Excitation and detection methods for analyzing biomolecules

Abstract

Methods for analyzing molecules are described, including method of analysis using luminophores with spectral overlap. The methods may comprise, for example, contacting the molecules with binders, wherein the binders are labeled with luminophores that have different but overlapping spectrums of emission and / or excitation wavelengths, and determining intensity characteristics from the luminophore's intensities to determine the binders and the corresponding bound targets in the molecules. In addition, the methods described herein are compatible with next-generation sequencing methods, such as sequencing-by-synthesis for the analysis of different nucleic acid analytes, including one or more nucleic acid templates to be sequenced.

Description

Docket No.: ESBI00007-1WOEXCITATION AND DETECTION METHODS FOR ANALYZING BIOMOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 655,440 filed June 3, 2024, which application is incorporated herein by reference herein as if set forth in full.FIELD OF THE INVENTION

[0002] The present disclosure relates in some aspects to methods and compositions for analyzing molecules, such as methods for detecting intensities of luminophores comprising overlapping spectra, including the use of such methods and compositions for nucleic acid sequencing.BACKGROUND

[0003] Luminescent labels are frequently used to detect and analyze molecules in biological and clinical contexts. Luminescence allows for a higher throughput in the detection and quantification of molecules. Types of luminescent properties include fluorescence, chemiluminescence, and phosphorescence. While luminescence intensity can provide information on a luminophore’s identity, it is challenging to distinguish luminophores with similar spectrums using an intensity alone. Distinct spectral properties are required to facilitate the identification of multiple luminophores. Additionally, detecting luminophores with distinct spectral properties may require a more complex optical system for excitation and emission detection. The ability to simplify optical systems to distinguish a larger number of luminophores is of great interest, as it can increase the number of luminophores detected by an optical system and facilitate the production of a more cost-optimized optical system. For example, nextgeneration sequencing is a widely used method that uses fluorescently labeled nucleotides to detect and distinguish nucleotide bases. Simplifying the detection of fluorescent signals in nextgeneration sequencing can provide several advantages. As such, methods and compositions improving the detection of luminophores with overlapping spectral properties are needed. The present disclosure addresses these needs.Docket No.: ESBI00007-1WGBRIEF SUMMARY

[0004] Disclosed herein are methods for analyzing molecules by detecting intensities of luminophores comprising overlapping spectrums based on using intensity characteristics to determine the identities of the luminophores and the corresponding molecules. Luminescence intensity provides information on a luminophore’s identity, but distinct spectral properties are needed to distinguish multiple luminophores in a group. It is challenging to distinguish luminophores with similar spectrums using an intensity alone. Detecting luminophores with distinct spectral properties requires a more complex optical system for excitation and emission detection. The ability to simplify and consolidate optical systems to distinguish a larger number of luminophores has several advantages. The components of an optical system can be costly. Thus, by reducing the number of detectors and / or excitation sources, an optical system can be more inexpensive than an analogous system used to detect an equivalent number of luminophores with distinct spectral properties. Additionally, in some aspects, methods detecting luminophores with overlapping spectrums can save time, as the measured intensities will provide information on all of the luminophores of interest, negating the need to perform measurements for each luminophore. Furthermore, in some aspects, relevant luminophores can be detected at a single wavelength, thus decreasing the demand on time and computing power required for detection. The methods described herein include emission and excitation detection methods that leverage intensities at multiple points along a luminophore’s spectra to obtain intensity characteristics and allow luminophores with overlapping spectrums to be identified with higher confidence.

[0005] In some embodiments, fluorophores with overlapping and similar emission spectra are used, and to detect emission intensities multiple detectors are used, with each detecting emission at a different wavelength. In some embodiments, fluorophores with overlapping and similar excitation spectra are used, and multiple light sources are used, with each light source providing light of a different excitation wavelength. In some embodiments, nucleotides of four different bases are used for nucleic acid sequencing, and the nucleotides of each different bases are labeled with a different fluorophore, wherein the four fluorophores have different but overlapping spectrums of emission wavelengths. In some embodiments, nucleotides of four different bases are used for nucleic acid sequencing, and the nucleotides of each different base are labeled with a different fluorophore, wherein the four fluorophores have different but overlapping spectrums of excitation wavelengths. In some embodiments, a base-line corrected intensity characteristic between emission intensities and / or between excitation intensities at twoDocket No.: ESBIO0007-1WO different wavelengths is detected for each of the four different fluorophores. In some embodiments, the base-line corrected intensity characteristic is an intensity ratio between emission intensities and / or between excitation intensities at two different wavelengths is detected for each of the four different fluorophores. In some embodiments, emission intensities and / or excitation intensities at three or more different wavelengths are detected for each of the four different fluorophores, and intensity values detected at one of the three or more different wavelengths are used as the base-line intensity values for generating four base-line corrected intensity characteristics, each of which is characteristic of one of the four different fluorophores and can be used to identify the fluorophores and the identities of their corresponding nucleotide base.

[0006] In some aspects, provided herein is a method for analyzing one or more molecules, comprising contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of emission wavelengths and / or different but overlapping spectrums of excitation wavelengths. In some aspects, the method comprises detecting: (i) a first intensity characteristic of emission intensities and / or of excitation intensities of the first luminophore at two or more different wavelengths, and (ii) a second intensity characteristic of emission intensities and / or of excitation intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.

