of the single-stranded target polynucleotide; (b) incorporating one type of nucleotide from the mixture into the primer polynucleotide to produce an extended primer polynucleotide (i.e., an extended primer polynucleotide / target polynucleotide complex); (c) performing a first imaging event using a first excitation light source and collecting a first emission signal from the extended primer polynucleotide / target polynucleotide complex with a first emission filter; (d) performing a second imaging event using a second excitation light source and collecting a second emission signal from the extended primer polynucleotide / target polynucleotide complex with a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first and second emission filters has a detection wavelength of approximately 415 nm to approximately 450 nm, and the other of the first and second emission filters has a detection wavelength of approximately 480 nm to approximately 525 nm.

[0034] In some embodiments of the methods described herein, the first excitation light source has a wavelength of about 350 nm to about 410 nm (e.g., about 405 nm), and the first emission filter has a detection wavelength of about 415 nm to about 450 nm. The second excitation light source has a wavelength of about 450 nm to about 460 nm (e.g., about 460 nm), and the second emission filter has a detection wavelength of about 480 nm to about 525 nm. In some other embodiments, the first excitation light source has a wavelength of about 450 nm to about 460 nm (e.g., about 460 nm), and the first emission filter has a detection wavelength of about 480 nm to about 525 nm. The second excitation light source has a wavelength of about 350 nm to about 410 nm (e.g., about 405 nm), and the second emission filter has a detection wavelength of about 415 nm to about 450 nm.

[0035] Labeled nucleotides in the incorporation mixture In some embodiments of the methods described herein, each type of nucleotide in the incorporation mixture is labeled. In some such embodiments, a first type of nucleotide is labeled with a first detectable label that is excitable by a first excitation light source and detectable by a first emission filter. In some further embodiments, a second type of nucleotide is labeled with a second detectable label that is excitable by a second excitation light source and detectable by a second emission filter, the second type of detectable label being spectrally distinguishable from the first type of detectable label. In some further embodiments, a third type of nucleotide is labeled with both a first detectable label and a second detectable label, and the third type of nucleotide is excitable by both a first excitation light source and a second excitation light source. In some other embodiments, the third type of nucleotides comprises a mixture of a third type of nucleotide labeled with a third label and a fourth type of nucleotide labeled with a fourth label, wherein the third label is excitable by a first excitation light source and detectable by a first emission filter, and the fourth label is excitable by a second excitation light source and detectable by a second emission filter. In further embodiments, the fourth type of nucleotide is unlabeled or labeled with a fluorescent moiety that does not emit light in either the first or second imaging event. In some examples, the fourth type of nucleotide comprises a G base (e.g., dGTP).

[0036] When a type of nucleotide is described as being labeled with two different labels, it includes the following two scenarios: In the first scenario, the nucleotide is a mixture of a nucleotide labeled with a first label and a nucleotide of the same type labeled with a second label; In the second scenario, the nucleotide has both a first label and a second label covalently attached thereto (i.e., two labels on the same molecule). In addition, a type of nucleotide described as being labeled with a first and a second label may also contain one or more additional detectable labels that are different from the first label and the second label.

[0037] As a first example, a first type of nucleotide can be labeled with a first dye that is excitable by a blue light source at approximately 450-460 nm (i.e., the first dye is a blue dye) and has an emission wavelength in the range of 480-525 nm. A second type of nucleotide can be labeled with a second dye that is excitable by a violet light source at approximately 400-405 nm (i.e., the second dye is a violet dye) and has an emission wavelength in the range of 415-450 nm. A third type of nucleotide can be a mixture of a third nucleotide labeled with a first dye and a third nucleotide labeled with a second dye. A fourth type of nucleotide is unlabeled. The first imaging event uses a blue light source having a wavelength of approximately 450-460 nm, and both the first type and the third type of nucleotide emit signals that can be detected or collected by an emission filter with a filter band encompassing 480-525 nm. The second imaging event uses a violet light source having a wavelength of approximately 400-405 nm, and both the second and third types of nucleotides emit signals that can be detected or collected by an emission filter with a filter band encompassing 415-450 nm. The fourth type of nucleotide is unlabeled, and therefore no signal is detected in either the first or second imaging event. Based on the signal detection patterns described herein, the identity of the nucleotide incorporated into the extended primer polynucleotide / target polynucleotide complex can be determined. In a further embodiment, the incorporation mixture includes the following: dATP labeled with blue dye A, dTTP labeled with purple dye B, dCTP labeled with blue dye A, dCTP labeled with purple dye B, and unlabeled dGTP (dark G). In one embodiment, the blue dye-labeled dATP has the following structure:

[0038] [ka] Such nucleotide dye conjugates are also called fully functionalized A nucleotides (ffAs).

[0039] In a second example, the first type of labeled nucleotide, the second type of labeled nucleotide, and the fourth type of unlabeled nucleotide are the same as those described in the first example. The third type of nucleotide can be labeled with both a third dye and a fourth dye (e.g., a mixture of a third type of nucleotide labeled with a third dye and a fourth type of nucleotide labeled with a fourth dye). The third dye has a similar fluorescence profile (i.e., absorption and emission spectrum) to the first dye, but can differ in emission intensity. The fourth dye has a similar fluorescence profile (i.e., absorption and emission spectrum) to the second dye, but can differ in emission intensity. In a further embodiment, the incorporation mixture includes the following: dATP labeled with blue dye A, dTTP labeled with purple dye B, dCTP labeled with blue dye C, dCTP labeled with purple dye D, and unlabeled dGTP (dark dye G).

[0040] purple pigment Fluorescent dyes excitable by a violet light source having a wavelength of about 350-405 nm can be used as the first or second detectable labels described herein. In further embodiments, particularly useful violet dyes can have emission spectra in the range of 410-460 nm or 415-450 nm. Non-limiting examples of violet dyes include:

[0041] [ka] These include the BD Horizon™ V450 and the BD Horizon™ V500.

[0042] blue pigment Fluorescent dyes excitable by a blue light source having a wavelength of about 450-460 nm can be used as the first or second detectable label described herein. In further embodiments, particularly useful blue dyes can have emission spectra in the range of 475-530 nm or 480-525 nm. Non-limiting examples of blue dyes include the coumarin dyes disclosed in U.S. Publication Nos. 2018 / 0094140A1, 2018 / 0201981A1, 2020 / 0277529A1, and 2020 / 0277670A1, which are incorporated herein by reference.

[0043] In some embodiments, non-limiting exemplary blue dyes include:

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] [ka] In one example, the blue dye used in sequencing includes

[0050] [ka] Additional exemplary dye compounds are disclosed in U.S. Application No. 17 / 385,232, which is incorporated herein by reference.

[0051] Antioxidant / Radical Scavenger In some embodiments, the methods described herein utilize a scan mixture containing one or more antioxidants / radical scavengers to reduce photodamage caused by blue and violet excitation. In particular, the extended primer polynucleotide / target polynucleotide complex is in a buffer containing one or more antioxidants during the first and second imaging events. Useful antioxidants include, but are not limited to, cyclooctatetraene (COT), taxifolin, quercetin, allylthiourea, dimethylthiourea, silibinin, ascorbic acid or a salt thereof (e.g., sodium ascorbate), polyphenolic compounds (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Troloc)), gallic acid and its lower alkyl esters, its monomethyl ether, and combinations of its lower alkyl esters and monomethyl esters, pyrogallol, and hydroquinones such as t-butyl hydroquinone (TBHQ), 2,4,5-trihydroxybutyrophenone (THBP), and optionally substituted derivatives and combinations thereof. In some further embodiments, the composition comprises cyclooctatetraene and quercetin, and optionally substituted derivatives and combinations thereof.

[0052] Alternatively, the blue / violet dyes described herein can also be covalently attached to a cyclooctatetraene photoprotective moiety. In some embodiments, the COT moiety that can be covalently attached to the blue or violet dyes described herein has the structure:

[0053] [ka] wherein R1A and R 2A each independently selected from H, hydroxyl, halogen, azide, thiol, nitro, cyano, optionally substituted amino, carboxyl, -C(O)OR 5A , -C(O)NR 6A R 7A , optionally substituted C 1~6 Alkyl, optionally substituted C 1~6 Alkoxy, optionally substituted C 1~6 Haloalkyl, optionally substituted C 2~6 Haloalkoxy, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl; X 1 and Y 1 each independently represents a bond, —O—, —S—, or —NR 3A -, -C(=O)-, -C(=O)-O-, -C(=O)-NR 4A -, -(O)2-, -NR 3A -C(=O)-NR 4A , -NR 3A -C(=S)-NR 4A -, optionally substituted C 1~6 alkylene or optionally substituted heteroalkylene in which at least one carbon atom is replaced with O, S, or N; Z is absent or optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 is alkynylene, R 3A and R 4A each independently selected from H, optionally substituted C 1~6 Alkyl or optionally substituted C 6~10 is aryl, R5A optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl; R 6A and R 7A each independently selected from H, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl;

[0054] [ka] In R 1A and R 2A is optionally substituted with O, S, or N, provided that when the carbon atom to which R is attached is substituted with O or S, 1A and R 2A When both are absent and the carbon atom is substituted with N, R 2A is absent, and m is an integer from 0 to 10. In some embodiments, X and Y are not both bonds.

