Methods, systems, and compositions for nucleic acid sequencing

Abstract

The present disclosure relates to a method, system, kit, and composition for nucleic acid sequencing applications. In particular, the method utilizes two imaging events with different excitation wavelengths and a single emission channel to collect the fluorescent signal patterns of different types of nucleotide conjugates and determine the identity of the incorporated nucleotide conjugates. The method described herein does not require chemical treatment of the nucleotide conjugates in the incorporation mixture between the two imaging events.

Description

[Technical Field]

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

[0002] Nucleic acid sequencing methods have evolved significantly from the chemical degradation methods used by Maxam and Gilbert and the strand extension methods used by Sanger. Several sequencing methodologies are now in use that allow for the parallel processing of thousands of nucleic acids, all in a single sequencing run. The equipment used to perform such methods is generally large and expensive because current methods typically rely on large amounts of expensive reagents and multiple sets of optical filters to record the incorporation of nucleic acids into the sequencing reaction.

[0003] The need for high-throughput, smaller, and cheaper DNA sequencing technologies has become evident to be beneficial in reaping the benefits of genome sequencing. Personalized medicine is moving forward and will benefit from such technologies. Sequencing an individual's genome to identify potential mutations and abnormalities is important in identifying a specific disease and subsequently identifying a next therapy tailored to that individual. To accommodate such efforts, sequencing technologies should not only have high-throughput capabilities, but also be scalable. Thus, there is a need for new sequencing methods that offer improved speed, error detection, and cost-effectiveness. Summary of the Invention

[0004] The present disclosure provides next-generation sequencing methods, systems, and compositions. Some embodiments 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 (i.e., an incorporation mixture) comprising one or more of four different types of nucleotide conjugates, wherein a first type of nucleotide conjugate comprises a first label, a second type of nucleotide conjugate comprises a second label, and a third type of nucleotide conjugate comprises a third label, wherein each of the first label, the second label, and the third label are spectrally distinct from one another, and wherein the primer polynucleotide is complementary to at least a portion of a target polynucleotide; (b) incorporating a nucleotide conjugate 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 to collect a first emission signal from the extended primer polynucleotide; (d) performing a second imaging event using a second excitation light source to collect a second emission signal from the extended primer polynucleotide; The first excitation light source and the second excitation light source have different wavelengths, and the first emission signal and the second emission signal are detected or collected in a single emission detection channel / filter. In some embodiments, the method does not include chemical modification of any of the nucleotide conjugates in the mixture after the first imaging event and before the second imaging event. In some embodiments, the mixture further includes a fourth type of nucleotide conjugate, where the fourth type of nucleotide conjugate is either unlabeled or labeled with a fluorescent moiety that does not emit a signal from either the first or second imaging event. In some further embodiments, one or more of the four different types of nucleotide conjugates in the incorporation mixture each include a 3' hydroxyl blocking group.

[0005] In the sequencing methods described herein, the identity of each incorporated nucleotide conjugate is determined based on the detection patterns of the first and second imaging events. For example, the incorporation of a first type of nucleotide conjugate is determined by the signal state in the first imaging event and the dark state in the second imaging event. The incorporation of a second type of nucleotide conjugate is determined by the dark state in the first imaging event and the signal state in the second imaging event. The incorporation of a third type of nucleotide conjugate is determined by the signal state in both the first and second imaging events. The incorporation of a fourth type of nucleotide conjugate is determined by the dark state in both the first and second imaging events. In some embodiments, the mixture in step (a) contains four different types of nucleotide conjugates (A, C, G, and T or U). In some further embodiments, three of the four types of nucleotide conjugates are each labeled with a spectrally distinct fluorophore, and one of the nucleotides is not labeled with a fluorophore. In further embodiments, steps (a)-(d) are performed in repeated cycles (e.g., at least 30, 50, 100, 150, 200, 250, or 300 times), and the method further comprises sequentially determining the identity of each incorporated nucleotide conjugate, thereby determining the sequence of at least a portion of the single-stranded polynucleotide.

[0006] An additional embodiment of the present disclosure relates to a kit for sequencing applications, the kit comprising: a first type of nucleotide conjugate comprising a first label; a second type of nucleotide conjugate comprising a second label; and a third type of nucleotide conjugate comprising a third label; The first label, the second label, and the third label are each spectrally distinct from one another, the first label and the third label are excitable using a first light source wavelength, the second label and the third label are excitable using a second light source wavelength different from the first light source wavelength, and each of the first label, the second label, and the third label has an emission spectrum that is detectable in a single detection channel. In some embodiments, the kit includes four different types of nucleotide conjugates (A, C, G, and T or U). In some further embodiments, three of the four types of nucleotide conjugates are each labeled with a spectrally distinct fluorophore, and one of the nucleotides is not labeled with a fluorophore. [Brief explanation of the drawings]

[0007] [Figure 1A] 1 shows a flow chart of standard Illumina 1 channel sequencing-by-synthesis (SBS) chemistry. [Figure 1B] Image 1 and Image 2 from standard one-channel SBS show how they are processed by image analysis software to identify which bases have been incorporated. [Figure 2] 1 illustrates an embodiment of the sequencing method described herein. [Figure 3A] 1 illustrates an embodiment of a detection system for the sequencing methods described herein. [Figure 3B] 1 illustrates an embodiment of a single emission detection channel as described herein. [Figure 4] 1 shows a scatter plot obtained on an iSeq™ 100 System according to an embodiment of the sequencing method described herein. DETAILED DESCRIPTION OF THE INVENTION

[0008] Illumina's next-generation sequencing system, the iSeq™ 100, uses complementary metal-oxide semiconductor (CMOS)-based technology to provide a simplified, accessible benchtop sequencing solution. A typical sequencing workflow, also referred to as single-channel sequencing, is shown in Figures 1A and 1B. Each sequencing cycle includes two chemical steps and two imaging steps. In Figure 1A, the first chemical step exposes the flow cell to a mixture of fluorescently labeled adenine and thymine nucleotides. During the first imaging step, the emission from each cluster is recorded by the CMOS sensor. The second chemical step removes the fluorescent label from the adenine and adds a fluorescent label to the cytosine. In both chemical steps, guanine is dark (unlabeled). A second image is recorded. In Figure 1B, the combination of Image 1 and Image 2 is processed by image analysis software to identify which base is incorporated at each cluster position. This sequencing cycle is repeated "n" times to create a read length of "n" bases. Unlike 4-channel SBS chemistry, where the sequencer uses a different dye for each nucleotide, the iSeq™ 100 system uses one dye per sequencing cycle. In 1-channel chemistry, adenine has a removable label and is labeled only in the first image. Cytosine is a linker group that can be attached to the label and is labeled only in the second image. Thymine has a permanent fluorescent label and is therefore labeled in both images, while guanine is permanently dark. Nucleotides are identified by analysis of the distinct emission patterns at each base across the two images.

[0009] The present disclosure provides an improved alternative solution to the single-channel sequencing described on Illumina's iSeq™ 100 platform. In particular, the present disclosure provides a method for determining the identity of nucleotide incorporations using two imaging steps and one emission detection filter / channel. The use of fewer filters allows for sequencing to be performed with a smaller footprint. Furthermore, the disclosed method eliminates the second chemical step described above. The methods and systems described herein reduce instrument hardware requirements, reducing instrument size, reagent usage, and cost while increasing data output speed and accuracy. For example, the methods described herein can be used with Illumina's iSeq™ sequencing system, providing rapid turnaround time and efficient sequencing.