[0007] In some aspects, provided herein is a method for analyzing one or more molecules, comprising (a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of emission wavelengths. In some aspects, the method comprises (b) detecting: (i) a first intensity characteristic of emission intensities of the first luminophore at two or more different wavelengths, and (ii) a second intensity characteristic of emission intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristicDocket No.: ESBI00007-1WG and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.

[0008] In some aspects, provided herein is a method for analyzing one or more molecules, comprising (a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of excitation wavelengths. In some aspects, the method comprises (b) detecting: (i) a first intensity characteristic of excitation intensities of the first luminophore at two or more different wavelengths, and (ii) a second intensity characteristic of excitation intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.

[0009] In any of the preceding embodiments, a first intensity characteristic can be about 1.25- fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold or more of the second intensity characteristic. In any of the preceding embodiments, a second intensity characteristic can be about 1.25-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold or more of the first intensity characteristic.

[0010] In any of the preceding embodiments, detecting in (b) can comprise (i) detecting emission intensities or excitation intensities of the first luminophore at three or more different wavelengths; and (ii) detecting emission intensities or excitation intensities of the second luminophore at the three or more different wavelengths. In any of the preceding embodiments, detecting in (b) can comprise (i) detecting first intensity characteristics of emission intensities or of excitation intensities of the first luminophore at three or more different wavelengths; and (ii) detecting second intensity characteristics of emission intensities or of excitation intensities of the second luminophore at the three or more different wavelengths.

[0011] In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be first baseline-corrected intensity ratio(s) and second baseline-corrected intensity ratio(s), respectively. In any of the preceding embodiments, the first baseline-corrected intensity ratio R 1 can be calculated using measured intensities of the firstDocket No.: ESBI00007-1WG luminophore at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, the measured intensities at XI, X2, and X3 are II, 12 and 13, respectively, and 7? 1 = (12 - / l) / (73 - II). In any of the preceding embodiments, the second baseline-corrected intensity ratio R2 can be calculated using measured intensities of the second luminophore at XI, X2, and X3, the measured intensities at XI, X2, and X3 are il, 12 and 13, respectively, and R2 = (12 - il) / (i3 - i 1 ).

[0012] In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be determined using polynomial regression or fitting. In any of the preceding embodiments, the polynomial regression or fitting can comprise using formula y = a + bx + ex2, wherein x is a wavelength and y is a measured intensity of the first or second luminophore at the wavelength x. In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be b and c which are determined using the polynomial regression or fitting. In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be b / c or c / b, wherein b and c are determined using the polynomial regression or fitting.

[0013] In any of the preceding embodiments, the one or more molecules can comprise a polynucleotide, a polypeptide, a carbohydrate, a lipid, a small molecule, or any combination thereof. In any of the preceding embodiments, the one or more molecules can be present as a separate molecule or in complex with one or more other molecules.

[0014] In any of the preceding embodiments, each of the one or more molecules can be present on an artificial substrate, in a biological sample, or in a solution, and wherein the first binder and the second binder are contacted with the artificial substrate, the biological sample, or the solution. In any of the preceding embodiments, the artificial substrate can be a patterned flow cell or a nonpatterned flow cell. In any of the preceding embodiments, the biological sample is a cell or a tissue sample. In any of the preceding embodiments, each of the one or more molecules can be present in and / or on a cell, an intracellular organelle, an extracellular vesicle, a virus, a viral-like particle (VLP), a lipid nanoparticle (LNP), a protein complex, a nucleic acid complex, or a protein-nucleic acid complex.

[0015] In any of the preceding embodiments, the one or more molecules can comprise a nucleic acid template to be sequenced. In some embodiments, the nucleic acid template can be a DNA or an RNA. In any of the preceding embodiments, the nucleic acid template can be present in aDocket No.: ESBIO0007-1WO clonal cluster of nucleic acid molecules each comprising a copy of a sequence to be sequenced; or the nucleic acid template is a rolling circle amplification product (RCP). In any of the preceding embodiments, the nucleic acid template can be a single molecule located on an artificial substrate at an optically resolvable distance from other nucleic acid templates on the artificial substrate.

[0016] In any of the preceding embodiments, the first binder and the second binder can be nucleotides that base pair with a nucleic acid residue in the nucleic acid template based on sequence complementarity, and wherein the different targets are nucleic acid residues of different bases. In any of the preceding embodiments, the nucleotides can comprise one or more reversibly terminated nucleotides, one or more irreversibly terminated nucleotides, and / or one or more nucleotides that are not terminated. In any of the preceding embodiments, the nucleotides can be independently incorporated or not incorporated into a priming strand that hybridizes to the nucleic acid template. In any of the preceding embodiments, each of the nucleotides can be an A nucleotide, a T / U nucleotide, a C nucleotide, or a G nucleotide.

[0017] In any of the preceding embodiments, the first binder and the second binder can be complexes each comprising: (i) a nucleotide, and (ii) the first luminophore and the second luminophore, respectively, wherein the nucleotides in the complexes base pair with a nucleic acid residue in the nucleic acid template based on sequence complementarity, and wherein the different targets are nucleic acid residues of different bases. In any of the preceding embodiments, each of the complexes can comprise: (i) a plurality of nucleotide moieties of the same base, and (ii) one or more copies of the first luminophore or the second luminophore.