[0055] In some embodiments, the cyclooctatetraene moiety has the structure

[0056] [ka] In some such embodiments, R 1A and R 2A In some further embodiments, at least one of R1A and R 2A and R are hydrogen. 1A is H and R 2A is an optionally substituted amino, carboxyl, or —C(O)NR 6A R 7A In some embodiments, m is 1, 2, 3, 4, 5, or 6, and R 1A and R 2A each independently represents hydrogen, optionally substituted amino, carboxyl, —C(O)NR 6A R 7A In some further embodiments, when m is 2, 3, 4, 5, or 6, one R 1A is amino, carboxyl, or -C(O)NR 6A R 7A and the remaining R 1A and R 2A is hydrogen. In some embodiments, R 1A and R 2A but

[0057] [ka] is substituted with O, S, or N. In some such embodiments,

[0058] [ka] At least one carbon atom in the formula (I) is replaced by an oxygen atom, and R bonded to the replaced carbon atom 1A and R 2A In some other embodiments, both of

[0059] [ka] One in When a carbon atom is substituted with a nitrogen atom, R bonded to the substituted carbon atom2A is absent, and R bonded to the substituted carbon atom 1A is hydrogen or C 1~6 It is alkyl. R 1A and R 2A In any embodiment of the present invention, R 1A or R 2A But -C(O)NR 6A R 7A If R 6A and R 7A are independently H, C 1~6 Alkyl or substituted C 1~6 Alkyl (e.g., -CO2H, -NH2, -SO3H, or -SO3 - C replaced with 1~6 alkyl).

[0060] In some further embodiments, the fluorescent dyes described herein have the following structure:

[0061] [ka] The COT moiety described herein may form an amide bond due to a reaction between a functional group (e.g., a carboxyl group) of a fluorescent dye described herein and an amino group of a COT derivative (the carbonyl group of the amide bond is not shown). Additional disclosure on COT-related antioxidants used in SBS chemistry can be found in U.S. Publication No. 2021 / 0155983 A1, which is incorporated herein by reference in its entirety.

[0062] Unlabeled nucleotides in the incorporation mixture in combination with the affinity reagent As an alternative to the above embodiment, a second aspect of the sequencing method described herein includes a secondary labeling step that can be used to reduce DNA damage and dye photobleaching caused by blue / violet excitation. In some embodiments, secondary labeling refers to a modification to standard sequencing methods in which unlabeled nucleotides are first incorporated into a primer polynucleotide, and then the incorporated unlabeled nucleotides bind to affinity reagents specific for the incorporated type of nucleotide, which contain one or more detectable labels that can be excited by blue or violet light and emit a signal that can be detected by an emission detection channel. Without being bound by theory, the size of the affinity reagents may shield DNA from ROS, thus reducing or mitigating photodamage caused by blue / violet light. In some embodiments of the methods described herein, one or more of the first, second, and third type nucleotides may be unlabeled. In one embodiment, each of the first, second, and third type nucleotides in the incorporation mixture is unlabeled, and the method further comprises contacting the extended primer polynucleotide / target polynucleotide complex with a set of affinity reagents prior to the first imaging event, wherein at least one affinity reagent in the set specifically binds to the incorporated first, second, or third type nucleotides. In some such embodiments, the set of affinity reagents comprises a first affinity reagent that specifically binds to the first type nucleotides and a second affinity reagent that specifically binds to the second type nucleotides. In some further embodiments, the first affinity reagent comprises one or more first detectable labels excitable by a first excitation light source and detectable by a second emission filter, and the second affinity reagent comprises one or more second detectable labels excitable by a second excitation light source and detectable by a second emission filter, wherein the first detectable labels are spectrally distinguishable from the second detectable labels. In some such embodiments, both the first affinity reagent and the second affinity reagent specifically bind to the third type nucleotides.In another embodiment, the set of affinity reagents further includes a third affinity reagent that specifically binds to a third type of nucleotide, the third affinity reagent including one or more third detectable labels that are excitable by a first excitation light source and detectable by a first emission filter, and one or more fourth detectable labels that are excitable by a second excitation light source and detectable by a second emission filter. The third dye has a similar fluorescence profile (i.e., absorption and emission spectrum) to the first dye, but may differ in emission intensity. The fourth dye has a similar fluorescence profile (i.e., absorption and emission spectrum) to the second dye, but may differ in emission intensity.

[0063] When an affinity reagent containing a detectable label binds to the incorporated nucleotide, the extended primer polynucleotide / target polynucleotide complex becomes a labeled extended primer polynucleotide / target polynucleotide complex that can be detected in the first and / or second imaging events. The purple and blue dyes described herein can be used in any embodiment of the modification methods described herein. Additionally, the labeled extended primer polynucleotide / target polynucleotide complex can be present in a scan mixture that includes one or more of the antioxidants and ROS scavengers described herein. The detectable label in the affinity reagent can also include a covalently attached photoprotective moiety as described herein.

[0064] Affinity Reagents As used herein, the term "affinity reagent" refers to a macromolecule, such as a protein or antibody, that specifically binds to an incorporated nucleotide in an extended primer polynucleotide / target polynucleotide complex. In some embodiments, the affinity reagent comprises a protein tag, an antibody (including, but not limited to, an antibody binding fragment, a single-chain antibody, a bispecific antibody, etc.), an aptamer, a knottin, an affimer, or any other known agent that binds to an incorporated nucleotide with suitable specificity and affinity. Each affinity reagent described herein may have a substantially higher affinity for certain types of nucleotides than for other types of nucleotides. Furthermore, the affinity reagent should bind to the incorporated nucleotide at the 3' end of the growing DNA strand (i.e., the extended primer polynucleotide), but not to nucleotides at other positions on the DNA strand. The affinity reagents described herein can be directly or indirectly labeled with one or more detectable labels, such as the blue and purple dyes described herein. In some embodiments, the one or more detectable labels are covalently attached to the affinity reagent via a cleavable linker.

[0065] In some embodiments of the methods described herein, the first type of nucleotide comprises a first hapten, and the first affinity reagent comprises a first hapten binding partner that specifically binds to the first hapten. In some such embodiments, the second type of nucleotide comprises a second hapten, and the second affinity reagent comprises a second hapten binding partner that specifically binds to the second hapten. Each of the first hapten and the second hapten may comprise or be selected from biotin, digoxigenin, dinitrophenol, or a chloroalkyl group. Each of the affinity reagents comprises a specific hapten binding partner that may be an anti-hapten antibody conjugated to one or more fluorescent moieties. In some further embodiments, the first hapten comprises a biotin moiety, the first hapten binding partner comprises streptavidin, and the streptavidin comprises one or more first detectable labels. In some further embodiments, the second hapten comprises a chloroalkyl group, the second hapten-binding partner comprises HaloTag®, and the HaloTag® comprises one or more second detectable labels. The first or second detectable labels may be conjugated to the affinity reagent (e.g., hapten-binding partner) via one or more cleavable linkers described herein. In some such embodiments, the third type of nucleotide comprises both the first hapten and the second hapten (e.g., a mixture of third type nucleotides comprising the first hapten and third type nucleotides comprising the second hapten), such that both the first affinity reagent and the second affinity reagent can specifically bind to the third type of nucleotide.

[0066] In other embodiments, when the set affinity reagents further comprise a third affinity reagent that specifically binds to a third type of nucleotide, the third type of nucleotide may comprise a third hapten, and the third affinity reagent comprises a third hapten binding partner. The third affinity reagent may be a mixture of a third affinity reagent comprising a third hapten binding partner and one or more third detectable labels, and a third affinity reagent comprising a third hapten binding partner and one or more fourth detectable labels.

[0067] For example, a first type of unlabeled nucleotide may include a first hapten containing a biotin moiety, while a first affinity reagent may include streptavidin conjugated to one or more first detectable labels, where the label is a blue dye excitable by a blue light source at approximately 450-460 nm (i.e., the first dye is a blue dye) and has an emission wavelength in the range of 480-525 nm. A second type of unlabeled nucleotide may include a second hapten containing a chloroalkyl group (e.g., CH2)6Cl), while a second affinity reagent may include HaloTag® conjugated to one or more second detectable labels, where the dye is a purple dye excitable by a violet light source at approximately 400-405 nm and has an emission wavelength in the range of 415-450 nm. A third type of unlabeled nucleotide may include both a first hapten and a second hapten that bind to both a first affinity reagent and a second affinity reagent. In some embodiments, the fourth type of nucleotide is unlabeled and does not bind to any affinity reagent. After contacting the extended primer polynucleotide / target nucleotide complex with the set of affinity reagents, the unbound affinity reagent is washed away. The first imaging event uses a blue light source having a wavelength of approximately 450-460 nm, and both the first type and the third type of nucleotide emit signals that can be detected or collected by an emission filter with a filter band encompassing 480-525 nm. The second imaging event uses a violet light source having a wavelength of approximately 400-405 nm, and both the second type and the third type of nucleotide (through detectable labels conjugated to the affinity reagents) emit signals that can be detected or collected by an emission filter with a filter band encompassing 415-450 nm. No signal is detected for the fourth type of nucleotide in either the first or second imaging event. Based on the signal detection patterns described herein, the identity of the nucleotide incorporated into the extended primer polynucleotide can be determined.In a further embodiment, the incorporation mixture comprises the following: dATP containing a chloroalkyl group, dTTP containing a biotin moiety, dCTP containing a biotin moiety, dCTP containing a chloroalkyl group, and unlabeled dGTP (dark G). Exemplary ffC containing a biotin moiety and ffA containing a chloroalkyl group are:

[0068] [ka] wherein n is 1, 2, or 3.