[0010] 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.

[0011] 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

[0012] 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 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, and 6,287,776. Nos. 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, EP742287, and EP799897.

[0013] 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.

[0014] In each instance where a single mesomeric form of a compound described herein is shown, the alternative mesomeric form is contemplated as well.

[0015] 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.

[0016] As used herein, "nucleotide conjugate" generally refers to a nucleotide labeled with a fluorescent moiety, optionally through a cleavable linker as described herein. In some embodiments, when a nucleotide conjugate is described as an unlabeled nucleotide conjugate, such a nucleotide does not include a fluorescent moiety. In some further embodiments, the unlabeled nucleotide conjugate also does not have a cleavable linker.

[0017] 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.

[0018] 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).

[0019] 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.

[0020] 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.

[0021] 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 contain 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.

[0022] 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.,

[0023] [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.

[0024] As will be appreciated by those skilled in the art, compounds such as the nucleotide conjugates described herein may be prepared using, for example, -CO2 - , -SO3 - , or -O - In other embodiments, the compounds may exist in ionized forms, including: Where the compounds contain positively or negatively charged substituents, 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.

[0025] 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.

[0026] 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 a primer polynucleotide with a mixture comprising one or more of four different types of nucleotide conjugates, wherein a first type of nucleotide conjugate comprises a first label, a second type of nucleotide conjugate comprises a second label, and a third type of nucleotide conjugate comprises a third label, wherein each of the first label, the second label, and the third label are spectrally distinct from one another, and wherein the primer polynucleotide is complementary to at least a portion of a target polynucleotide; (b) incorporating a nucleotide conjugate 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 to detect / collect a first emission signal from the extended primer polynucleotide; (d) performing a second imaging event using a second excitation light source and detecting / collecting a second emission signal from the extended primer polynucleotide; The first excitation light source and the second excitation light source have different wavelengths, and the first emission signal and the second emission signal are detected or collected in a single emission detection channel.

[0027] In some embodiments of the sequencing methods described herein, the incorporation mixture further comprises a fourth type of nucleotide, wherein the fourth type of nucleotide is labeled with a fluorescent moiety that does not emit a detectable signal from either the first or second imaging event. In some embodiments of the method, the first emission signal comprises a signal from a first label of the first type of nucleotide conjugate or a third label of the third type of nucleotide conjugate. In some embodiments of the method, the second emission signal comprises a signal from a second label of the second type of nucleotide conjugate or a third label of the third type of nucleotide conjugate. In some embodiments, the incorporation of the first type of nucleotide conjugate is determined by a signal state in the first imaging event and a dark state in the second imaging event. In some embodiments, the incorporation of the second type of nucleotide conjugate is determined by a dark state in the first imaging event and a signal state in the second imaging event. In some embodiments, the incorporation of the third type of nucleotide conjugate is determined by a signal state in both the first imaging event and the second imaging event. In some embodiments, the incorporation of the fourth type of nucleotide conjugate is determined by the dark state in both the first imaging event and the second imaging event. In one embodiment, the incorporation of the first type of nucleotide conjugate is determined by the signal state in the first imaging event and the dark state in the second imaging event, the incorporation of the second type of nucleotide conjugate is determined by the dark state in the first imaging event and the signal state in the second imaging event, the incorporation of the third type of nucleotide conjugate is determined by the signal state in the first imaging event and the second imaging event, and the incorporation of the fourth type of nucleotide conjugate is determined by the dark state in the first imaging event and the second imaging event.

[0028] The term "signal state," when used with respect to an imaging event, refers to the state of a labeled nucleotide conjugate such that a specific emission signal is generated by such imaging event, and the emission signal is detected or collected in a single detection channel / filter (i.e., one-channel detection) as described herein. For example, a fluorescent moiety in a nucleotide conjugate can be excited at a specific wavelength by a light source (e.g., a laser) and emit a fluorescent signal that is collected or detected in a single emission detection channel / filter, indicating a "signal state" in such imaging event.

[0029] When used with respect to an imaging event, the term "dark state" refers to a state of a labeled nucleotide conjugate in which either no specific emission signal is generated by such an imaging event or no emission signal is collected or detected by a single emission detection channel / filter. The "dark state" of a nucleotide conjugate can result from a variety of circumstances. In one scenario, such a nucleotide conjugate lacks a fluorescent moiety and, as a result, does not emit any fluorescent signal. In another scenario, the nucleotide conjugate is labeled with a fluorescent moiety that cannot be excited by a light source at a specific wavelength and therefore cannot emit any signal or emits minimal fluorescence (e.g., a red-emitting dye cannot be excited by wavelengths in the blue or purple region). In a third scenario, the nucleotide conjugate is labeled with a fluorescent moiety and emits a signal as a result of an imaging event. However, the wavelength of such emission signal is outside the single detection channel and therefore cannot be detected (e.g., the dye emits a blue signal but the detection channel is in the green-red region). Dark state detection can also include background fluorescence in which no fluorescent label may be present. For example, some reaction components may exhibit minimal fluorescence when excited at a particular wavelength. Thus, even in the absence of a fluorescent moiety, background fluorescence from such components may be present. Furthermore, background fluorescence may be due to light scattering, e.g., from adjacent sequencing reactions, and may be detected by a detector. In addition, background fluorescence may be caused by impure clusters (e.g., due to multiple template seeding, phasing, or prephasing events during cluster amplification). Thus, the "dark state" may include such background fluorescence when a fluorescent moiety is specifically not included, such as when nucleotides lacking a fluorescent label are utilized in the methods described herein. However, such background fluorescence is considered distinguishable from the signal state, such that nucleotide incorporation of unlabeled nucleotides (or "dark" nucleotides) is still discernible.

[0030] In one example of the methods described herein, the "T" nucleotide conjugate is determined by the signal state in the first imaging event and the dark state in the second imaging event, the "C" nucleotide conjugate is determined by the dark state in the first imaging event and the signal state in the second imaging event, the "A" nucleotide conjugate is determined by the signal state in the first imaging event and the signal state in the second imaging event, and the "G" nucleotide conjugate is determined by the dark state in the first imaging event and the dark state in the second imaging event. In another example, the "C" nucleotide conjugate is determined by the signal state in the first imaging event and the dark state in the second imaging event, the "T" nucleotide conjugate is determined by the dark state in the first imaging event and the signal state in the second imaging event, the "A" nucleotide conjugate is determined by the signal state in the first imaging event and the signal state in the second imaging event, and the "G" nucleotide conjugate is determined by the dark state in the first imaging event and the dark state in the second imaging event. A non-limiting example is shown in Table A below. In each instance, "T" can also be replaced by "U".

[0031] [Table 1-1]

[0032] [Table 1-2]

[0033] In some embodiments of the methods described herein, the nucleotide conjugates in the mixture in step (a) comprise a nucleotide type selected from the group consisting of A, C, G, T, and U, and non-natural nucleotide analogs thereof. In further embodiments, the mixture in step (a) comprises four different types of nucleotide conjugates (A, C, G, and T or U), or non-natural nucleotide analogs thereof. In further embodiments, the four different types of nucleotide conjugates are dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogs thereof. In some further embodiments, three of the four types of nucleotide conjugates are each labeled with a fluorophore that is spectrally distinct from one another, and one of the nucleotide conjugates is either not labeled with a fluorophore or is labeled with a fluorophore but is unable to emit a signal in either the first imaging or the second imaging event. In further embodiments, each of the four types of nucleotide conjugates in the incorporation mixture comprises a 3' hydroxyl blocking group.