[0018] In any of the preceding embodiments, the one or more molecules can comprise different nucleic acid analytes. In any of the preceding embodiments, the different nucleic acid analytes can comprise a genomic DNA, an RNA, a cDNA, a nucleic acid probe, a reporter oligonucleotide conjugated to a binding moiety that binds to a non-nucleic acid analyte, a rolling circle amplification product (RCP), or any combination thereof. In any of the preceding embodiments, the first binder and the second binder can be nucleic acid probes, and the different targets are the different nucleic acid analytes. In any of the preceding embodiments, the nucleic acid probes can hybridize to the different nucleic acid analytes. In any of the preceding embodiments, the one or more molecules can comprise different protein analytes. In any of the preceding embodiments, the first binder and the second binder can be antibodies or aptamers and the different targets are the different protein analytes.Docket No.: ESBIO0007-1WO

[0019] In any of the preceding embodiments, the first luminophore and the second luminophore can be fluorophores.

[0020] In any of the preceding embodiments, a single light source can be used to excite the different luminophores, and a different detector is used to detect the emission intensities at each different emission wavelength. In any of the preceding embodiments, a separate light source can be used to excite each different luminophore at each different excitation wavelength, and a single detector can be used to detect the excitation intensities of the different luminophores. In any of the preceding embodiments, the emission intensities at the different emission wavelengths can be simultaneously detected by the different detectors. In any of the preceding embodiments, the excitation intensities at the different excitation wavelengths can be sequentially detected by the single detector. In any of the preceding embodiments, the single light source or each of the separate light sources can be independently a light emitting diode (LED).

[0021] In some aspects, provided herein is a method for analyzing a nucleic acid template comprising (a) contacting the nucleic acid template with: (i) a first nucleotide which is of a first base and labeled with a first fluorophore, and (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, wherein the first base and the second base are different bases, and wherein the first fluorophore and the second fluorophore have different but overlapping spectrums of emission wavelengths and / or different but overlapping spectrums of excitation wavelengths. In some aspects, the method comprises (b) allowing nucleotide binding to the nucleic acid template, wherein the first nucleotide or the second nucleotide binds to a complementary nucleic acid residue in the nucleic acid template, optionally wherein the bound nucleotide is incorporated into a sequencing strand hybridized to the nucleic acid template. In some aspects, the method comprises, (c) imaging the bound and optionally incorporate nucleotide, wherein (i) or (ii) is determined based on the imaging: (i) a first intensity characteristic of emission intensities and / or of excitation intensities of the first fluorophore at two or more different wavelengths, (ii) a second intensity characteristic of emission intensities and / or of excitation intensities of the second fluorophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first nucleotide and the second nucleotide, respectively, thereby identifying the bound and optionally incorporate nucleotide and the complementary nucleic acid residue in the nucleic acid template.Docket No.: ESBI00007-1WG

[0022] In any of the preceding embodiments, the imaging in (c) can comprise imaging the bound and optionally incorporated nucleotide at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, and at each of XI, X2, and X3, measuring a first, second, and third fluorescence intensity, respectively, wherein a baseline- corrected intensity ratio R is determined according to R = (value of the second fluorescence intensity - value of the first fluorescence intensity ) / (value of the third fluorescence intensity - value of the first fluorescence intensity), and wherein the baseline-corrected intensity ratio R is the first intensity characteristic or the second intensity characteristic, thereby identifying the bound and optionally incorporate nucleotide as the first nucleotide or the second nucleotide. In any of the preceding embodiments, the baseline-corrected intensity ratio R can be a first baseline-corrected intensity ratio R 1 corresponding to the first nucleotide, wherein the measured fluorescence intensities of the first nucleotide at XI, X2, and X3 are 71, 72 and 73, respectively, and 7? 1 = (72 -71) / (73 - 71). In any of the preceding embodiments, the baseline-corrected intensity ratio 7? can be a second baseline-corrected intensity ratio 7?2 corresponding to the second nucleotide, wherein the measured fluorescence intensities of the first nucleotide at XI, X2, and X3 are i , i2 and z‘3 , respectively, and 7?2 = (z’2 - z 1 ) (z‘3 - zl ).

[0023] In any of the preceding embodiments, the imaging in (c) can comprise imaging the bound and optionally incorporated nucleotide at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, and at each of XI, X2, and X3, measuring a first, second, and third fluorescence intensity, respectively, subjecting the measured fluorescence intensities and XI, X2, and X3 to polynomial regression or fitting, thereby generating formula y = a + bx + ex2, wherein x is a wavelength andy is a fluorescence intensity at the wavelength x, and wherein (i) b and c and / or (ii) b / c or c / b is the first intensity characteristic or the second intensity characteristic, thereby identifying the bound and optionally incorporate nucleotide as the first nucleotide or the second nucleotide.

[0024] In any of the preceding embodiments, the contacting in (a) can comprise contacting a plurality of different nucleic acid templates with: (i) a first nucleotide which is of a first base and labeled with a first fluorophore, (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, (iii) a third nucleotide which is of a third base and labeled with a third fluorophore, and (iv) a fourth nucleotide which is of a fourth base and labeled with a fourth fluorophore, wherein the plurality of different nucleic acid templates comprise the nucleic acid template, and the first base, the second base, the third base, and the fourth base are different bases. In any of the preceding embodiments, the first fluorophore, the second fluorophore, theDocket No.: ESBI00007-1WG third fluorophore, and the fourth fluorophore can have different but overlapping spectrums of emission wavelengths. In any of the preceding embodiments, the imaging in (c) can comprise using a single light source to excite the fluorophores and using multiple detectors each for detecting an emission intensity at a different wavelength. In any of the preceding embodiments, the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore can have different but overlapping spectrums of excitation wavelengths. In any of the preceding embodiments, the imaging in (c) can comprise using a single detector and using multiple light sources each for exciting the fluorophores at a different wavelength.