[0069] A third aspect of the sequencing method described herein also includes the use of secondary labeling with an affinity reagent, but only one of the first, second, or third type of nucleotides is unlabeled. In some embodiments, the first type of nucleotide is labeled with a first detectable label, the second type of nucleotide is unlabeled, and the third type of nucleotide is both unlabeled and labeled with a first detectable label, the first detectable label being excitable by a first excitation light source and detectable by a first emission filter. The method further includes contacting the extended primer polynucleotide with an affinity reagent prior to the first imaging event, wherein the affinity reagent specifically binds to the second type of unlabeled nucleotide and / or the third type of unlabeled nucleotide. In some such embodiments, the affinity reagent includes one or more second detectable labels excitable by a second excitation light source and detectable by a second emission filter. In some further embodiments, the affinity reagent comprises streptavidin conjugated to one or more second detectable labels, and both the second type of nucleotides and the third type of unlabeled nucleotides comprise biotin moieties. In some other embodiments, the first type of nucleotides are unlabeled, the second type of nucleotides are labeled with a second detectable label, and the third type of nucleotides are both unlabeled and labeled with a second detectable label, the second detectable label being excitable by a second excitation light source and detectable by a second emission filter. The method further comprises contacting the extended primer polynucleotide with an affinity reagent prior to the first imaging event, wherein the affinity reagent specifically binds to the first type of unlabeled nucleotides and / or the third type of unlabeled nucleotides. In some such embodiments, the affinity reagent comprises one or more first detectable labels excitable by a first excitation light source and detectable by a first emission filter.In some further embodiments, the affinity reagent comprises streptavidin conjugated to one or more first detectable labels, and both the first type of nucleotides and the third type of unlabeled nucleotides comprise biotin moieties. In any such embodiments, the fourth type of nucleotides is unlabeled and does not bind to the affinity reagent or emit any signal during the first and second imaging events.

[0070] In another example, the first type of nucleotide is labeled with a blue dye, excitable at approximately 450-460 nm with a blue light source and having an emission wavelength in the range of 480-525 nm. The second type of nucleotide is unlabeled and contains a biotin moiety. The affinity reagent includes streptavidin conjugated to one or more violet dyes, excitable at approximately 400-405 nm with a violet light source and having an emission wavelength in the range of 415-450 nm. The third type of nucleotide is a mixture of both unlabeled third type nucleotides containing a biotin moiety and third type nucleotides labeled with the same blue dye as the first type nucleotides. The fourth type of nucleotide is unlabeled and does not bind to any affinity reagent or emit any signal in the first or second imaging events. After contacting the extended primer polynucleotide / target nucleotide complex with the affinity reagent, the unbound affinity reagent is washed away. The first imaging event uses a blue light source having a wavelength of approximately 450-460 nm, and both the first and third type nucleotides emit signals that can be detected or collected by an emission filter with a filter band encompassing 480-525 nm. The second imaging event uses a violet light source having a wavelength of approximately 400-405 nm, and both the second and third type nucleotides (via a violet dye conjugated to streptavidin) emit signals that can be detected or collected by an emission filter with a filter band encompassing 415-450 nm. No signal is detected for the fourth type nucleotide in either the first or second imaging event. Based on the signal detection patterns described herein, the identity of the nucleotide incorporated into the extended primer polynucleotide can be determined. In a further embodiment, the incorporation mixture includes the following: dATP labeled with a blue dye, dTTP containing a biotin moiety, dCTP containing a biotin moiety, dCTP labeled with a blue dye, and unlabeled dGTP (dark G).

[0071] In an alternative embodiment of the third aspect of the sequencing method described herein, the first type of nucleotides are labeled with a first detectable label, the second type of nucleotides are unlabeled, and the third type of nucleotides are both unlabeled and labeled with a third detectable label, both of which are excitable by a first excitation light source and detectable by a first emission filter (e.g., the third label has a similar fluorescence profile to the first label but may differ in emission intensity). The method further includes contacting the extended primer polynucleotide with a set of affinity reagents prior to the first imaging event, wherein at least one affinity reagent specifically binds to the second type of unlabeled nucleotides and at least one affinity reagent specifically binds to the third type of unlabeled nucleotides. In some such embodiments, the affinity reagents that specifically bind to the second type of nucleotides comprise one or more second detectable labels that are excitable by a second excitation light source and detectable by a second emission filter. The affinity reagents that specifically bind to the third type of nucleotides include one or more fourth detectable labels that are excitable by a second excitation light source and detectable by a second emission filter (e.g., the fourth labels may have a similar fluorescence profile as the second labels but differ in emission intensity). In some further embodiments, the set affinity reagents may include streptavidin conjugated to one or more second detectable labels and a second antibody / protein conjugated to one or more fourth detectable labels. The second type of unlabeled nucleotides includes a biotin moiety, and the third type of unlabeled nucleotides includes a hapten specific for the second antibody / protein conjugated to a fourth label.

[0072] Another alternative embodiment of the third aspect of the sequencing method described herein includes using an incorporation mixture, wherein a first type of nucleotide is unlabeled, a second type of nucleotide is labeled with a second detectable label, and a third type of nucleotide is both unlabeled and labeled with a fourth detectable label, both of which are excitable by a second excitation light source and detectable by a second emission filter (e.g., the third label has a similar fluorescence profile to the first label but may differ in emission intensity). The method further includes contacting the extended primer polynucleotide with a set of affinity reagents prior to the first imaging event, wherein at least one affinity reagent specifically binds to the first type of unlabeled nucleotide and at least one affinity reagent specifically binds to the third type of unlabeled nucleotide. In some such embodiments, the affinity reagent that specifically binds to the first type of nucleotide comprises one or more first detectable labels. The affinity reagent that specifically binds to the third type of nucleotide comprises one or more third detectable labels. Both the first and third detectable labels are excitable by a first excitation light source and detectable by a first emission filter (e.g., the third label may have a similar fluorescence profile as the first label but differ in emission intensity).

[0073] In any embodiment of the methods described herein, the nucleotides in the mixture in step (a) include four different types of nucleotides (A, C, G, and T or U), or non-natural nucleotide analogs thereof. In further embodiments, the four different types of nucleotides are dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogs thereof. In some further embodiments, three of the four types of nucleotides are each labeled with a detectable label, and one of the nucleotides is either not labeled with a fluorophore or is labeled with a fluorophore but cannot be excited to emit a signal in either the first or second imaging event. In other embodiments, the detectable labels in one, two, or three of the four types of nucleotides are added using a secondary labeling step described herein, in which the unlabeled nucleotide is first incorporated into a primer polynucleotide, and then an affinity reagent specific for the incorporated type of nucleotide is introduced into the primer polynucleotide, the affinity reagent comprising one or more detectable labels capable of emitting a signal during the first and / or second imaging events. In a further embodiment, each of the four types of nucleotides in the incorporation mixture contains a 3' hydroxyl blocking group. Such a 3' hydroxyl blocking group ensures that only a single base can be added to the 3' end of the primer polynucleotide by the polymerase. After incorporation of the nucleotide in step (b), any remaining unincorporated nucleotides are washed away.

[0074] In some embodiments of the methods described herein, the method further comprises step (e): removing the 3' hydroxyl blocking group from the incorporated nucleotide after the second imaging event and before the next sequencing cycle. In further embodiments, any detectable label attached to the incorporated nucleotide (either directly to the incorporated nucleotide via a cleavable linker or indirectly via an affinity reagent) is also removed before the next sequencing cycle. In some such embodiments, the detectable label and the 3' hydroxyl blocking group are removed in a single step (e.g., under the same chemical reaction conditions). In other embodiments, the label and the 3' hydroxyl blocking group are removed in two separate steps (e.g., the label and the 3' blocking group are removed under two separate chemical reaction conditions). In some further embodiments, a post-cleavage wash step is used after the label and the 3' blocking group are removed. In further embodiments, steps (a)-(e) are performed in repeated cycles (e.g., at least 30, 50, 100, 150, 200, 250, 300, 400, or 500 times), and the method further comprises sequentially determining the sequence of at least a portion of the single-stranded target polynucleotide based on the identity of each successively incorporated nucleotide. In some such embodiments, steps (a)-(e) are repeated for at least 50 cycles. In some further embodiments, the incorporation of nucleotides from the incorporation mixture is performed by a polymerase (e.g., a DNA polymerase). Exemplary polymerases include, but are not limited to, Pol812, Pol1901, Pol1558, or Pol963. The amino acid sequences of Pol812, Pol1901, Pol1558, or Pol963 DNA polymerase are described, for example, in U.S. Patent Publication Nos. 2020 / 0131484A1 and 2020 / 0181587A1, both of which are incorporated herein by reference.

[0075] In some embodiments of the methods described herein, the first excitation light source used in the first imaging event and the second excitation light source used in the second imaging event each comprise a laser, a light emitting diode (LED), or a combination thereof.

[0076] In some embodiments, the combined emission detection from the first and second imaging events is processed by image analysis software to determine the identity of the incorporated base at each immobilized primer polynucleotide / target polynucleotide complex position, hi some such embodiments, image analysis is processed after repeated cycles of incorporation (after at least 50, 100, 150, 200, 250, or 300 runs).

[0077] In any embodiment of the methods described herein, the single-stranded target polynucleotide may be immobilized on a solid support. In some such embodiments, the solid support comprises a plurality of immobilized single-stranded target polynucleotides. The primer polynucleotide is complementary to at least a portion of the target polynucleotide. In some such embodiments, the primer polynucleotide hybridizes to at least a portion of the target polynucleotide to form a primer polynucleotide / target polynucleotide complex. The solid support may comprise clustered primer polynucleotide / target polynucleotide complexes. In some embodiments, the solid support comprises a flow cell, e.g., a patterned flow cell comprising a plurality of nanowells, each spaced apart. In some further embodiments, each nanowell comprises one immobilized cluster therein. In some embodiments, the density of nanowells on the patterned flow cell is about 100K / mm 2 ~approx. 500K / mm 2 , about 200K / mm 2 ~approx. 400K / mm 2 , or about 250K / mm 2 ~about 350K / mm 2In some embodiments, the density of immobilized single-stranded target polynucleotides (or clusters formed from hybridization with primer polynucleotides) on the solid support is about 80K / mm 2 ~approx. 400K / mm 2 , about 100K / mm 2 ~about 300K / mm 2 , or about 150K / mm 2 ~about 250K / mm 2 In some embodiments, the sequencing methods described herein allow for up to a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in cluster density compared to dual-channel sequencing methods using red / green or green / blue excitation with similar or equivalent optical resolution.

[0078] Additional exemplary embodiments of the method are described below.