[0034] In some embodiments of the methods described herein, the method further comprises step (e): cleaving or removing the fluorescent label from the incorporated nucleotide conjugate prior to the next sequencing cycle (e.g., after the second imaging event and before the start of the next sequencing cycle). In further embodiments, the incorporated nucleotide conjugate has a 3' hydroxy blocking group, and the 3' hydroxy blocking group is also removed prior to the next sequencing cycle. In some such embodiments, the label and the 3' hydroxy blocking group are removed in a single step (e.g., under the same chemical reaction conditions). In other embodiments, the label and the 3' hydroxy 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 target polynucleotide based on the identity of each successively incorporated nucleotide conjugate. In some such embodiments, steps (a)-(e) are repeated for at least 50 cycles. In some such embodiments, four different types of nucleotide conjugates are simultaneously present and compete for incorporation during each cycle. In some further embodiments, the incorporation of the nucleotide conjugates 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.

[0035] 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. In some embodiments, the first excitation light source has a shorter wavelength than the second excitation light source. In some such embodiments, the first excitation light source has a wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or about 450 nm to about 460 nm (i.e., "blue light"). In one embodiment, the first excitation light source has a wavelength of about 450 nm. The second excitation light source has a wavelength of about 490 nm to about 550 nm, about 500 nm to about 540 nm, or about 510 nm to about 530 nm (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520 nm. In other embodiments, the first excitation light source has a longer wavelength than the second excitation light source. In some such embodiments, the first excitation light source has a wavelength of about 490 nm to about 550 nm, about 500 nm to about 540 nm, or about 510 nm to about 530 nm (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520 nm. The second excitation light source has a wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or about 450 nm to about 460 nm (i.e., "blue light"). In one embodiment, the second excitation light source has a wavelength of about 450 nm.

[0036] In some embodiments of the methods described herein, the single emission detection channel has a spectral detection range of greater than about 560 nm, greater than about 570 nm, greater than about 580 nm, greater than about 590 nm, or greater than about 600 nm. In further embodiments, the single emission detection has a spectral detection range of less than about 700 nm, less than about 690 nm, less than about 680 nm, less than about 670 nm, less than about 660 nm, or less than about 650 nm. In some further embodiments, the single emission detection channel has a spectral detection range of about 560 nm to about 690 nm, about 570 nm to about 680 nm, or about 580 nm to about 650 nm. As used herein, the term "single emission detection channel" refers to a detection channel or filter that only allows light to be detected in a specific region of the spectrum or within a specific wavelength range, without detecting any emissions outside of that spectral region. In particular, the first label, the second label, and the third label each have overlapping emission spectra in a particular wavelength range such that emission from any of the first label, the second label, and the third label can be detected by the same detection channel or filter. In some such embodiments, the emission spectra can be detected by a single emission detection channel between about 560 nm and about 690 nm, between about 570 nm and about 680 nm, or between about 580 nm and about 650 nm.

[0037] Figure 2 shows an embodiment of an imaging event described in a sequencing method. The incorporation mixture contains four nucleotides: dCTP (ffC) labeled with a first dye excitable by a blue light source having a wavelength of approximately 450 nm; dTTP (ffT) labeled with a second dye excitable by a green light source having a wavelength of approximately 520 nm; dATP (ffA) labeled with a third dye ("dual dye") excitable by both a blue light source at 450 nm and a green light source at 540 nm; and unlabeled dGTP (ffG). The emission spectra of ffC, ffT, and ffA are detected in a CMOS collection region above 560 nm (e.g., from approximately 570 nm to approximately 650 nm). If the first imaging event uses a blue light source and the second imaging event uses a green light source, the identity of the incorporated nucleotide conjugate can be determined as follows:

[0038] [Table 2]

[0039] Similarly, if the first imaging event uses a green light source and the second imaging event uses a blue light source, the identity of the incorporated nucleotide conjugate can be determined as follows:

[0040] [Table 3]

[0041] The dual excitation, single channel detection sequencing methods described herein require fluorescent dyes that not only have strong fluorescence and chemical stability, but also tailored absorption and long Stokes shifts. In some embodiments, nucleotide conjugates with dual dyes that are excitable by both a first and a second light source (e.g., 450 nm and 520 nm, or 520 nm and 450 nm) have absorbance maxima (A) between about 480 nm and 510 nm, or between about 490 nm and 500 nm. maxNucleotide conjugates with dyes that are excitable only at shorter wavelengths (either the first or second light source) have an A of about 450-460 nm. max Nucleotide conjugates with dyes that are excitable only at longer wavelengths (either the first light source or the second light source) have an A that is greater than about 520 nm. max In a further embodiment, the emission maximum (E max ) is greater than 550 nm or greater than 560 nm. The Stokes shift of such labeled nucleotide conjugates is greater than 60 nm. In some further embodiments, the E of nucleotide conjugates having dyes that are excitable only at shorter wavelengths (shown in a signal state with a shorter wavelength light source and in a dark state with a longer wavelength light source) is greater than 550 nm or greater than 560 nm. max The Stokes shift of such labeled nucleotide conjugates is greater than 100 nm. In some further embodiments, the E of nucleotide conjugates bearing dyes that are excitable only at longer wavelengths is greater than 560 nm. max The Stokes shift of such nucleotide conjugates is greater than about 30 nm, or greater than about 40 nm. A of 450-460 nm, 480-510 nm, or 490-500 nm can be used in the sequencing methods described herein. max Non-limiting examples of coumarin dyes having an A of greater than 520 nm that can be used in the sequencing methods described herein include those disclosed in U.S. Publication Nos. 2018 / 0094140A1, 2018 / 0201981A1, 2020 / 0277529A1, and 2020 / 0277670A1, and US 63 / 057,758, which are incorporated by reference. max Non-limiting examples of polymethine dyes having the formula (I) include those disclosed in International Patent Publication Nos. WO2013 / 041117, WO2014 / 135221, WO2015 / 170102, WO2016 / 189287, and WO2017 / 051201.

[0042] 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. In some such embodiments, image analysis is processed after repeated cycles of incorporation (after at least 50, 100, 150, 200, 250, or 300 runs). In further embodiments, multiple immobilized primer polynucleotide / target polynucleotide complexes (clusters) are detected and sequenced in parallel.

[0043] Figure 3A shows a perspective view of a system used for single-channel detection. A solid support (e.g., a flow cell) contains clustered target polynucleotide templates immobilized on its surface and primer polynucleotides hybridized to at least a portion of the templates to form clustered primer polynucleotide / target polynucleotide complexes. The flow channel contains individual nanowells, each containing an immobilized primer polynucleotide / target polynucleotide complex. The incorporated nucleotide conjugates are exposed to green and blue light source excitation, respectively. Depending on the fluorescent moiety attached to the incorporated nucleotide conjugate, it can absorb either blue / green light, both blue / green light, or neither blue / green light (e.g., if the incorporated nucleotide conjugate does not contain a fluorescent moiety). The fluorescent moiety's emission (e.g., red light) is detected in a single detection channel. The workflow is simplified to merely illustrate the imaging events and detection mechanism. This workflow does not include additional steps of the sequencing method described herein, such as cleavage of the fluorescent moiety after the second imaging event and optional post-cleavage wash steps.