[0025] In some aspects, provided herein is a kit or composition, comprising (i) a first nucleotide which is of a first base and labeled with a first fluorophore, (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, (iii) a third nucleotide which is of a third base and labeled with a third fluorophore, and (iv) a fourth nucleotide which is of a fourth base and labeled with a fourth fluorophore, wherein the first base, the second base, the third base, and the fourth base are different bases, and the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore have different but overlapping emission spectrums and / or different but overlapping excitation spectrums.INCORPORATION BY REFERENCE

[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Various aspects of the disclosed methods are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed methods will be obtained by reference to the following detailed description of illustrative embodiments and the accompanying drawings, of which:

[0028] FIG. 1 provides an exemplary flowchart for nucleic acid sequencing, according to some embodiments described herein. The nucleotides labeled with fluorophores consist of fluorophores with different but overlapping emission spectrums.Docket No.: ESBIG0007-1WO

[0029] FIG. 2 provides an exemplary flowchart for nucleic acid sequencing, according to some embodiments described herein. The nucleotides labeled with fluorophores consist of fluorophores with different but overlapping excitation spectrums.

[0030] FIG. 3A provides an exemplary diagram of emission intensities measured at three wavelengths for four fluorophores. Each of the four fluorophores corresponds to one of the four nucleotide bases, i.e., A, T / U, C, and G.

[0031] FIG. 3B provides an exemplary optical system diagram for the use of detecting fluorophores with different but overlapping emission spectrums. This exemplary system relies on one light source to excite all of the four fluorophores.

[0032] FIG. 4A provides an exemplary diagram of excitation intensities measured at three wavelengths for four fluorophores. Each of the four fluorophores corresponds to one of the four nucleotide bases, i.e., A, T / U, C, and G.

[0033] FIG. 4B provides an exemplary optical system diagram for the use of detecting fluorophores with different but overlapping excitation spectrums. This exemplary system relies on one detector to detect all of the four fluorophores.DETAILED DESCRIPTION

[0034] Methods for analyzing molecules are described herein. That is, different molecules are contacted with binders labeled with luminophores. The binders bind directly or indirectly to different targets comprised of one or more molecules. The luminophores that label the binders are different but have overlapping spectrums of emission wavelengths or overlapping spectrums of excitation wavelengths. The different luminophores used to label the binders can also have both overlapping spectrums of excitation wavelengths and overlapping spectrums of emission wavelengths. Intensities at two or more wavelengths are detected for a luminophore. The intensities determined can correspond to the luminophore ’s emission intensities and / or excitation intensities. Since the luminophores have overlapping spectrums, the two or more wavelengths used to determine the intensities will provide information regarding all of the luminophores. The determined intensities are then used to determine an intensity characteristic for each luminophore of interest. The intensity characteristic will correspond to a specific luminophore, which corresponds to a specific binder, thereby identifying the different targets in one or more molecules. Furthermore, intensity ratios determined between emission intensities orDocket No.: ESBI00007-1WG between excitation intensities can incorporate a baseline correction. A measured intensity can serve as a baseline intensity and the intensities used to determine the ratios can incorporate this baseline intensity, thus providing a ratio that accounts for background luminescence and technical variations. The intensity ratio or ratios obtained for each luminophore provides a way to distinguish luminophores with overlapping spectrums.

[0035] Multicolor detection of luminophores can be challenging. Luminescence intensity provides information on a luminophore ’s identity, but typically, to distinguish multiple luminophores in a group, distinct spectral properties are needed. The availability of spectrally separable luminophores can limit the number of potential luminophores that can be used when using intensity values alone to distinguish luminophores. This is especially relevant since luminophores can have spectral profiles several hundred nanometers wide. Additionally, optical systems can be constrained by the number and specificity of available excitation wavelengths, and the number of detectors can limit the detectable emission wavelengths. Further, various considerations have to be taken for luminophore selection when relying on spectrally distinguishable luminophores. Preferred luminophores have narrow emission peaks and / or larger Stokes shifts (i.e., the separation between excitation and emission maxima). A balance of emission intensities is also required, as luminophores with significant differences in intensities can overwhelm the less intense luminophores. Optimization of image acquisition parameters is also needed to maximize spectral separation. Parameters to optimize include the choice of lasers, excitation lines, filter combinations, and detection bandwidths.

[0036] Thus, using intensity characteristics to detect luminophores with overlapping spectrums can simplify multicolor luminophore detection. Users can select from a greater number of luminophores for detection by using luminophores with spectral overlap. A user need only decide which luminophore will be assigned to represent a molecule, and provided that a unique profile can be constructed from the characteristics for each luminophore, the user can conduct and interpret their detection of luminophores and further identification of molecules. Additionally, a consolidated optical system can be used to distinguish a larger number of luminophores, as the same excitation and / or emission settings can be used to detect multiple luminophores. Existing optical platforms can readily incorporate the methods described herein.

[0037] In addition, the methods described herein are compatible with next-generation sequencing methods, such as the analysis of different nucleic acid analytes, including one or more nucleic acid templates to be sequenced. For example, a binder can correspond to aDocket No.: ESBI00007-1WG nucleotide that base pairs with a nucleic acid residue in the nucleic acid template based on sequence complementarity. The different targets of the binders are different nucleotide bases. Binders can comprise an A nucleotide, a T / U nucleotide, a C nucleotide, or a G nucleotide. The binders can be labeled with fluorophores that have spectral overlap, but not all binders need to be labeled. This method is applicable to nucleotides that are reversibly terminated nucleotides, irreversibly terminated nucleotides, and nucleotides that are not terminated. The nucleotides can also be incorporated or not incorporated into a priming strand that hybridizes to the nucleic acid template. The described methods are readily compatible with most existing sequencing platforms and schemes.