[0079] In certain embodiments, a synthesis step is performed, and may optionally include incubating a template or target polynucleotide strand with a reaction mixture containing the fluorescently labeled nucleotides of the present disclosure. A polymerase may also be provided under conditions that allow the formation of a phosphodiester bond between a free 3'OH group on a polynucleotide strand annealed to the template or target polynucleotide strand and a 5' phosphate group on the labeled nucleotide. Thus, the synthesis step may include the formation of a polynucleotide strand achieved by complementary base pairing of nucleotides to the template / target strand.

[0080] In all embodiments of the present method, the detection step can be performed while the polynucleotide strand incorporating the labeled nucleotide is annealing to the target strand, or after the denaturation step in which the two strands are separated. Additional steps, such as chemical or enzymatic reaction steps or purification steps, can be included between the synthesis step and the detection step. Specifically, the polynucleotide strand incorporating the labeled nucleotide can be isolated or purified, and then further processed or used for subsequent analysis. For example, a target polynucleotide incorporating the labeled nucleotide described herein in the synthesis step can then be used as a labeled probe or primer. In other embodiments, the product of the synthesis step described herein can be subjected to additional reaction steps, and if desired, the product of these subsequent steps can be purified or isolated.

[0081] Suitable conditions for the synthesis step are well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step resembles a standard primer extension reaction using nucleotide precursors containing labeled nucleotides as described herein to form an extended polynucleotide strand (primer nucleotide strand) complementary to the target strand in the presence of a suitable polymerase enzyme. In another embodiment, the synthesis step may itself be part of an amplification reaction, generating a labeled double-stranded amplification product consisting of an annealed complementary strand derived from copying the primer target polynucleotide strand. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random-primed DNA labeling, and the like. Polymerase enzymes particularly useful for the synthesis step are those capable of catalyzing the incorporation of labeled nucleotides as described herein. A variety of naturally occurring or mutant / modified polymerases can be used. For example, thermostable polymerases can be used in synthesis reactions performed using thermal cycling conditions, but thermostable polymerases may not be desirable for isothermal primer extension reactions. Suitable thermostable polymerases capable of incorporating labeled nucleotides according to the present disclosure include those described in WO2005 / 024010 or WO06 / 120433, each of which is incorporated herein by reference. For synthesis reactions performed at low temperatures, such as 37°C, the polymerase enzyme does not necessarily need to be a thermostable polymerase; therefore, the choice of polymerase will depend on several factors, such as reaction temperature, pH, and strand displacement activity.

[0082] In certain non-limiting embodiments, the present disclosure includes methods for nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, as well as any other application involving the detection of modified nucleotides or nucleosides labeled with the dyes described herein when incorporated into a polynucleotide.

[0083] In certain embodiments, the present disclosure provides for the use of labeled nucleotides containing dye moieties according to the present disclosure in polynucleotide sequencing-by-synthesis reactions. Sequencing-by-synthesis generally involves sequentially adding one or more nucleotides or oligonucleotides to a growing polynucleotide chain in a 5' to 3' direction using a polymerase or ligase to form an extended polynucleotide chain complementary to the template / target nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotides can be determined in a detection or "imaging" step. The identity of the added base can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using conventional Watson-Crick base-pairing rules. Using nucleotides labeled with the dyes described herein to determine the identity of a single base can be useful, for example, in scoring single nucleotide polymorphisms, and such single-base extension reactions are within the scope of the present disclosure.

[0084] In an embodiment of the present disclosure, the sequence of a target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the target polynucleotide to be sequenced through detection of a fluorescent label attached to the incorporated nucleotide. Sequencing of the target polynucleotide can be primed with a suitable primer (or a primer prepared as a hairpin structure containing the primer as part of the hairpin), and the nascent strand is extended stepwise by adding nucleotides to the 3' end of the primer in a polymer-catalyzed reaction.

[0085] In certain embodiments, each of the different nucleotide triphosphates (A, T, G, and C) may be labeled with a unique fluorophore and contain a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates the nucleotide into the nascent strand complementary to the template / target polynucleotide, but the blocking group prevents further incorporation of the nucleotide. Any unincorporated nucleotides can be washed away, and the fluorescent signal pattern from each incorporated nucleotide can be optically "read" by suitable means, such as a charge-coupled device using optical excitation and suitable emission filters. The 3'-blocking group and fluorescent dye compound can then be removed (cleaved) (simultaneously or sequentially) to expose the nascent strand for incorporation of additional nucleotides. Typically, the identity of the incorporated nucleotide is determined after each incorporation step, although this is not strictly required. Similarly, US Pat. No. 5,302,509 (hereby incorporated by reference in its entirety) discloses a method for sequencing polynucleotides immobilized on a solid support.

[0086] The method, as exemplified above, incorporates 3'-blocked nucleotides A, G, C, and T into a growing strand complementary to an immobilized polynucleotide in the presence of a DNA polymerase. The polymerase incorporates the base complementary to the target polynucleotide but is prevented from further addition by a 3'-hydroxyl blocking group. The label of the incorporated nucleotide can then be determined, after which the blocking group can be removed by chemical cleavage to allow further polymerization. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction can be any polynucleotide for which sequencing is desired. Nucleic acid templates for sequencing reactions typically contain a double-stranded region with a free 3' OH group that serves as a primer or starting point for the addition of additional nucleotides in the sequencing reaction. The region of the template to be sequenced has this free 3' OH group overhanging on the complementary strand. The overhang region of the template to be sequenced may be single-stranded, but can also be double-stranded, so long as a "nick" exists on the strand complementary to the target strand to be sequenced, providing a free 3'OH group for initiating the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer having a free 3'OH group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand that can form an intramolecular duplex, such as a hairpin loop structure. Hairpin polynucleotides and methods by which they can be attached to a solid support are disclosed in PCT Publications WO 01 / 57248 and WO 2005 / 047301, each of which is incorporated herein by reference. Nucleotides can be added sequentially to the growing primer to synthesize a polynucleotide chain in the 5' to 3' direction. The nature of the added base may, but need not, be determined particularly after each nucleotide addition, and such determination provides sequence information about the nucleic acid template.Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by linking the nucleotide to the free 3' OH group of the nucleic acid strand through the formation of a phosphodiester bond with the 5' phosphate group of the nucleotide.

[0087] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template may contain naturally occurring and / or non-naturally occurring nucleotides and naturally occurring or non-naturally occurring backbone linkages, as long as they do not prevent copying of the template in the sequencing reaction.

[0088] In certain embodiments, the target polynucleotide to be sequenced can be attached to the solid support via any suitable linking method known in the art, for example, via a covalent bond. In certain embodiments, the target polynucleotide can be directly attached to the solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support can be modified in some way to allow either direct covalent attachment of the target polynucleotide or immobilization of the target polynucleotide through a hydrogel or polyelectrolyte layer (which itself is attached to the solid support by a method other than a covalent bond).

[0089] Arrays in which polynucleotides are directly attached to a support (e.g., silica-based supports such as those disclosed in WO 00 / 006770, incorporated herein by reference) are immobilized to the glass support by reaction between pendant epoxide groups on the glass and internal amino groups of the polynucleotide. Additionally, polynucleotides can be attached to a solid support by reaction of sulfur-based nucleophiles with the solid support, as described, for example, in WO 2005 / 047301, incorporated herein by reference. Yet further examples of solid-supported template polynucleotides are those in which the template polynucleotide is attached to a hydrogel supported on a silica-based or other solid support, as described, for example, in WO 00 / 31148, WO 01 / 01143, WO 02 / 12566, WO 03 / 014392, U.S. Pat. No. 6,465,178, and WO 00 / 53812, each of which is incorporated herein by reference.

[0090] Specific surfaces onto which template polynucleotides can be immobilized include polyacrylamide hydrogels. Polyacrylamide hydrogels are described in the above-cited references and WO2005 / 065814, which is incorporated herein by reference. Specific hydrogels that can be used include those described in WO2005 / 065814 and U.S. Publication No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)).

[0091] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Patent No. 6,172,218 (incorporated herein by reference). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared, with each bead containing a different DNA sequence. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005), Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of such bead arrays using the nucleotides described herein is within the scope of this disclosure.

[0092] The template to be sequenced may form part of an "array" on a solid support, where the array may take any convenient form. Thus, the method of the present disclosure is applicable to all types of high-density arrays, including single molecule arrays, clustered arrays, and bead arrays. Nucleotides labeled with the dye compounds of the present disclosure may be used to sequence templates on essentially any type of array, including, but not limited to, those formed by immobilizing nucleic acid molecules on a solid support.

[0093] However, the nucleotides labeled with the dye compounds of the present disclosure are particularly advantageous in the context of clustered array sequencing. In clustered arrays, distinct regions (often called sites or features) on the array contain multiple polynucleotide template molecules. Generally, multiple polynucleotide molecules are not individually resolvable by optical means, but are instead detected as an ensemble. Depending on how the array is formed, each site on the array may contain multiple copies of one individual polynucleotide molecule (e.g., the site is homogeneous for a specific single-stranded or double-stranded nucleic acid species), or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules can be generated using techniques commonly known in the art. By way of example, WO 98 / 44151 and WO 00 / 18957, each incorporated herein by reference, describe methods for amplifying nucleic acids, in which both the template and the amplification product remain immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with the dye compounds of the present disclosure.

[0094] Nucleotides labeled with the dye compounds of the present application are also useful for sequencing templates on single molecule arrays. As used herein, the term "single molecule array" or "SMA" refers to a collection of polynucleotide molecules distributed (or arrayed) on a solid support, where the separation distance of any individual polynucleotide from all other polynucleotides in the collection is such that each individual polynucleotide molecule can be individually distinguished. Thus, in some embodiments, target nucleic acid molecules immobilized on the surface of a solid support can be distinguished by optical means. This means that one or more distinct signals, each representing a single polynucleotide, are generated within an area that can be distinguished by the particular imaging device used.