[0044] FIG. 3B is a perspective view of an exemplary sequencing and detection mechanism 300 used in the sequencing methods described herein. 300 can be integrated into a solid support described herein. Initially, a patterned flow cell 310 includes multiple individual nanowells 315 separated by an intermediate region. Sequencing reactions (e.g., nucleotide incorporation, deprotection of the 3' block of the incorporated nucleotide conjugate, and cleavage of the label after detection) occur within each of the nanowells. The flow cell can also include a protective surface that is transparent, allowing blue and green light illumination to pass through. 320 are multiple optical filters embedded in light-blocking element 325, allowing only luminescence signals generated from incorporated nucleotide conjugates in a specific wavelength range to pass through and be detected by the CMOS image sensor.

[0045] In any embodiment of the methods described herein, a target polynucleotide, e.g., a single-stranded target polynucleotide, can be immobilized on a solid support. In some such embodiments, the solid support comprises a plurality of immobilized target polynucleotides, e.g., single-stranded target polynucleotides. In further embodiments, each target polynucleotide hybridizes to a primer polynucleotide that is complementary to at least a portion of the target polynucleotide, thus forming a primer nucleotide / target nucleotide complex. The solid support can comprise clustered primer nucleotide / target nucleotide complexes. In some embodiments, the solid support comprises a flow cell, e.g., a patterned flow cell comprising a plurality of nanowells, each separated from one another. In some further embodiments, each nanowell comprises one immobilized cluster therein. In some embodiments, the solid support further comprises a CMOS chip. For example, the patterned flow cell is assembled on top of the CMOS chip and separated by a plurality of optical filters and light-blocking elements, so that luminescence signals generated from imaging events are filtered through the optical filters and detected by the CMOS image sensor. In yet another embodiment, each of the plurality of nanowells is directly aligned over a respective CMOS photodiode (pixel).

[0046] In any embodiment described herein, the method does not include any chemical modification of any of the nucleotide conjugates in the mixture after the first imaging event and before the second imaging event. Chemical modification may utilize chemical reagents such as cleavage reagents, binding partner-fluorescent moiety conjugates, or other reagents that can directly or indirectly cause an identifiable and measurable change in fluorescence from the first imaging event to the second imaging event. For example, chemical modification may include cleaving a label from one or more types of nucleotide conjugates and / or adding a label to one or more different types of nucleotide conjugates. In particular, the methods described herein eliminate the second chemical step required for the iSeq™ 100 sequencer to remove a fluorescent label from one nucleotide conjugate and add a fluorescent label to a different type of nucleotide conjugate. As a result, the sequencing methods described herein provide substantial reductions in cycle time and reduce the volumes of required reagents (e.g., THP reagents and wash solutions). In some embodiments, the sequencing methods described herein provide at least a 5%, 10%, 15%, 20%, 25%, or 30% reduction in cycle time compared to the standard cycle time on an Illumina iSeq™ 100 using a single dye, one detection method is shown in FIG. 1A.

[0047] Additional exemplary embodiments are described below.

[0048] In some embodiments, a method for sequencing a nucleic acid involves the use of one fluorescent label for the direct or indirect detection of three different nucleotide types, and one nucleotide type that is not detected by the presence of a fluorescent signal, but instead is detected by the absence or absence of a fluorescent signal. In some embodiments, a method for sequencing a nucleic acid involves the use of two or more different fluorescent labels with the same or similar excitation / emission spectra for the direct or indirect detection of three different nucleotide types, and one nucleotide type that is not detected by the presence of a fluorescent signal, but instead is detected by the absence or absence of a fluorescent signal. The same or similar excitation and emission spectra are such that a light source excites the two or more different fluorescent labels, and optical filters capture their emitted fluorescent signals. Detection of fluorescence to determine the sequence of a nucleic acid sample is performed, for example, at different time intervals during a sequencing reaction (i.e., before and after changes in reaction conditions, such as enzymatic cleavage, changes in environmental pH, or the addition of additional reagents), providing a pattern of fluorescence, such as a fluorescence transition pattern, and their cumulative pattern, which determines the sequence of the nucleic acid target. Thus, the methods described herein are time- and cost-efficient and allow for simplification of associated sequencing equipment.

[0049] An exemplary application that utilizes differences in fluorescence patterns over time intervals to determine target nucleic acid sequences is sequence-by-synthesis (SBS) methodologies and techniques. Accordingly, the embodiments described herein find particular utility in sequence by synthesis fluorescence applications. While the embodiments described herein are examples of innovative methods of fluorescent sequencing, embodiments of the present disclosure also find utility in a variety of other applications in which detection of two or more analytes (i.e., nucleotides, proteins, or fragments thereof) in a sample is desired.

[0050] In some embodiments, sequencing is performed on a substrate such as glass, plastic, semiconductor chips, or composite-derived substrates. In some embodiments, one nucleic acid species is provided on the substrate, for example, for single-target sequencing. In other embodiments, sequencing can also be in a multiplex format, where multiple nucleic acid targets are detected and sequenced in parallel, for example, in a flow cell or array-type format. The embodiments described herein are particularly advantageous when performing parallel or massively parallel sequencing. Platforms for performing fluorescent parallel sequencing include, but are not limited to, those provided by Illumina, Inc. (e.g., HiSeq®, Genome Analyzer, MiSeq™, MiniSeq™, NextSeq™, iSeq™, and NovaSeq™ platforms), Life Technologies (e.g., SOLiD), Helicos Biosciences (e.g., Heliscope), 454 / Roche Life Sciences (Branford, Conn.), and Pacific Biosciences (e.g., SMART). Flow cells, chips, and other types of surfaces that can accommodate multiple nucleic acid species are exemplary substrates used for parallel sequencing. In multiplex formats, where multiple nucleic acid species are sequenced in parallel, clonally amplified target sequences (e.g., via emulsion PCR (emPCR) or bridge amplification) are typically covalently immobilized on a substrate. For example, when performing emulsion PCR, the target of interest is immobilized on a bead, while clonally amplified targets are immobilized on channels of a flow cell or specific locations on an array or chip.

[0051] Flow cells for use in the compositions and methods described herein can be used for sequencing in a number of ways. For example, a DNA sample, such as a DNA library, can be applied to a flow cell or fluidic device containing one or more etched flow channels, which can further contain a population of probe molecules covalently bound to its surface. The bound probes in the flow cell channels can be conveniently arranged at different addressable locations within the channels, and DNA library molecules can be added to the flow cell channel to which complementary sequences bind (as described herein and further in WO 2012 / 096703, which is incorporated herein by reference in its entirety). Another example of a flow cell for use in this application includes a CMOS flow cell, such as those described in U.S. Patent Nos. 8,906,320 and 9,990,381, which are incorporated herein by reference in their entirety. Bridge amplification can be performed as described herein, followed by sequencing using the synthesis methods and compositions described herein. Methods for making and utilizing flow cells for sequencing are known in the art and are provided herein, all of which are incorporated herein by reference in their entirety. It is intended that the methods and compositions described herein are not limited to any particular manufacture or method of flow cell-directed sequencing.

[0052] Sequencing using the methods and compositions described herein can also be performed in microtiter plates, e.g., high-density reaction plates or slides (Margulies et al., 2005, Nature 437(7057): 376-380, incorporated herein by reference in its entirety). For example, genomic targets can be prepared by emPCR technology. The reaction plate or slide can be made from fiber optic material that can capture and record light generated from the reaction, e.g., from a fluorescent or luminescent reaction. The core material can be etched to provide separate reaction wells that can hold at least one emPCR reaction bead. Such slides / plates can contain over 1.6 million wells. Target sequencing reaction emPCR beads can be added to the prepared slide / plate and then mounted in an instrument, where sequencing reagents are provided and sequencing is performed.