[0038] Methods for analyzing a molecule are described. In some aspects, a method for molecule analysis can comprise: (a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of emission wavelengths and / or different but overlapping spectrums of excitation wavelengths; and (b) determining: (i) a first intensity characteristic between emission intensities and / or between excitation intensities of the first luminophore at two different wavelengths, and (ii) a second intensity characteristic between emission intensities and / or between excitation intensities of the second luminophore at the two different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.I. Definitions

[0039] Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.

[0040] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0041] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees ofDocket No.: ESBI00007-1WG error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0042] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0043] As used herein, the terms "comprising" (and any form or variant of comprising, such as "comprise" and "comprises"), "having" (and any form or variant of having, such as "have" and "has"), "including" (and any form or variant of including, such as "includes" and "include"), or "containing" (and any form or variant of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, un-recited additives, components, integers, elements, or method steps.

[0044] As used herein, ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0045] A binder refers to a molecule that interacts with another molecule and results in a stable association in which the molecules are then in close proximity to each other. It may involveDocket No.: ESBI00007-1WG chemical bonding, but it is not required. A binder can be a nucleotide, a nucleic acid probe, an antibody, or an aptamer. A binder can bind to targets individually or in complex with other binders. The binding of binders to a target can occur in biological complexes and in artificial substrates.

[0046] A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art.

[0047] A “probe” or a “target,” when used in reference to a molecule or a nucleic acid or sequence of a nucleic acids, is intended as a semantic identifier for the molecule or the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the molecule or the nucleic acid or sequence beyond what is expressly indicated.

[0048] The terms “oligonucleotide” and “polynucleotide” are used interchangeably to refer to a single-stranded multimer of nucleotides from about 2 to about 500 nucleotides in length. Oligonucleotides can be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method. Oligonucleotides can include ribonucleotide monomers (e.g., can be oligoribonucleotides) and / or deoxyribonucleotide monomers (e.g., oligodeoxyribonucleotides). In some examples, oligonucleotides can include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). An oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400-500 nucleotides in length, for example. Oligonucleotides can include one or more functional moieties that are attached (e.g., covalently or non-covalently) to the multimer structure. For example, an oligonucleotide can include one or more detectable labels (e.g., a radioisotope or fluorophore).

[0049] The terms “detectable label,” “optical label,” and “label” are used interchangeably herein to refer to a directly or indirectly detectable moiety that is coupled to or may be coupled toDocket No.: ESBI00007-1WG another moiety, for example, a binder, a nucleotide, or a nucleotide analog. The detectable label can be directly detectable by itself (e.g., fluorescent labels). The label can emit a signal or alter a signal delivered to the label so that the presence or absence of the label can be detected. In some cases, coupling may be via a linker, which may be cleavable, such as photo-cleavable (e.g., cleavable under ultra-violet light), chemically-cleavable (e.g., via a reducing agent, such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP)) or enzymatically cleavable (e.g., via an esterase, lipase, peptidase, or protease).

[0050] In some embodiments, a detectable label is or includes a luminophore. Exemplary luminophores include, but are not limited to, fluorescein derivatives; Rhodamine B derivatives; BODIPY derivatives; Pdots based on polyfluorene including polyfluorene (PFO), poly(fluorene benzothiadiazole) (PFBT), polyfluorene -6-poly(thiophene benzothiadiazole) (PFTBT5); quantum dots.

[0051] In some embodiments, a detectable label is or includes a fluorophore. Exemplary fluorophores include, but are not limited to, fluorescent nanocrystals; d-Rhodamine acceptor dyes including dichlorofRl 10], dichloro [R6G], dichloro [TAMRA], dichlorofROX] or the like; fluorescein donor dye including fluorescein, 6-FAM, or the like; Cyanine dyes such as Cy3B; Atto dyes such as atto 647N which forms a FRET pair with Cy3B and the like. Fluorophores include, but are not limited to, MDCC (7-diethylamino-3-[([(2- maleimidyl)ethyl]amino)carbonyl]coumarin), TET, HEX, Cy3, TMR, ROX, LC red 705 and LC red 640, Alexa Fluor dyes, including Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750; Brilliant dyes, including Brilliant Ultra Violet 395, Brilliant Ultra Violet 496, Brilliant Ultra Violet 563, Brilliant Ultra Violet 615, Brilliant Ultra Violet 661, Brilliant Ultra Violet 737, Brilliant Ultra Violet 805, Brilliant Violet 421, Brilliant Violet 480, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 786, Brilliant Violet 605; eFluor dyes including eFluor 450, eFluor 506, eFluor 660; Expressed fluorescent proteins including Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP); NovaFluor dyes including, NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-120S, NovaFluor Blue 660-40S, NovaFluor Blue 690, NovaFluor Blue 725, NovaFluor Blue 760, NovaFluor Red 660, NovaFluor Red 685, NovaFluor Red 700, NovaFluor Red 710, NovaFluor Red 725, NovaFluor Red 755, NovaFluor Yellow 570, NovaFluor Yellow 590, NovaFluor Yellow 610,Docket No.: ESBI00007-1WGNovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755; Organic dyes including, BODIPY FL, Coumarin and Coumarin Derivatives, Cy3, Cy5, Fluorescein (FITC), Oregon Green 488, Pacific Blue, Pacific Green, Pacific Orange, Tetramethylrhodamine (TRITC), Texas Red (and Texas Red-X); PE, APC, and related tandems including APC-eFluor 780 Allophycocyanin (APC), PE-Cyanine7, PE-eFluor 610, PerCP-Cyanine5.5, PerCP-eFluor 710, R-phycoerythrin (R-PE); Qdots including Qdot 525 Probe, Qdot 565 Probe, Qdot 605 Probe, Qdot 655 Probe, Qdot 705 Probe, Qdot 800 Probe; Super Bright dyes including Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, and Super Bright 780.