[0095] Single molecule detection can be achieved where the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more specifically at least 250 nm, even more specifically at least 300 nm, and even more specifically at least 350 nm, such that each molecule is individually resolvable and detectable as a single molecule fluorescent dot, and the fluorescence from this single molecule fluorescent dot also exhibits single-step photobleaching.

[0096] The terms "individually resolved" and "individual resolution" are used herein to designate the ability to distinguish one molecule on an array from its neighboring molecules when visualized. The separation between individual molecules on an array is determined, in part, by the particular technique used to resolve the individual molecules. General characteristics of single molecule arrays can be understood by reference to WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. One application of the labeled nucleotides of the present disclosure is in sequencing-by-synthesis reactions, although the utility of each nucleotide is not limited to such methods. Indeed, the labeled nucleotides described herein can be advantageously used in any sequencing method requiring detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0097] kit Some aspects of the present disclosure relate to kits for the blue / purple dual channel sequencing methods described herein. In some embodiments, the kit for sequencing applications comprises: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with a first detectable label and a second detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label being excitable by a first light source and detectable by a first emission filter, and the second detectable label being excitable by a second light source and detectable by a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm. In some embodiments, the third type of nucleotides is a mixture of a third type of nucleotide labeled with a first detectable label and a third type of nucleotide labeled with a second detectable label. As a specific example, the kit can include dATP labeled with a blue dye, dTTP containing a biotin moiety, dCTP containing a biotin moiety, dCTP labeled with a blue dye, and unlabeled dGTP (dark G).

[0098] In a second embodiment of the kit for sequencing applications, the kit comprises: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with a third detectable label and a fourth detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label being excitable by a first light source and detectable by a first emission filter, and the second detectable label being excitable by a second light source and detectable by a second emission filter; the third detectable label and the fourth detectable label are spectrally distinguishable from one another, the third detectable label being excitable by a first light source and detectable by a first emission filter, and the fourth detectable label being excitable by a second light source and detectable by a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; One of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm. In some embodiments, the third type of nucleotides is a mixture of third type nucleotides labeled with a third detectable label and third type nucleotides labeled with a fourth detectable label.

[0099] In a third embodiment of the kit described herein, one or more types of nucleotides can be unlabeled. In some examples, the kit for sequencing applications includes: a first type of unlabeled nucleotide; a second type of unlabeled nucleotide; and a third type of unlabeled nucleotide; and and a set of affinity reagents including a first affinity reagent that specifically binds to a first type of unlabeled nucleotides and a second affinity reagent that specifically binds to a second type of unlabeled nucleotides, wherein the first affinity reagent comprises one or more first detectable labels excitable by a first excitation light source and detectable by a first emission filter, and the second affinity reagent comprises one or more second detectable labels excitable by a second excitation light source and detectable by a second emission filter, wherein the first detectable labels are spectrally distinguishable from the second detectable labels. In some such embodiments, the first type of nucleotides comprises a first hapten, and the first affinity reagent comprises a first hapten binding partner that specifically binds to the first hapten. In further embodiments, the first hapten comprises or consists of a biotin moiety, and the first hapten binding partner comprises or consists of streptavidin, optionally conjugated to one or more first detectable labels through a cleavable linker. In some further embodiments, the second type of nucleotide comprises a second hapten, and the second affinity reagent comprises a second hapten binding partner that specifically binds to the second hapten. In further embodiments, the second hapten comprises a chloroalkyl group (e.g., —(CH2)6Cl), and the second hapten binding partner comprises or consists of HaloTag®, optionally conjugated to one or more second detectable labels through a cleavable linker. In some further embodiments, the third type of nucleotide comprises a mixture of the first hapten and the second hapten. Additional haptens can also be used with the unlabeled nucleotides described herein, including, but not limited to, digoxigenin and dinitrophenol. The corresponding affinity reagent contains an anti-digoxigenin binding partner or an anti-dinitrophenol binding partner. In some embodiments, both the first affinity reagent and the second affinity reagent specifically bind to a third type of unlabeled nucleotide.In other embodiments, the set of affinity reagents further includes a third affinity reagent that specifically binds to a third type of nucleotide, the third affinity reagent comprising one or more third detectable labels excitable by a first excitation light source and detectable by a first emission filter, and one or more fourth detectable labels excitable by a second excitation light source and detectable by a second emission filter. In some such embodiments, the third type of nucleotide comprises a first hapten, and the third affinity reagent comprises a third hapten binding partner. In further embodiments, the third affinity reagent comprises a mixture of a third affinity reagent comprising a third hapten binding partner and one or more third detectable labels, and a third affinity reagent comprising a third hapten binding partner and one or more fourth detectable labels.

[0100] In a fourth embodiment of the kit for sequencing applications, the kit comprises: a first type of nucleotide, either unlabeled or labeled with a first detectable label; a second type of nucleotide that is either unlabeled or labeled with a second detectable label, wherein one of the first type of nucleotide and the second type of nucleotide is unlabeled; and a third type of unlabeled nucleotide, and a third type of nucleotide labeled with the same detectable label as either the first or second type of nucleotide, wherein the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label is excitable by a first light source and detectable by a first emission filter, and the second detectable label is excitable by a second light source and detectable by a second emission filter; and an affinity reagent comprising either a third type of unlabeled nucleotides and a first affinity reagent that specifically binds to the first type of nucleotides when the first type of nucleotides are unlabeled, or a third type of unlabeled nucleotides and a second affinity reagent that specifically binds to the second type of nucleotides when the second type of nucleotides are unlabeled, wherein the first affinity reagent comprises one or more first detectable labels and the second affinity reagent comprises one or more second detectable labels. In some embodiments, the first type of nucleotides are unlabeled, the second type of nucleotides are labeled with a second detectable label, and the third type of nucleotides are both unlabeled and labeled with a second detectable label, and the affinity reagent is the first affinity reagent that specifically binds to the first type of nucleotides and the third type of unlabeled nucleotides, and the first affinity reagent comprises one or more first detectable labels. In other embodiments, the first type of nucleotides are labeled with a first detectable label, the second type of nucleotides are unlabeled, and the third type of nucleotides are both unlabeled and labeled with a second detectable label; the affinity reagent is a second affinity reagent that specifically binds to the second type of nucleotides and the third type of unlabeled nucleotides, and the second affinity reagent comprises one or more second detectable labels. In further embodiments, when either the first type or the second type of nucleotides are unlabeled, such unlabeled nucleotides independently comprise a hapten. In further embodiments, the affinity reagent comprises a hapten binding partner that specifically binds to the hapten in the unlabeled nucleotide. In one example, the hapten comprises a biotin moiety, and the hapten binding partner comprises streptavidin. The streptavidin is optionally conjugated to one or more first detectable labels via a cleavable linker. In another example, the hapten comprises a chloroalkyl group, and the hapten binding partner comprises HaloTag®.The HaloTag® is optionally conjugated to one or more second detectable labels through a cleavable linker.

[0101] In an alternative to the fourth aspect of the kit described herein, the kit comprises: a first type of nucleotide, either unlabeled or labeled with a first detectable label; a second type of nucleotides that are either unlabeled or labeled with a second detectable label, wherein one of the first type of nucleotides and the second type of nucleotides is unlabeled; and a third type of nucleotide, which may comprise (i) a mixture of unlabeled third type nucleotides and third type nucleotides labeled with a third detectable label when the second type nucleotides are unlabeled, or (ii) a mixture of unlabeled third type nucleotides and third type nucleotides labeled with a fourth detectable label when the first type nucleotides are unlabeled; a third type of nucleotide, wherein the first detectable label and the second detectable label are spectrally distinguishable from one another, the first and third detectable labels are both excitable by a first light source and detectable by a first emission filter, and the second and fourth detectable labels are both excitable by a second light source and detectable by a second emission filter; a set of affinity reagents that may include (iii) a mixture of a first affinity reagent that specifically binds to a first type of nucleotide and a third affinity reagent that specifically binds to a third type of unlabeled nucleotide when the first type of nucleotide is unlabeled, or (iv) a mixture of a second affinity reagent that specifically binds to a second type of nucleotide and a third affinity reagent that specifically binds to a third type of unlabeled nucleotide when the second type of nucleotide is unlabeled, (iii) in which the first affinity reagent comprises one or more first detectable labels and the third affinity reagent comprises one or more third detectable labels; (iv) A set of affinity reagents, wherein the second affinity reagent comprises one or more second detectable labels and the third affinity reagent comprises one or more fourth detectable labels.

[0102] In any embodiment of the kits described herein, the kit may further comprise a fourth type of nucleotide, wherein the fourth type of nucleotide is unlabeled (dark). Additionally, any of the blue and purple dyes disclosed herein may be used as a primary or secondary label for the nucleotides or affinity reagents described in this section.

[0103] As one particular example, the kit may include the following nucleotide set: dATP labeled with blue dye A, dTTP labeled with purple dye B, dCTP labeled with blue dye A, dCTP labeled with purple dye B, and unlabeled dGTP (dark G).

[0104] As another specific example, the kit may include the following set of nucleotides: dATP labeled with blue dye A, dTTP labeled with purple dye B, dCTP labeled with blue dye C, dCTP labeled with purple dye D, and unlabeled dGTP (dark G).

[0105] As another specific example, the kit may include the following set of nucleotides: dATP labeled with blue dye A, dTTP containing a biotin moiety, dCTP containing a biotin moiety, dCTP labeled with blue dye A, and unlabeled dGTP (dark G). Additionally, the kit may optionally further include an affinity reagent comprising streptavidin labeled with one or more purple dyes B via a cleavable linker.

[0106] As another specific example, the kit may include the following set of nucleotides: dATP labeled with blue dye A, dTTP containing a first hapten that is a biotin moiety, dCTP containing a second hapten moiety, dCTP labeled with blue dye B, and unlabeled dGTP (dark G). Additionally, the kit may further include a set of affinity reagents including streptavidin conjugated to one or more purple dyes C and a second hapten binding partner conjugated to one or more purple dyes D, each optionally through a cleavable linker.