[0053] An example of a substrate arrayed for sequencing targets using the compositions and methods disclosed herein is provided by performing patterned substrates containing DNA nanoballs on chips or slides, as performed by Complete Genomics (Mountain View, Calif.). As reported in Drmanac et al., 2010, Science 327(5961): 78-81, silicon wafers can be layered with silicon dioxide and titanium, and then patterned using photolithography and dry etching techniques. The wafers can be treated with HMDS and coated with a photoresist layer to define distinct areas for silylation and subsequent covalent attachment of DNA nanoballs for sequencing. Those skilled in the art will understand that there are many ways to create slides / chips with distinct locations for immobilizing nucleic acids for use in sequencing methodologies, and the present method is not limited by the method by which the substrate is prepared for sequencing.

[0054] For purposes of illustration and not intended to limit the embodiments described herein, the general strategy of a sequencing cycle can be described by a series of steps: While the following examples are based on sequence by synthesis sequencing reactions, the methods described herein are not limited to any particular sequencing reaction methodology.

[0055] Alternatively, the sequencing method described herein can also be performed using unlabeled nucleotides while using the same two imaging events and single-channel emission detection described above. For example, one, two, three, or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) in the incorporation mixture in step (a) can be unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3' hydroxyl blocking group to ensure that only a single base can be added to the 3' end of the primer polynucleotide by polymerase. After incorporation of the unlabeled nucleotide in step (b), the remaining unincorporated nucleotides are washed away. An affinity reagent is then introduced to specifically recognize and bind to the incorporated dNTP, providing a labeled extension product containing the incorporated dNTP. The use of unlabeled nucleotides and affinity reagents in sequencing by synthesis is disclosed in WO2018 / 129214 and WO2020 / 097607. The modified sequencing method of the present disclosure using unlabeled nucleotides comprises the following: (a') contacting a primer polynucleotide with a mixture comprising one or more of four different types of unlabeled nucleotides, wherein the primer polynucleotide is complementary to at least a portion of a single-stranded target polynucleotide; (b') incorporating one unlabeled nucleotide from the mixture into the primer polynucleotide to generate an extended primer polynucleotide; (c') contacting the extended primer polynucleotide with a set of different affinity reagents under conditions where one affinity reagent specifically binds to the incorporated unlabeled nucleotide to provide a labeled extended primer polynucleotide; (d') performing a first imaging event using a first excitation light source to detect a first emission signal from the labeled extended primer polynucleotide; (e') performing a second imaging event using a second excitation light source and detecting a second emission signal from the labeled extended primer polynucleotide; The first excitation light source and the second excitation light source have different wavelengths, and the first emission signal and the second emission signal are detected or collected into a single emission detection filter or channel. In some embodiments of the modified sequencing methods described herein, each of the unlabeled nucleotides in the incorporation mixture includes a 3' hydroxyl blocking group. In further embodiments, the 3' hydroxyl blocking group of the incorporated nucleotide is removed before the next sequencing cycle. In still further embodiments, the method further includes removing the affinity reagent from the incorporated nucleotide. In yet another embodiment, the 3' hydroxyl blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the set of affinity reagents can include a first affinity reagent that specifically binds to a first type of nucleotide, 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 nucleotide. In some further embodiments, each of the first, second, and third affinity reagents includes a detectable label. In some embodiments, the affinity reagent comprises a protein tag, an antibody (including but not limited to, a binding fragment of an antibody, a single chain antibody, a bispecific antibody, etc.), an aptamer, a knottin, an affimer, or any other known agent that binds with suitable specificity and affinity to the incorporated nucleotide. In one embodiment, the affinity reagent is a protein tag or an antibody. In another embodiment, the affinity reagent is a protein tag or an antibody that comprises one or more detectable labels that are fluorescent labels.

[0056] Modified nucleotides designed for sequencing reactions, typically three of the four nucleotide types A, C, T, and G, with 3' hydroxyl blocking groups fluorescently labeled, are simultaneously added to a location where a desired template sequence (also referred to interchangeably as a desired target polynucleotide sequence) will be located, along with other reaction components, and the sequencing reaction occurs (e.g., a flow cell, chip, slide, etc.). After incorporation of the nucleotide into a growing sequencing nucleic acid strand based on the target sequence, the reaction is exposed to light, and any emission signal is captured and recorded, constituting a first imaging event and a first fluorescence detection pattern. Following the first imaging event, the sample is irradiated with a second light source having a different wavelength from the first light source, illuminating the reaction location once again, and any change in fluorescence is captured and recorded, constituting a second imaging event (i.e., a second fluorescence detection pattern). Following the second imaging event, the 3' blocking group on the incorporated nucleotide is removed and washed, along with any other reagents present, in preparation for the next sequencing cycle. In some embodiments, the disclosed methods do not involve the use of any chemical reagents that can directly cause an identifiable and measurable change in fluorescence from the first imaging event to the second imaging event. The fluorescence patterns from the two imaging events are compared, and the incorporation of nucleotides, and therefore the sequence of the target nucleic acid, is determined for that particular cycle.

[0057] In one embodiment, at least one nucleotide is incorporated into a polynucleotide (such as a single-stranded primer polynucleotide described herein) during synthesis by the action of a polymerase enzyme. However, other methods of joining nucleotides to a polynucleotide can be used, such as chemical oligonucleotide synthesis or ligation of a labeled oligonucleotide to an unlabeled oligonucleotide. Thus, the term "incorporating," when used with respect to nucleotides and polynucleotides, can encompass chemical and enzymatic polynucleotide synthesis.

[0058] 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 linkage 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.

[0059] 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.

[0060] Suitable conditions for the synthesis step are well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step can resemble a standard primer extension reaction using nucleotide precursors containing labeled nucleotides as described herein to form an extended polynucleotide strand (primer polynucleotide strand) complementary to the target strand in the presence of a suitable polymerase enzyme. In another embodiment, the synthesis step can 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.

[0061] 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.

[0062] 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.

[0063] In embodiments 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 that is sequenced through detection of a fluorescent label(s) attached to the incorporated nucleotide(s). 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.

[0064] 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.

[0065] The method, as exemplified above, incorporates fluorescently labeled 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 Publication Nos. 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 attaching the nucleotide to the free 3' OH group of the nucleic acid strand through the formation of a phosphodiester linkage with the 5' phosphate group of the nucleotide.

[0066] 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.

[0067] 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).

[0068] 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 target polynucleotides are those in which template polynucleotides are attached to hydrogels supported on silica-based or other solid supports, 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.

[0069] 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)).

[0070] 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 reported 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.

[0071] 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.

[0072] 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, the 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, 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.

[0073] 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.

[0074] 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.

[0075] 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 published applications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. One use 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 methodology requiring detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0076] In particular, nucleotide conjugates labeled with the dye compounds of the present disclosure can be used in automated fluorescent sequencing protocols, particularly fluorescent dye terminator cycle sequencing, which is based on the chain termination sequencing method of Sanger and coworkers. Such methods generally incorporate fluorescently labeled dideoxynucleotides in primer extension sequencing reactions using enzymes and cycle sequencing. The so-called Sanger sequencing method and related protocols (Sanger-type) utilize randomized chain terminations with labeled dideoxynucleotides.