[0052] In some embodiments, a detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze chemiluminescent reactions given the appropriate substrates (e.g., an oxidizing reagent plus a chemiluminescent compound. A number of compound families are known to provide chemiluminescence under a variety of conditions. Non-limiting examples of chemiluminescent compound families include 2,3- dihydro-l,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base. Other examples of chemiluminescent compound families include, e.g., 2,4,5-triphenylimidazoles, para-dimethylamino and - methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters. In some embodiments, a detectable label is or includes a metal-based or mass-based label.

[0053] The terms “hybridizing,” “hybridize,” “annealing,” and “anneal” are used interchangeably in this disclosure, and refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.Docket No.: ESBI00007-1WG

[0054] A “primer” is a single-stranded nucleic acid sequence having a 3 ’ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence.

[0055] A “nucleic acid extension” generally involves incorporation of one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, or derivatives thereof) into a molecule (such as, but not limited to, a nucleic acid sequence) in a template-dependent manner, such that consecutive nucleic acids are incorporated by an enzyme (such as a polymerase or reverse transcriptase), thereby generating a newly synthesized nucleic acid molecule. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase. For example, a primer that hybridizes to a complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule by using the complementary nucleic acid sequence as a template for nucleic acid synthesis. Similarly, a 3’ polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence can be used as a template for singlestrand synthesis of a corresponding cDNA molecule. Furthermore, a poly (dT) sequence may be used as a sequencing primer for sequencing RNA molecules comprising poly(A) tails.

[0056] A “non-terminating nucleotide” or “incorporating nucleotide” can include a nucleic acid moiety that can be attached to a 3' end of a polynucleotide using a polymerase or transcriptase, and that can have another non-terminating nucleic acid attached to it using a polymerase or transcriptase without the need to remove a protecting group or reversible terminator from the nucleotide. Naturally occurring nucleic acids are a type of non-terminating nucleic acid. Nonterminating nucleic acids may be labeled or unlabeled.

[0057] A “PCR amplification” refers to the use of a polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for implementing PCR are described, for example, in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of each of which areDocket No.: ESBIO0007-1WO incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers that are employed in a primer extension reaction, and reagents for the reaction. The oligonucleotide primers are of sufficient length to provide for hybridization to complementary genetic material under annealing conditions. The length of the primers generally depends on the length of the amplification domains, but will typically be at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be as long as 40 bp or longer, where the length of the primers will generally range from 18 to 50 bp. The genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending upon whether primer extension, linear or exponential amplification of the genetic material is desired.

[0058] In some embodiments, the PCR amplification process uses a DNA polymerase enzyme. The DNA polymerase activity can be provided by one or more distinct DNA polymerase enzymes. In some embodiments, the DNA polymerase enzyme is from a bacterium, e.g., the DNA polymerase enzyme is a bacterial DNA polymerase enzyme. For instance, the DNA polymerase can be from a bacterium of the genus Escherichia, Bacillus, Thermophilus, or Pyrococcus .

[0059] In some embodiments, PCR amplification can include reactions such as, but not limited to, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, an isothermal amplification reaction, and / or a loop-mediated amplification reaction.

[0060] In some embodiments, PCR amplification uses a single primer that is complementary to the 3’ tag of target DNA fragments. In some embodiments, PCR amplification uses a first and a second primer, where at least a 3 ’ end portion of the first primer is complementary to at least a portion of the 3’ tag of the target nucleic acid fragments, and where at least a 3’ end portion of the second primer exhibits the sequence of at least a portion of the 5 ’ tag of the target nucleic acid fragments. In some embodiments, a 5’ end portion of the first primer is non-complementary to the 3’ tag of the target nucleic acid fragments, and a 5’ end portion of the second primer does not exhibit the sequence of at least a portion of the 5’ tag of the target nucleic acid fragments. In some embodiments, the first primer includes a first universal sequence and / or the second primer includes a second universal sequence.Docket No.: ESBI00007-1WG

[0061] The term “DNA polymerase” includes not only naturally-occurring enzymes but also all modified derivatives thereof, including also derivatives of naturally-occurring DNA polymerase enzymes. For instance, in some embodiments, the DNA polymerase can have been modified to remove 5 ’-3’ exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerase enzymes that can be used include, but are not limited to, mutants that retain at least some of the functional, e.g., DNA polymerase activity of the wild-type sequence. Mutations can affect the activity profile of the enzymes, e.g., enhance or reduce the rate of polymerization, under different reaction conditions, e.g., temperature, template concentration, primer concentration, etc. Mutations or sequence-modifications can also affect the exonuclease activity and / or thermostability of the enzyme.

[0062] Suitable examples of DNA polymerases that can be used include, but are not limited to: E.coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNA polymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNA polymerase, OneTaq® DNA polymerase, OneTaq® Quick- Load® DNA polymerase, Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNA polymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, KI enow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNA polymerase enzymes.