[0107] In yet another example, the kit may include the following set of nucleotides: dATP with a chloroalkyl group (e.g., -(CH2)6Cl), dTTP with a biotin moiety, dCTP with a biotin moiety, dCTP with a chloroalkyl group, and unlabeled dGTP (dark G). The kit may optionally further include a first affinity reagent including streptavidin labeled with one or more purple dyes via a cleavable linker. The kit may optionally further include a second affinity reagent including HaloTag® labeled with one or more blue dyes via a cleavable linker.

[0108] In yet another example, a kit can include the following set of nucleotides: dATP comprising a first hapten comprising a chloroalkyl group (e.g., —(CH)Cl), dTTP comprising a second hapten comprising a biotin moiety, dCTP comprising a third hapten, and unlabeled dGTP (dark G). The kit can optionally further include a first affinity reagent comprising streptavidin labeled with one or more purple dyes B through a cleavable linker. The kit can optionally further include a second affinity reagent comprising HaloTag® labeled with one or more blue dyes A through a cleavable linker. The kit can further include a third affinity reagent comprising a third hapten binding partner and one or more blue dyes C. The kit can further include a third affinity reagent comprising a third hapten binding partner and one or more purple dyes D.

[0109] In some embodiments of the kits described herein, the first excitation light source has a wavelength of about 350 nm to about 410 nm (e.g., about 405 nm), and the first emission filter has a detection wavelength of about 415 nm to about 450 nm. The second excitation light source has a wavelength of about 450 nm to about 460 nm (e.g., about 460 nm), and the second emission filter has a detection wavelength of about 480 nm to about 525 nm. In some other embodiments, the first excitation light source has a wavelength of about 450 nm to about 460 nm (e.g., about 460 nm), and the first emission filter has a detection wavelength of about 480 nm to about 525 nm. The second excitation light source has a wavelength of about 350 nm to about 410 nm (e.g., about 405 nm), and the second emission filter has a detection wavelength of about 415 nm to about 450 nm.

[0110] In addition to the above examples, the kit may also include at least one additional component. The additional component may be one or more of the components identified in the methods described herein or in the Examples section below. Some non-limiting examples of components that can be combined into the kits of the present disclosure are described below. In some embodiments, the kit further includes a DNA polymerase (such as a mutant DNA polymerase) and one or more buffer compositions. The kit may also include one or more antioxidants and / or ROS scavengers described herein. The antioxidants and / or ROS scavengers may be in a buffer or composition that can be used to protect DNA (target polynucleotide and / or primer polynucleotide) and dyes from photodamage during detection. The additional buffer composition may include a reagent that can be used to cleave the 3' hydroxyl blocking group and / or the cleavable linker. For example, a water-soluble phosphine or water-soluble transition metal catalyst formed from a transition metal and an at least partially water-soluble ligand, such as a palladium complex. Various components of the kit may be provided in a concentrated form that is diluted before use. In such embodiments, a suitable dilution buffer may also be included. In further embodiments, the kit may include one or more solid supports. In some such embodiments, the solid support may include a plurality of oligonucleotides immobilized thereon. In some embodiments, the solid support includes a flow cell, e.g., a patterned flow cell including a plurality of nanowells.

[0111] In some embodiments of the kits described herein, detectable labels (e.g., blue and purple dyes) can be covalently attached to the nucleotide via the nucleotide base. In some such embodiments, the labeled nucleotide can have a label attached to the C5 position of a pyrimidine base or to the C7 position of a 7-deazapurine base, optionally through a linker moiety. For example, the nucleobase can be 7-deazaadenine, and a dye attached to the 7-deazaadenine at the C7 position, optionally through a linker. The nucleobase can be 7-deazaguanine, and a dye attached to the 7-deazaguanine at the C7 position, optionally through a linker. The nucleobase can be cytosine, and a dye attached to the cytosine at the C5 position, optionally through a linker. As another example, the nucleobase can be thymine or uracil, and a dye attached to the thymine or uracil at the C5 position, optionally through a linker. In any embodiment of the nucleotide or nucleotide conjugate described herein, the nucleotide or nucleotide conjugate may include a 3' hydroxyl blocking group. In other embodiments, when the nucleotide is unlabeled and a secondary labeling method is used, one or more blue or purple dyes may be conjugated to the affinity reagent described herein, optionally via a cleavable linker. For example, a single streptavidin may be labeled with two, three, four, five, or six molecules of the same purple dye to increase the fluorescence intensity of the incorporated nucleotide to be detected.

[0112] In any embodiment of the methods and kits described herein, when a label is excitable by a light source and detectable by an emission filter, it also refers to a nucleotide conjugated to such a label (either direct labeling or secondary labeling via an affinity reagent) that is excitable by such light source and detectable by such emission filter.

[0113] 3' hydroxyl blocking group In any embodiment of the methods and kits described herein, the nucleotides used in the incorporation mixture can have a blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide. In certain embodiments, the blocking group is at the 3'OH position of the deoxyribose sugar of the nucleotide. Various 3'OH blocking groups are disclosed in WO 2004 / 018497 and WO 2014 / 139596, which are incorporated herein by reference. For example, the blocking group can be azidomethyl (-CHN) or substituted azidomethyl (e.g., -CH(CHF)N or CH(CHF)N), or an allyl attached to the 3' oxygen atom of the ribose or deoxyribose sugar. In some embodiments, the 3' blocking group is azidomethyl, forming 3'-OCHN with the 3' carbon of the ribose or deoxyribose.

[0114] In some other embodiments, the 3' blocking group and the 3' oxygen atom are attached to the 3' carbon of the ribose or deoxyribose.

[0115] [ka] forming a covalently bonded acetal group of the structure shown above, In the formula, each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl; Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano, or halogen; Alternatively, R 1a and R 2a together with the atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclyl group, R Fis H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted (C1-C6 alkylene)Si(R 3a )3, Each R 3a are independently H, C1-C6 alkyl, or optionally substituted C6-C 10 It is aryl.

[0116] Additional 3'OH blocking groups are disclosed in U.S. Publication No. 2020 / 0216891A1, which is incorporated herein by reference in its entirety. Acetal Blocking Groups

[0117] [ka] are non-limiting examples of ribose and deoxyribose, respectively, which are covalently attached to the 3' carbon of ribose or deoxyribose.

[0118] Deprotection of the 3' hydroxyl blocking group In some embodiments, the azidomethyl 3' hydroxyl protecting group can be removed or deprotected by using a water-soluble phosphine reagent. Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP), or tris(hydroxylpropyl)phosphine (THP or THPP). The 3'-acetal blocking groups described herein can be removed or cleaved under a variety of chemical conditions. Acetal blocking groups containing vinyl or alkenyl moieties

[0119] [ka] In

[0010] , non-limiting cleavage conditions include Pd(II) complexes such as Pd(OAc)2 or allylPd(II) chloride dimer in the presence of a phosphine ligand, such as tris(hydroxymethyl)phosphine (THMP) or tris(hydroxylpropyl)phosphine (THP or THPP). For those blocking groups containing alkynyl groups (e.g., ethynyl), they can also be removed by Pd(II) complexes (e.g., Pd(OAc)2 or allylPd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).

[0120] Palladium Cleavage Reagent In some embodiments, the 3' hydroxyl blocking groups described herein can be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, the Pd(0) complex is a Pd(0) complex (e.g., tris(3,3',3"phosphinidinetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some cases, Pd(0) can be generated in situ from the reduction of a Pd(II) complex with a reagent such as an alkene, alcohol, amine, phosphine, or metal hydride. Suitable palladium sources include NaPdCl, Pd(CHCN)Cl, (PdCl(C3H5)), [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)]Cl, Pd(OAc), Pd(Ph), Pd(dba), Pd(Acac), PdCl(COD), and Pd(TFA). In one such embodiment, the Pd(0) complex is In another embodiment, the palladium source is allylpalladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated in aqueous solution by mixing the Pd(II) complex with a phosphine. Suitable phosphines include water-soluble phosphines such as tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt, tris(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3',3"-trisulfonic acid trisodium salt.

[0121] In some embodiments, Pd(0) is prepared in situ by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP. The molar ratio of Pd(II) complex to THP can be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some further embodiments, one or more reducing agents, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate), may be added. In some embodiments, the cleavage mixture may contain additional buffering reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate, a phosphate, or a borate, or a combination thereof. In some further embodiments, the buffering reagent comprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2-diethylethanolamine (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED), or N,N,N',N'-tetraethylethylenediamine (TEEDA), or a combination thereof. In one embodiment, the buffering reagent is DEEA. In another embodiment, the buffering reagent contains one or more inorganic salts, such as carbonate, phosphate, or borate, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0122] Linker The fluorescent label can be covalently attached to the nucleotide via a cleavable linker. The use of the term "cleavable linker" does not imply that the entire linker needs to be removed. The cleavage site can be located on the linker to ensure that a portion of the linker remains attached to the dye and / or substrate moiety after cleavage. The cleavable linker can be, by way of non-limiting example, an electrophilically cleavable linker, a nucleophilically cleavable linker, a photocleavable linker, a linker cleavable under reducing conditions (e.g., a disulfide- or azide-containing linker), a linker cleavable under oxidative conditions, a linker cleavable by the use of a safety linker, or a linker cleavable by a removal mechanism. Using a cleavable linker to attach the dye compound to the substrate moiety allows for label removal after detection, if necessary, to avoid any interfering signals in downstream processes.

[0123] Useful linker groups are described in PTC Publication No. WO2004 / 018493 (incorporated herein by reference), and examples include linkers that can be cleaved using a water-soluble phosphine or water-soluble transition metal catalyst formed from a transition metal and at least a partially water-soluble ligand. In aqueous solution, the latter forms at least a partially water-soluble transition metal complex. Such cleavable linkers can be used to connect the base of a nucleotide to a label, such as the dyes described herein.