[0077] kitSome aspects of the present disclosure relate to kits for the sequencing methods described herein. In particular, the kits may include a first type of nucleotide conjugate comprising a first fluorescent moiety (i.e., a first label), a second type of nucleotide conjugate comprising a second fluorescent moiety (i.e., a second label), and a third type of nucleotide conjugate comprising a third fluorescent moiety (i.e., a third label), wherein the first label, the second label, and the third label are each spectrally distinct from one another, the first label and the third label are excitable by a first light source, and the second label and the third label are excitable by a second light source, the first light source and the second light source have different excitation wavelengths, and the first label, the second label, and the third label each have an emission spectrum that is detectable in a single detection channel. In further embodiments, the kits further include a fourth type of nucleotide conjugate. In some such embodiments, the fourth type of nucleotide conjugate is unlabeled. The four different types of nucleotide conjugates are A, C, G, and T or U, or non-natural nucleotide analogs thereof. In further embodiments, the four different types of nucleotide conjugates are dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogs thereof. In some embodiments, the first excitation light source has a wavelength of about 490 nm to about 550 nm, about 510 to about 540 nm, or about 520 to about 530 nm (e.g., 520 nm). The second light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (e.g., 450 nm). In alternative embodiments, the first light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (e.g., 450 nm). The second excitation light source has a wavelength of about 490 nm to about 550 nm, about 500 to about 540 nm, or about 510 to about 530 nm (e.g., 520 nm). In further embodiments, the first label, second label, and third label each have an emission spectrum that is greater than 550 nm or 560 nm that can be collected on a single emission filter or channel.In further embodiments, the single emission detection channel has a detection range greater than 560 nm and / or less than 700 nm (eg, about 565 nm to about 690 nm, about 570 nm to about 670 nm, or about 580 nm to about 650 nm).

[0078] The compounds, nucleotide conjugates, or kits described herein may be used to detect, measure, or identify biological systems (e.g., including processes or components thereof). Exemplary techniques in which the compounds, nucleotides, or kits can be used include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assays (e.g., cell binding or cell function analysis), or protein assays (e.g., protein binding assays or protein activity assays). Their use may also be in automated instruments for performing specific techniques, such as automated sequencing instruments. Sequencing devices may include two light sources (i.e., a first excitation light source and a second excitation light source) operating at different wavelengths.

[0079] In certain embodiments, the labeled nucleotide conjugates described herein can be supplied in combination with unlabeled or natural nucleotides, or any combination thereof. The nucleotide combination can be provided as separate individual components (e.g., one nucleotide type per container or tube) or as a nucleotide mixture (e.g., two or more nucleotides mixed in the same container or tube). In further embodiments, the nucleotide mixture can contain all four types of nucleotides.

[0080] As used herein, the term "spectrally distinct" fluorescent dyes or labels refers to fluorescent dyes that absorb light energy at different wavelengths and / or have different Stokes shifts that can be distinguished by a fluorescence detection device when two or more such dyes are present in a sample. When two labeled nucleotide conjugates are provided in kit form, a feature of some embodiments is that the spectrally distinct fluorescent dyes can be excited at different wavelengths, e.g., by two different light sources. When four nucleotides labeled with fluorescent dye compounds are provided in kit form, a feature of some embodiments is that two of the spectrally distinct fluorescent dyes can both be excited at one wavelength and two spectrally distinct dyes can both be excited at another wavelength. Specific excitation wavelengths for the dyes are 450-460 nm, 490-500 nm, or 520 nm or longer (e.g., 532 nm).

[0081] In some embodiments of the kits described herein, the nucleotide conjugate having a first label has an absorbance maximum (A) in the range of about 450-460 nm. max The nucleotide conjugate having a second label that is excitable only by a second light source has an A above 520 nm. max Nucleotide conjugates having a third label that is excitable by both the first and second light sources (e.g., 450 nm and 520 nm, or 520 nm and 450 nm) have an A of about 480 nm to 510 nm, or about 490 nm to 500 nm. max Alternatively, the nucleotide conjugate having a first label that is excitable only by one light source has an A above 520 nm. max The nucleotide conjugate having a second label that is excitable only by the second light source has an A of about 450-460 nm. maxNucleotide conjugates having a third label that is excitable by both the first and second light sources (e.g., 450 nm and 520 nm, or 520 nm and 450 nm) have an A of about 480 nm to 510 nm, or about 490 nm to 500 nm. max In a further embodiment, the emission maximum (E max ) is greater than 550 nm or greater than 560 nm, and the Stokes shift of such a labeled nucleotide conjugate is greater than 60 nm. max is greater than 560 nm, and the Stokes shift of such a labeled nucleotide conjugate is greater than 100 nm. max is greater than 560 nm, and the Stokes shift of such a nucleotide conjugate is greater than about 30 nm or greater than about 40 nm. Alternatively, the E of a nucleotide conjugate having a third label max is greater than 550 nm or greater than 560 nm, and the Stokes shift of such a labeled nucleotide conjugate is greater than 60 nm. max is greater than 560 nm, and the Stokes shift of such a nucleotide conjugate is greater than about 30 nm or greater than about 40 nm. max is greater than 560 nm and the Stokes shift of such a labeled nucleotide conjugate is greater than 100 nm.

[0082] In one example, the first light source has an excitation wavelength of 450-460 nm, and the second light source has an excitation wavelength of 520 nm. In another example, the first light source has an excitation wavelength of 520 nm, and the second light source has an excitation wavelength of 450-460 nm. In some such embodiments, the first type of nucleotide conjugate is a C nucleotide (dCTP), the second type of nucleotide conjugate is a T nucleotide (dTTP), the third type of nucleotide conjugate is an A nucleotide (dATP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP). In some other embodiments, the first type of nucleotide conjugate is a T nucleotide (dTTP), the second type of nucleotide conjugate is a C nucleotide (dCTP), the third type of nucleotide conjugate is an A nucleotide (dATP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP). In some other embodiments, the first type of nucleotide conjugate is a C nucleotide (dCTP), the second type of nucleotide conjugate is an A nucleotide (dATP), the third type of nucleotide conjugate is a T nucleotide (dTTP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP). In still other embodiments, the first type of nucleotide conjugate is an A nucleotide (dATP), the second type of nucleotide conjugate is a C nucleotide (dCTP), the third type of nucleotide conjugate is a T nucleotide (dTTP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP). In still other embodiments, the first type of nucleotide conjugate is an A nucleotide (dATP), the second type of nucleotide conjugate is a T nucleotide (dTTP), the third type of nucleotide conjugate is a C nucleotide (dCTP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP).In yet other embodiments, the first type of nucleotide conjugate is a T nucleotide (dTTP), the second type of nucleotide conjugate is an A nucleotide (dATP), the third type of nucleotide conjugate is a C nucleotide (dCTP), and the fourth type of nucleotide is an unlabeled G nucleotide (dGTP). Alternatively, the G or dGTP nucleotide can be labeled with a spectrally distinct label described herein, and one of the other three nucleotide conjugates can be unlabeled.

[0083] Although the kits are exemplified herein in terms of configurations having different nucleotides labeled with different dye compounds, it will be understood that the kits may include two, three, four or more different nucleotides having the same dye compound.