[0063] In some embodiments, genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more distinct reverse transcriptase enzymes, suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™, ThermoScript™, and SuperScript® I, II, III, and IV enzymes. “Reverse transcriptase” includes not only naturally occurring enzymes, but all such modified derivatives thereof, including also derivatives of naturally-occurring reverse transcriptase enzymes.

[0064] In addition, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptase enzymes, including mutants that retain at least some of the functional, e.g., reverse transcriptase, activity of the wildtype sequence. The reverse transcriptase enzyme can be provided as part of a composition that includes other components, e.g., stabilizing components that enhance or improve the activity ofDocket No.: ESBI00007-1WG the reverse transcriptase enzyme, such as RNase inhibitor(s), inhibitors of DNA-dependent DNA synthesis, e.g., actinomycin D. Many sequence-modified derivative or mutants of reverse transcriptase enzymes, e.g., M-MLV, and compositions including unmodified and modified enzymes are commercially available, e.g., ArrayScript™, MultiScribe™, ThermoScript™, and SuperScript® I, II, III, and IV enzymes.

[0065] Certain reverse transcriptase enzymes (e.g., Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase) can synthesize a complementary DNA strand using both RNA (cDNA synthesis) and singlestranded DNA (ssDNA) as a template. Thus, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that is capable of using both RNA and ssDNA as the template for an extension reaction, e.g., an AMV or MMLV reverse transcriptase.

[0066] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be readily apparent to those persons skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiment shown but is to be accorded the widest scope consistent with the principles and features described herein.

[0067] The figures illustrate processes according to various embodiments. In the exemplary processes, some blocks are, optionally, combined, the order of some blocks is, optionally, changed, and some blocks are, optionally, omitted. In some examples, additional steps may be performed in combination with the exemplary processes. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature, and, as such, should not be viewed as limiting.II. Overview

[0068] The methods described herein provide the above advantages by leveraging intensity characteristics to allow the detection and identification of luminophores with overlapping spectrums. More specifically, FIG. 1 provides an exemplary schematic showing a general process 100 for nucleic acid sequencing, according to some embodiments described herein. The method can include: labeling the nucleotides with fluorophores consisting of fluorophores withDocket No.: ESBI00007-1WG different but overlapping emission spectrums. The template nucleic acid sequence is contacted with nucleotides, which bind and optionally incorporate to the nucleotides in the template nucleic acid sequence (102). Two or more nucleotides are labeled with fluorophores with overlapping emission spectrums and the emission intensity is determined at two or more wavelengths using an excitation wavelength (104, 106). The emission intensity is optionally determined for a third fluorophore and a fourth fluorophore, which label a third nucleotide and a fourth nucleotide, respectively (108, 110). The same excitation wavelength is used to obtain an emission intensity for all of the fluorophores of interest. The determined emission intensities at two or more wavelengths are then used to obtain an intensity characteristic for each fluorophore, and the intensity detected at a wavelength can also serve as a baseline value to obtain a baseline corrected emission intensity ratio (112). Once an emission intensity ratio or ratios are determined for each fluorophore, the fluorophore is identified, and the identity of the corresponding labeled nucleotide is confirmed (114). In some instances, the method can further comprise being iterated over iterations, with respect to the (102) contacting of the template nucleic acid sequence and (104 - 114) determining the fluorophore and corresponding nucleotide identity. In some instances, the iterated method can further comprise identifying a nucleic acid sequence comprising the identified bases.

[0069] Furthermore, FIG. 2 provides another exemplary schematic showing a general process 200 for nucleic acid sequencing, according to some embodiments described herein. The method can include: labeling the nucleotides with fluorophores consisting of fluorophores with different but overlapping excitation spectrums. The template nucleic acid sequence is contacted with nucleotides, which bind and optionally incorporate to the nucleotides in the template nucleic acid sequence (202). Two or more nucleotides are labeled with fluorophores with overlapping excitation spectrums and the excitation intensity is determined at two or more wavelengths for one or more fluorophore (204, 206). The excitation intensity is optionally determined for a third fluorophore and a fourth fluorophore, which label a third nucleotide and a fourth nucleotide, respectively (208, 210). Various excitation wavelengths can be used to obtain an emission intensity for all of the fluorophores of interest. The determined excitation intensities at two or more wavelengths are then used to obtain an intensity ratio for each fluorophore, and a wavelength intensity can also serve as a baseline value to obtain a baseline corrected excitation intensity ratio (212). Once an excitation intensity ratio or ratios are determined for each fluorophore, the fluorophore is identified, and the identity of the corresponding labeled nucleotide is confirmed (214). In some instances, the method can further comprise being iteratedDocket No.: ESBI00007-1WO over iterations, with respect to the (202) contacting of the template nucleic acid sequence and (204 - 214) determining the fluorophore and corresponding nucleotide identity. In some instances, the iterated method can further comprise identifying a nucleic acid sequence comprising the identified bases.

[0070] In process (100 and 200), some blocks are, optionally, combined, the order of some blocks is, optionally, changed, and some blocks are, optionally, omitted. In some examples, additional steps may be performed in combination with the process 100 and the process 200. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

[0071] At 102 in FIG. 1, a plurality of template nucleic acid sequences can be sequenced, wherein of the nucleotides contacting the template, two or more are labeled with fluorophores with different but overlapping emission spectrums. The emission spectra for the multiple different labels should be largely overlapping, e.g., emission spectra peaks between the multiple different labels are close enough such that a measured wavelength will provide information on the emission intensities of all of the fluorophores. A single light source can readily excite the different fluorophores and initiate emission intensities for all of the different types of labels. The different labels can comprise fluorophores. A single light source can be a light emitting diode (LED). Different detectors can detect the emission intensities at each different emission wavelength of interest. The emission intensities at the different emission wavelengths can also be simultaneously detected by the different detectors.