[0124] Particular linkers include those disclosed in PCT Publication No. WO2004 / 018493 (hereby incorporated by reference), which have the formula:

[0125] [ka] wherein X is selected from the group including O, S, NH, and NQ, Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group including O, S, NH, and N(allyl), and T is hydrogen or a C1-C 10 is a substituted or unsubstituted alkyl group,* In some embodiments, the linker connects the base of the nucleotide to the label.

[0126] Additional examples of linkers include those disclosed in U.S. Patent Application Publication No. 2016 / 0040225 (herein incorporated by reference), such as those containing a moiety of the formula:

[0127] [ka] (In the formula, * indicates the position where the moiety is attached to the remainder of the nucleotide). The linker moieties shown herein can comprise a full or partial linker structure between the nucleotide and the label. The linker moieties shown herein can comprise a full or partial linker structure between the nucleotide and the label.

[0128] Additional examples of linkers include those of the formula:

[0129] [ka] wherein B is a nucleobase, Z is -N3 (azido), -O-C1-C6 alkyl, -O-C2-C6 alkenyl, or -O-C2-C6 alkynyl, and F1 comprises a dye moiety that may contain an additional linker structure. One skilled in the art will appreciate that the dye compounds described herein are covalently attached to a linker by reacting a functional group (e.g., carboxyl) of the dye compound with a functional group (e.g., amino) of the linker. In one embodiment, the cleavable linker is

[0130] [ka] (an "AOL" linker moiety), where Z is -O-allyl.

[0131] The dye may be attached to any position on the nucleotide base, for example, via a linker. In certain embodiments, Watson-Crick base pairing can still be performed on the resulting analog. Specific nucleobase labeling sites include the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases.

[0132] In some embodiments, when the nucleotide is unlabeled upon incorporation and relies on an affinity reagent to add a detectable label to the extended primer polynucleotide, the unlabeled nucleotide may still include a cleavable linker for attaching a hapten. The cleavable linkers described herein may also be used to attach a dye to the affinity reagent when a secondary labeling method is used to add a label to the extended primer polynucleotide / target polynucleotide complex using an affinity reagent.

[0133] Labeled nucleotides The dye-labeled nucleotides described herein have the formula:

[0134] [ka] wherein Dye is a dye compound (label) moiety as described herein (after covalent attachment between the functional group of the dye and the functional group of the linker "L"); B is a nucleobase, such as, for example, uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, 7-deazaguanine, etc.; L is an optional linker group that may or may not be present; R' can be H; -OR' is a monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analog, -O- attached to a reactive phosphorus-containing group, or -O- protected by a blocking group; R" is H or OH; R'" is H, a 3'OH blocking group as described herein, or -OR'" forms a phosphoramidite, where -OR'" is a phosphoramidite and R' is an acid-cleavable hydroxyl protecting group that allows for subsequent monomer coupling under automated synthesis conditions. In some further embodiments, B is

[0135] [ka] or optionally substituted derivatives and analogs thereof. In some further embodiments, the labeled nucleobase has the structure

[0136] [ka] Includes:

[0137] In certain embodiments, the blocking group is separate and independent from the dye compound, i.e., not attached to the dye compound. Alternatively, the dye may include all or part of the 3'-OH blocking group. Thus, R''' can be a 3'-OH blocking group that may or may not include a dye compound.

[0138] In yet another alternative embodiment, there is no blocking group on the 3' carbon of the pentose sugar, and for example, the dye attached to the base (or the dye and linker structure) may be of sufficient size or structure to act as a block to the incorporation of additional nucleotides. Thus, whether or not the dye is attached to the 3' position of the sugar, the block may be due to steric hindrance or a combination of size, charge, and structure.

[0139] In yet another alternative embodiment, the blocking group is present on the 2' or 4' carbon of the pentose sugar and can be of sufficient size or structure to act as a block to the incorporation of additional nucleotides.

[0140] In some embodiments, both the linker (between the dye and the nucleotide) and the blocking group are present and are separate moieties. In certain embodiments, the linker and the blocking group are both cleavable under the same or substantially similar conditions. Thus, the deprotection and deblocking process may be more efficient because only a single treatment is required to remove both the dye compound and the blocking group. However, in some embodiments, the linker and the blocking group do not need to be cleavable under similar conditions, instead being individually cleavable under separate conditions.

[0141] The present disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA composed of deoxyribonucleotides or ribonucleotides, respectively, joined by phosphodiester linkages. Polynucleotides can include naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof, in combination with at least one modified nucleotide as described herein (e.g., labeled with a dye compound). Polynucleotides according to the present disclosure can also include non-naturally occurring backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures composed of a mixture of ribonucleotides and deoxyribonucleotides containing at least one labeled nucleotide are also contemplated.

[0142] Non-limiting labeled nucleotide conjugates described herein include:

[0143] [ka] wherein L represents a linker and R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety substituted at the 5' position with a monophosphate, diphosphate, or triphosphate.

[0144] In some embodiments, a non-limiting exemplary fully functionalized nucleotide conjugate comprising a cleavable linker and a fluorescent moiety is as follows:

[0145] [ka]

[0146] [ka]

[0147] [ka] wherein PG represents a 3'OH blocking group as described herein, p is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and k is 0, 1, 2, 3, 4, or 5. In one embodiment, -O-PG is AOM. In another embodiment, -O-PG is -O-azidomethyl. In one embodiment, k is 5. In some further embodiments, p is 1, 2, or 3 and k is 5.

[0148] [ka] refers to the attachment point of the Dye with a cleavable linker as a result of a reaction between the amino group of the linker moiety and the carboxyl group of the Dye (i.e., a blue or purple dye described herein). In any embodiment of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate. Alternatively, if the ffN is unlabeled, the Dye moiety can be replaced with a functional moiety (e.g., a hapten) that can enable binding of the unlabeled nucleotide to an affinity reagent described herein. Example

[0149] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims.

[0150] Example 1. Evaluation of DNA photodamage using violet radiation at 405 nm In this example, we evaluated DNA damage induced by 405 nm violet light. Single-stranded DNA in Tris buffer (pH = 8, 100 mM) was covalently linked to the violet dye DY405 or irradiated for 2 hours under a violet LED in the presence of DY405 in the buffer. When DY405 was covalently linked to the 5' end of the DNA, photodamage to the DNA induced by irradiation was observed to be substantially increased compared to when the DNA was mixed in a buffer containing DY405. The results are shown in Figure 1.

[0151] Example 2. Sequencing by synthesis using a blue / purple dual-channel MiSeq® system In this example, sequencing by synthesis was performed on a MiSeq® instrument configured as a two-channel blue / purple. Standard sequencing reagents were used. The incorporation mixture for standard SBS is summarized in Table 1. The sequenced library shown in these data is PhiX.

[0152] In the standard SBS incorporation mixture, the purple dye used is DY405. Both ffT and ffC were labeled with DY405. Blue DyeA was used to label ffA and ffC. Green ffN was also introduced to reduce the signal intensity in the purple and blue channels, resulting in a square scatter plot with a desirable shape after analysis. ffG was unlabeled ("dark G"). The structures of the nucleotides in the incorporation mixture are shown below. Both ffC-sPA-DY405 and ffT-LN3-DY405 were prepared using the standard ffN coupling reaction by reacting pppC-sPA-NH2 or pppT-LN3-NH2 with Dy405-NHS (5 mg).

[0153] [Table 1]

[0154] [ka]

[0155] [ka]

[0156] Preparation of DY405-labeled streptavidinFirst, DY405 was converted to DY405-NHS by reacting it with N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (1.5 equivalents) in anhydrous DMA in the presence of Hunig's base and TSTU for 30 minutes. Second, streptavidin powder was dissolved in water and NaHCO3 buffer. DY405-NHS prepared from the first step was transferred to the streptavidin solution and incubated at room temperature for 1 hour with occasional mixing. A 5M NaCl solution was then added to the reaction mixture. The reaction product was purified by removing excess dye using a Thermo Fisher dye removal column. Quantification of the reaction product indicated that the final dye / protein ratio was approximately 3.1–3.4.

[0157] For secondary-labeled SBS, secondary labels were used for dTTP and dCTP. In the secondary-labeled SBS incorporation mixture, ffT was unlabeled and contained a biotin moiety. ffC was both unlabeled and labeled with blue dye A, and ffA was labeled with blue dye A. The incorporation mixtures are summarized in Table 2. For secondary-labeled SBS, an additional step was required for standard post-incorporation sequencing. After incorporation of one nucleotide, a solution of DY405-labeled streptavidin was flushed through the flow cell and incubated at 60°C for 25 seconds, followed by a buffer wash before the first and second imaging events. The streptavidin-DY405 solution contained 5 μg / ml streptavidin-DY405, NaCl, EDTA, and Tween® 20 (polysorbate 20) in 5 mM Tris, pH 7.5. A commercially available MiSeq® flow cell was used for this experiment. Figure 2 shows a scatter plot obtained using secondary label SBS, demonstrating the utility of this sequencing method.

[0158] [Table 2]

[0159] [ka]

[0160] [Table 3]

[0161] Table 3 shows the primary sequencing metrics for both secondary labeled SBS and standard SBS (R1 = secondary labeled SBS and R2 = standard SBS). The results show that blue / purple dual channel sequencing is comparable to the modified method with purple dye secondary labeling.

[0162] However, the % fading and % signal decay observed for the 50-cycle run using the blue / violet (B / V) channel is much higher than in standard blue / green (B / G) sequencing (Table 4).

[0163] [Table 4]

[0164] Further experiments were performed to understand the causes of signal decay related to nucleotide incorporation time and violet illumination time. It was discovered that both increasing the violet light irradiance and increasing the violet light exposure time worsened signal decay. However, by increasing the nucleotide incorporation time, the % fading was substantially reduced, and as a result, signal decay was also improved. Furthermore, signal decay was also improved by using a brighter flow cell with reduced violet light loss and shorter exposure times (e.g., shortening the violet exposure time from 250 ms to 170 ms).