[0084] In addition to labeled nucleotides, 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. One buffer composition may include an antioxidant, such as ascorbic acid or sodium ascorbate, which can be used to protect the dye compound from photodamage during detection. An additional buffer composition may include a reagent that can be used to cleave the 3' 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. Again, one or more of the components identified in the methods described herein can be included in the kits of the present disclosure.

[0085] In some embodiments of the kits described herein, the fluorescent dye compound may be covalently attached to the nucleotide via the nucleotide base. In some such embodiments, the labeled nucleotide may have a dye 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 may be 7-deazaadenine, and the dye is attached to the 7-deazaadenine at the C7 position, optionally through a linker. The nucleobase may be 7-deazaguanine, and the dye is attached to the 7-deazaguanine at the C7 position, optionally through a linker. The nucleobase may be cytosine, and the dye is attached to the cytosine at the C5 position, optionally through a linker. As another example, the nucleobase may be thymine or uracil, and the dye is 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.

[0086] 3' hydroxyl blocking group The labeled nucleotide conjugate used in the incorporation mixture can also have a blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide. The blocking group may be attached to any position on the ribose or deoxyribose sugar. In certain embodiments, the blocking group is at the 3'OH position of the ribose or 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 moiety. In some embodiments, the 3' blocking group is azidomethyl, forming 3'-OCHN with the 3' carbon of the ribose or deoxyribose.

[0087] In some other embodiments, the 3' blocking group and the 3' oxygen atom are covalently attached to the 3' carbon of the ribose or deoxyribose.

[0088] [ka] to form an acetal group of the formula In the formula, each R 1a and R 1b are independently H, C1-C6 alkyl, C 1~ C6 haloalkyl, C 1~ C6 alkoxy, C 1~ 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 F is 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.

[0089] Additional 3'OH blocking groups are disclosed in U.S. Publication No. 2020 / 0216891A1, which is incorporated by reference in its entirety. Acetal Blocking Groups

[0090] [ka] (AOM),

[0091] [ka] are non-limiting examples of ribose and deoxyribose, respectively, which are covalently attached to the 3' carbon of ribose or deoxyribose.

[0092] Deprotection of the 3'-OH blocking group In some embodiments, the azidomethyl 3' hydroxy 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(hydroxypropyl)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

[0093] [ka] In the case of these blocking groups containing 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 phosphine ligands (e.g., THP or THMP).

[0094] 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 (e.g., tris(3,3',3''phosphinidinetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate) is used. 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, and the like. 2,Examples of suitable palladium sources include (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), and Pd(TFA)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4. 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 a 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.

[0095] 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.

[0096] 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 lock 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.

[0097] 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.

[0098] Particular linkers include those disclosed in PCT Publication No. WO2004 / 018493 (herein incorporated by reference), such as those containing a moiety of the formula:

[0099] [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 C1-C 10A linker is a substituted or unsubstituted alkyl group, and the * indicates where the moiety is attached to the remainder of the nucleotide or nucleoside. In some embodiments, the linker connects the base of the nucleotide to a label, such as, for example, a dye compound described herein.

[0100] 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:

[0101] [ka] (where * indicates where the moiety is attached to the remainder of the nucleotide or nucleoside.) The linker moiety shown herein may include the entire or partial linker structure between the nucleotide / nucleoside and the label. The linker moiety shown herein may include the entire or partial linker structure between the nucleotide / nucleoside and the label.

[0102] Additional examples of linkers include those of the formula:

[0103] [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

[0104] [ka] ("AOL" linker moiety), where Z is -O-allyl. Further examples of cleavable linkers are disclosed in U.S. Application No. 17 / 353,512, which is incorporated herein by reference in its entirety.

[0105] In certain embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be modified, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base via other linkages known in the art. Exemplary linkers and their properties are described in PCT Publication No. WO 2007 / 020457 (incorporated herein by reference). Such linker designs, particularly their increased length, can allow for improved brightness of fluorophores attached to the guanine base of guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the dye is intended for use in any analytical method requiring the detection of a fluorescent dye label attached to a guanine-containing nucleotide, the linker may be a linker of the formula -((CH2)2O) n It is advantageous to include a spacer group of the formula - (where n is an integer from 2 to 50).

[0106] 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. As noted above, a linker group can be used to covalently attach the dye to the nucleoside or nucleotide.

[0107] The dye-labeled nucleotides described herein have the formula:

[0108] [ka]

[0109] where 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

[0110] [ka] or

[0111] [ka] or optionally substituted derivatives and analogs thereof. In some further embodiments, the labeled nucleobase has the structure

[0112] [ka] Includes.

[0113] 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.

[0114] In yet another alternative embodiment, there is no blocking group on the 3' carbon of the pentose sugar, and the dye attached to the base (or the dye and linker structure) may be of sufficient size or structure to act, for example, 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 may be due to a combination of size, charge, and structure.

[0115] 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.

[0116] The use of blocking groups allows for control of polymerization, such as by terminating elongation when a labeled nucleotide is incorporated. If the blocking effect is reversible, for example, but not limited to, by changing the chemical conditions or removing the chemical block, elongation can be stopped at a specific point and then allowed to continue.

[0117] In certain embodiments, both the linker (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.

[0118] 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.

[0119] Non-limiting labeled nucleotide conjugates described herein include:

[0120] [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.

[0121] In some embodiments, a non-limiting exemplary fully functionalized nucleotide conjugate comprising a cleavable linker and a fluorescent moiety is as follows:

[0122] [ka]

[0123] [ka]

[0124] [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.

[0125] [ka] refers to the point of attachment of the dye with the cleavable linker as a result of the reaction between the amino group of the linker moiety and the carboxyl group of the dye. In any embodiment of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate. [Example]

[0126] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims.

[0127] Example 1. Sequencing experiments on an Illumina iSeq™ 100 instrument In this example, the dual-channel excitation and single-channel detection sequencing method described herein was used on an Illumina iSeq™ 100 instrument, configured to acquire the first image with green excitation light (approximately 520 nm) and the second image with blue excitation light (approximately 450 nm). The sequencing procedure was modified to perform 1×300 standard SBS cycles (incorporation, followed by imaging, followed by cleavage). The incorporation mixture contained the following four ffNs: ffA labeled with chromenoquinoline dye A, excitable with both green light at 520 nm and blue light at 450 nm; ffC labeled with coumarin dye B, excitable with blue light at 450 nm; ffT labeled with polymethine dye C, excitable with green light; and unlabeled ffG (dark dGTP), in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO, and DNA polymerase. The structures of ffC, ffA, ffT, and ffG are shown below.

[0128] [ka]

[0129] Figure 4 shows the scatter plot obtained for the embedded mix at cycle 26. The scatter plot showed good separation of the clouds and the usefulness of the ffN set.

[0130] The same ffN set was then used in a 2 × 300 cycle run on an Illumina iSeq™ 100 instrument. The instrument was configured to capture the first image with green excitation light and the second image with blue excitation light, and the run was modified to perform 2 × 300 cycles of standard SBS cycling (acquisition, followed by imaging, followed by cleavage). Sequencing metrics are summarized in Table 1 below. Phasing and prephasing values ​​were observed to be improved compared to those obtained with standard iSeq™ 100 reagents. Additionally, with this ffN set, the sequencing run used only half the volumes of wash and scan buffer compared to the standard iSeq™ 100 run. These features enabled the extended read length of 2 × 300 cycles to be achieved with low error rates and high Q30% values, and a total run time of approximately 28 hours.

[0131] [Table 4]

[0132] Example 2. Sequencing experiments on an Illumina iSeq™ 100 instrument In this example, the dual-channel excitation and single-channel detection sequencing method described herein was used on an Illumina iSeq™ 100 instrument, configured to acquire the first image with green excitation light (approximately 520 nm) and the second image with blue excitation light (approximately 450 nm). Using standard sequencing procedures, two 300 SBS cycles (incorporation, followed by imaging, followed by cleavage) were performed. The incorporation mixture contained the following four ffNs: ffA labeled with chromenoquinoline dye A, excitable with both green light at 520 nm and blue light at 450 nm; ffC labeled with coumarin dye B, excitable with blue light at 450 nm; ffT labeled with polymethine dye C, excitable with green light; and unlabeled ffG (dark dGTP), in 50 mM glycine buffer, pH 9.8, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO, and DNA polymerase. The cleavage solution used in these experiments contained 100 mM diethylethanolamine buffer pH 9.5, 100 mM tris(hydroxypropyl)phosphine, 10 mM [AllylPdCl], 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20. The structures of ffC, ffA, ffT, and ffG are shown below.

[0133] [ka]

[0134] Table 2 shows the cycle time, phasing, prephasing, PhiX error rate, and Q30% metrics for 2 × 150-cycle runs and 2 × 300-cycle runs on an Illumina iSeq™ 100 instrument using the ffN set described in this example. It was observed that using this ffN set, the cycle time for the 2 × 150 run could be reduced to 60 seconds, approximately half the cycle time of the standard iSeq™ cartridge and run (approximately 141 seconds), while maintaining good sequencing metrics. In addition, sequencing runs using this ffN set use only half the volumes of wash and scan buffers compared to standard iSeq™ 100 runs (1-Ex chemistry). These features enabled extended read lengths for 2 × 300 cycles to be achieved with low error rates and high Q30% values ​​using cycle times equivalent to those used in standard iSeq™ 100 runs using standard iSeq™ cartridges. The total time for a full 2 ​​x 300 cycle run with this ffN set on the iSeq™ 100 was approximately 28 hours, a 2-fold reduction compared to the 2 x 300 cycle runs (approximately 56 hours) performed on an Illumina MiSeq® using standard reagents and procedures.

[0135] [Table 5]

[0136] Table 3 shows cycle time, phasing, prephasing, PhiX error rate, and Q30% metrics for 2 x 150 cycle runs on an Illumina iSeq™ 100 instrument using the ffN set described in this example, with the flow cell pixel size reduced from 1.75 μm to 1 μm. The change in flow cell pixel size results in a smaller pitch and shorter light pipe for better signal, but also carries potential concerns of lower signal, higher background noise, and higher crosstalk. Sequencing metrics were observed to be of very good quality overall with the 1 μm flow cell, comparable to those obtained from the 1.75 μm flow cell.

[0137] [Table 6]

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 four different types of nucleotide conjugates, wherein a first type of nucleotide conjugate comprises a first label, a second type of nucleotide conjugate comprises a second label, and a third type of nucleotide conjugate comprises a third label, each of the first label, the second label, and the third label being spectrally distinct from one another, and wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating a nucleotide conjugate 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 to collect a first luminescence signal from the extended primer polynucleotide; (d) performing a second imaging event using a second excitation light source to collect a second luminescent signal from the extended primer polynucleotide; the first excitation light source and the second excitation light source have different wavelengths, and the first emission signal and the second emission signal are collected in a single emission detection channel; A method, wherein a fourth type of nucleotide conjugate is unlabeled or labeled with a fluorescent moiety that does not emit a detectable signal from either the first imaging event or the second imaging event.

2. i. incorporation of the first type of nucleotide conjugate is determined by a signal state in the first imaging event and a dark state in the second imaging event; and / or ii. the incorporation of the second type of nucleotide conjugate is determined by a dark state in the first imaging event and a signal state in the second imaging event; and / or iii. the incorporation of the third type of nucleotide conjugate is determined by a signal state in both the first imaging event and the second imaging event; and / or iv. The method of claim 1, wherein the incorporation of the nucleotide conjugate of the fourth type is determined by dark conditions in both the first imaging event and the second imaging event.

3. 4. The method of any one of claims 1 to 3, wherein the four types of nucleotide conjugates comprise dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogues thereof, and optionally each of the four types of nucleotide conjugates in the mixture has a 3' hydroxyl blocking group.

4. 4. The method of claim 1, further comprising: (e) removing the 3' hydroxyl blocking group and label from the incorporated nucleotide conjugate after the second imaging event and before a next sequencing cycle.

5. repeating steps (a)-(e) for a number of cycles; 5. The method of claim 4, further comprising determining a sequence of the target polynucleotide based on the sequentially incorporated nucleotide conjugates.

6. 6. The method of any one of claims 1 to 5, wherein the first excitation light source has a wavelength of about 400 nm to about 480 nm, and optionally the first excitation light source has a wavelength of about 450 nm to about 460 nm.

7. 7. The method of any one of claims 1 to 6, wherein the second excitation light source has a wavelength of from about 490 nm to about 550 nm, and optionally the second excitation light source has a wavelength of from about 510 nm to about 530 nm.

8. 6. The method of any one of claims 1 to 5, wherein the first excitation light source has a wavelength of from about 490 nm to about 550 nm, and optionally the first excitation light source has a wavelength of from about 510 nm to about 530 nm.

9. 9. The method of any one of claims 1 to 5, wherein the second excitation light source has a wavelength of about 400 nm to about 480 nm, and optionally the second excitation light source has a wavelength of about 450 nm to about 460 nm.

10. The method of any one of claims 1 to 9, wherein the single emission detection channel has a detection spectral range above about 560 nm.

11. 11. The method of any one of claims 1 to 10, wherein the method does not include chemical modification of any nucleotide conjugates in the mixture between the first imaging event and the second imaging event.

12. The method of any one of claims 1 to 11, wherein the target polynucleotide is immobilized on a solid support, optionally the solid support comprising a plurality of immobilized target polynucleotides.

13. 13. The method of claim 12, wherein the solid support comprises a patterned flow cell, optionally the patterned flow cell comprising a plurality of nanowells, each nanowell comprising an immobilized target polynucleotide.

14. A kit for sequencing applications comprising: a first type of nucleotide conjugate comprising a first label; a second type of nucleotide conjugate comprising a second label; and a third type of nucleotide conjugate comprising a third label; each of the first label, the second label, and the third label are spectrally distinct from one another, the first label and the third label being excitable using a first light source wavelength, and the second label and the third label being excitable using a second light source wavelength different from the first light source wavelength; The kit, wherein the first label, the second label, and the third label each have an emission spectrum that is detectable in a single detection channel.

15. 15. The kit of claim 14, further comprising a fourth type of nucleotide, said fourth type of nucleotide being unlabeled (dark).

16. 16. The kit of claim 14 or 15, wherein the first light source has a wavelength of about 400 nm to about 480 nm, and optionally the first light source has a wavelength of about 450 nm to about 460 nm.

17. 17. The kit of any one of claims 14 to 16, wherein the second light source has a wavelength of about 490 nm to about 550 nm, and optionally the second light source has a wavelength of about 510 nm to about 530 nm.

18. The kit of any one of claims 14 to 17, wherein the single emission detection channel has a detection spectral range above about 560 nm.

19. The kit of any one of claims 14 to 18, further comprising a DNA polymerase and one or more buffer compositions.