Claims

1. 1. A method for determining a sequence of a target polynucleotide, comprising: (a) contacting a primer polynucleotide with a mixture comprising one or more of a first type of nucleotides, a second type of nucleotides, a third type of nucleotides, and a fourth type of nucleotides, wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating one type of nucleotide from the mixture into the primer polynucleotide to generate an extended primer polynucleotide; (c) performing a first imaging event using a first excitation light source and collecting a first emission signal from the extended primer polynucleotide with a first emission filter; (d) performing a second imaging event using a second excitation light source and collecting a second emission signal from the extended primer polynucleotide with a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm; the first type of nucleotides are labeled with a first detectable label that is excitable by the first excitation light source and detectable by the first emission filter; the second type of nucleotides are labeled with a second detectable label that is excitable by the second excitation light source and detectable by the second emission filter, the second type of detectable label being spectrally distinguishable from the first type of detectable label; and i. the third type of nucleotides are labeled with both a first detectable label and a second detectable label, and the third type of nucleotides are excitable by both the first excitation light source and the second excitation light source; or ii. The method, wherein the third type of nucleotides comprises a mixture of a third type of nucleotides labeled with a third label and a third type of nucleotides labeled with a fourth label, wherein the third label is excitable by the first excitation light source and detectable by the first emission filter, and the fourth label is excitable by the second excitation light source and detectable by the second emission filter.

2. A method for determining the sequence of a target polynucleotide, comprising: (a) contacting a primer polynucleotide with a mixture comprising one or more of a first type of nucleotides, a second type of nucleotides, a third type of nucleotides, and a fourth type of nucleotides, wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating one type of nucleotide from the mixture into the primer polynucleotide to generate an extended primer polynucleotide; (c) performing a first imaging event using a first excitation light source and collecting a first emission signal from the extended primer polynucleotide with a first emission filter; (d) performing a second imaging event using a second excitation light source and collecting a second emission signal from the extended primer polynucleotide with a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm; each of the first type, the second type, and the third type of nucleotide is unlabeled, and the method comprises contacting the extended primer polynucleotide with a set of affinity reagents prior to the first imaging event, at least one affinity reagent in the set specifically binds to the incorporated first type, second type, or third type of nucleotide; the set of affinity reagents comprises a first affinity reagent that specifically binds to the first type of nucleotide and a second affinity reagent that specifically binds to the second type of nucleotide; the first affinity reagent comprises one or more first detectable labels excitable by the first excitation light source and detectable by the first emission filter, the second affinity reagent comprises one or more second detectable labels excitable by the second excitation light source and detectable by the second emission filter, the first detectable labels being spectrally distinguishable from the second detectable labels, and i. both the first affinity reagent and the second affinity reagent specifically bind to the third type of nucleotide; or ii. The method, wherein the set of affinity reagents further comprises a third affinity reagent that specifically binds to the third type of nucleotide, the third affinity reagent comprising one or more third detectable labels excitable by the first excitation light source and detectable by the first emission filter, and one or more fourth detectable labels excitable by the second excitation light source and detectable by the second emission filter.

3. i. the first type of nucleotide comprises a first hapten, the first affinity reagent comprises a first hapten binding partner that specifically binds to the first hapten, and optionally the first hapten comprises a biotin moiety and the first hapten binding partner comprises streptavidin; and / or ii. The method of claim 2, wherein the second type of nucleotide comprises a second hapten and the second affinity reagent comprises a second hapten binding partner that specifically binds to the second hapten.

4. the first type of nucleotides are labeled with a first detectable label, the second type of nucleotides are unlabeled, and the third type of nucleotides are both unlabeled and labeled with the first detectable label, the first label being excitable by the first excitation light source and detectable by the first emission filter, the method comprising contacting the extended primer nucleotides with an affinity reagent prior to the first imaging event, the affinity reagent specifically binding to the second type of unlabeled nucleotides or the third type of unlabeled nucleotides, Optionally, the affinity reagent comprises one or more second detectable labels excitable by the second excitation light source and detectable by the second emission filter; 3. The method of claim 2, further optionally, wherein the affinity reagent comprises streptavidin and both the second type of nucleotides and the third type of unlabeled nucleotides comprise a biotin moiety.

5. the first type of nucleotides are unlabeled, the second type of nucleotides are labeled with the second detectable label, the third type of nucleotides are both unlabeled and labeled with the second detectable label, the second detectable label being excitable by the second excitation filter and detectable by the second emission filter, the method comprising contacting the extended primer polynucleotides with an affinity reagent prior to the first imaging event, the affinity reagent specifically binding to the first type of unlabeled nucleotides or the third type of unlabeled nucleotides, Optionally, the affinity reagent comprises one or more first detectable labels excitable by the first excitation light source and detectable by the first emission filter; 3. The method of claim 2, further optionally, wherein the affinity reagent comprises streptavidin and both the first type of nucleotides and the third type of unlabeled nucleotides comprise a biotin moiety.

6. 6. The method of any one of claims 1 to 5, wherein the fourth type of nucleotide is unlabeled (dark) or labeled with a fluorescent moiety that has no emission from either the first imaging event or the second imaging event.

7. the four types of nucleotides include dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogs thereof; Optionally, each of the four types of nucleotides in the mixture has a 3' hydroxyl blocking group; 7. The method of any one of claims 1 to 6, further optionally comprising: (e) removing the 3' hydroxyl blocking group from the incorporated nucleotide after the second imaging event and prior to a next sequencing cycle.

8. Repeating steps (a)-(e) for a number of cycles; and 8. The method of claim 7, further comprising determining a sequence of the target polynucleotide based on consecutively incorporated nucleotides.

9. 9. The method of claim 1, wherein each of the first excitation light source and the second excitation light source comprises a laser, a light emitting diode (LED), or a combination thereof.

10. i. the first excitation light source has a wavelength of about 350 nm to about 410 nm, the first emission filter has a detection wavelength of about 415 nm to about 450 nm, the second excitation light source has a wavelength of about 450 nm to about 460 nm, and the second emission filter has a detection wavelength of about 480 nm to about 525 nm, or ii. the first excitation light source has a wavelength of about 450 nm to about 460 nm and the first emission filter has a detection wavelength of about 480 nm to about 525 nm; 10. The method of claim 1, wherein the second excitation light source has a wavelength of about 350 nm to about 410 nm and the second emission filter has a detection wavelength of about 415 nm to about 450 nm.

11. the target polynucleotide is immobilized on a solid support; Optionally, the solid support comprises a plurality of immobilized target polynucleotides; 11. The method of any one of claims 1 to 10, further optionally, wherein the solid support comprises a flow cell comprising a plurality of nanowells, each nanowell comprising one immobilized target polynucleotide.

12. The density of the immobilized target polynucleotides on the solid support is about 100 k / mm ２ ~Approx. 300k / mm ２ The method of claim 11, wherein

13. A kit for sequencing applications comprising: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with said first detectable label; a third type of nucleotide labeled with said second detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label is excitable by a first light source and detectable by a first emission filter, and the second detectable label is excitable by a second light source and detectable by a second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm.

14. A kit for sequencing applications comprising: a first type of nucleotide labeled with a first detectable label; a second type of nucleotide labeled with a second detectable label; a third type of nucleotide labeled with a third detectable label; and a third type of nucleotide labeled with a fourth detectable label; the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label is excitable by a first light source and detectable by a first emission filter, and the second detectable label is excitable by a second light source and detectable by a second emission filter; the third detectable label and the fourth detectable label are spectrally distinguishable from one another, the third detectable label is excitable by the first light source and detectable by the first emission filter, and the fourth detectable label is excitable by the second light source and detectable by the second emission filter; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm.

15. A kit for sequencing applications comprising: a first type of unlabeled nucleotide; a second type of unlabeled nucleotide; and a third type of unlabeled nucleotide; and A set of affinity reagents comprising: a first affinity reagent that specifically binds to the first type of unlabeled nucleotides; and a second affinity reagent that specifically binds to the second type of unlabeled nucleotides; a set of affinity reagents, wherein the first affinity reagent comprises one or more first detectable labels excitable by a first excitation light source and detectable by a first emission filter, and the second affinity reagent comprises one or more second detectable labels excitable by a second excitation light source and detectable by a second emission filter, and the first detectable labels are spectrally distinguishable from the second detectable labels; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm.

16. A kit for sequencing applications comprising: a first type of nucleotide, either unlabeled or labeled with a first detectable label; a second type of nucleotides that are either unlabeled or labeled with a second detectable label, wherein one of the first type of nucleotides and the second type of nucleotides is unlabeled; and a third type of unlabeled nucleotide, and a third type of nucleotide labeled with the same detectable label as either the first or second type of nucleotide, wherein the first detectable label and the second detectable label are spectrally distinguishable from one another, the first detectable label is excitable by a first light source and detectable by a first emission filter, and the second detectable label is excitable by a second light source and detectable by a second emission filter; an affinity reagent comprising either the third type of unlabeled nucleotides and a first affinity reagent that specifically binds to the first type of nucleotides when the first type of nucleotides are unlabeled, or a second affinity reagent that specifically binds to the third type of unlabeled nucleotides and the second type of nucleotides when the second type of nucleotides are unlabeled, wherein the first affinity reagent comprises one or more first detectable labels and the second affinity reagent comprises one or more second detectable labels; one of the first excitation light source and the second excitation light source has a wavelength of about 350 nm to about 410 nm, and the other of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm; one of the first emission filter and the second emission filter has a detection wavelength of about 415 nm to about 450 nm, and the other of the first emission filter and the second emission filter has a detection wavelength of about 480 nm to about 525 nm.

Images