[0072] At 202 in FIG. 2, a plurality of template nucleic acid sequences can be sequenced, wherein of the nucleotides contacting the template, two or more are labeled with fluorophores with different but overlapping excitation spectrums. The excitation spectra for the multiple different labels should be largely overlapping, e.g., excitation spectra peaks between the multiple different labels are close enough such that an excitation intensity at a particular wavelength will provide information on the excitation intensities of all of the fluorophores. The multiple different labels can comprise fluorophores. A different light source (or the same light source but with different dichroic mirrors) is used to excite each of the different fluorophores, and a single detector is used to detect the emission intensities at the selected emission wavelengths. The light sources can be light emitting diode (LED). The excitation intensities at the different excitation wavelengths can be sequentially detected by the single detector.Docket No.: ESBIO0007-1WO

[0073] After contacting the template nucleic acid with nucleotides, including nucleotides labeled with fluorophores with different but overlapping excitation spectrums, the intensity characteristic or characteristics are determined for each fluorophore. The difference between a determined intensity characteristic for a first fluorophore and a determined intensity characteristic for a second fluorophore can be interpreted as a fold change. The first intensity characteristic can be about 1.25-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold or more than the second intensity characteristic. Conversely, the second intensity characteristic can be about 1.25-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold or more than the first intensity characteristic.

[0074] The biological sample can be a cell or a tissue sample. One or more molecules can be nucleic acid analytes to be sequenced. The nucleic acid analytes can comprise a genomic DNA, an RNA, a cDNA, a nucleic acid probe, a reporter oligonucleotide conjugated to a binding moiety that binds to a non-nucleic acid analyte, a rolling circle amplification product (RCP), or any combination thereof.

[0075] For nucleic acid sequencing, a plurality of different polynucleotide templates with nucleotides of the four different bases is contacted, wherein the nucleotides comprise nucleotides labeled with multiple different binders. The plurality of different polynucleotide templates can be present on an artificial substrate, in a biological sample, or in a solution. The artificial substrate can be a patterned flow cell or a nonpatterned flow cell. The plurality of different polynucleotide templates can comprise one or more clusters of different polynucleotide templates. The one or more clusters can comprise an ordered array of clusters on the artificial substrate. The one or more clusters can be formed via bridge amplification. The one or more clusters can comprise multiple molecules comprising: i) one or more adapter sequences and / or one or more primer binding sequences and ii) the same template sequence or complement thereof. The one or more clusters can be formed via rolling circle amplification (RCA). The one or more clusters can each comprise one or more RCA products (RCPs) each comprising: i) one or more adapter sequences and / or one or more primer binding sequences and ii) the same template sequence. The one or more clusters can each comprise one or more RCA products (RCPs) each comprising: i) one or more adapter sequences and / or one or more primer binding sequences and ii) the same template sequence. The one or more clusters can comprise an intermediate from a sequencing by synthesis method or a sequencing by avidity method.Docket No.: ESBI00007-1WG

[0076] The method can further comprise being iterated over iterations, with respect to the contacting of the template nucleic acid sequence and the identification of the fluorophore and the corresponding nucleotide base. In some instances, the iterated method can further comprise identifying a nucleic acid sequence comprising the identified bases.III. Biological Samples and Analytes

[0077] The molecules used as analytes in the methods described herein may be obtained from any suitable biological source, for example a tissue sample, a blood sample, a plasma sample, a saliva sample, a fecal sample, or a urine sample. The molecules may also be present in and / or on a cell, a virus, or a viral-like particle (VLP). In some embodiments, the molecules may also be present in and / or on an intracellular organelle, an extracellular vesicle, a lipid nanoparticle (LNP), a protein complex, a nucleic acid complex, or a protein-nucleic acid complex.

[0078] The molecules may be a polynucleotide, a polypeptide, a carbohydrate, a lipid, a small molecule, or any combination thereof. The polynucleotides may be DNA or RNA molecules. In some embodiments, RNA molecules are reverse transcribed into DNA molecules prior to hybridizing the polynucleotide to a sequencing primer. In some embodiments, RNA molecules are not reverse transcribed and are hybridized to a sequencing primer for direct RNA sequencing. In some embodiments, the nucleic acid molecule is a cell-free DNA (cfDNA), such as a circulating tumor DNA (ctDNA) or a fetal cell-free DNA. The polypeptides may be oligopeptides, including dipeptides, tripeptides, and tetrapeptides. In some embodiments, the polypeptides are bound to ligands including coenzymes and cofactors, or to polynucleotides. The carbohydrates may be polysaccharides. In some embodiments, the carbohydrates may be linear or branched. The lipids may be hydrophobic or amphiphilic small molecules. In some embodiments, the lipids are fats, waxes, sterols, and others. In some embodiments, the small molecules may be chemically derived. In some embodiments, the molecules are present as individual molecules or in complex with other molecules.

[0079] Examples of nucleic acid molecules include DNA molecules such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.Docket No.: ESBI00007-1WG

[0080] Examples of nucleic acid molecules also include RNA molecules such as various types of coding and non-coding RNA, including viral RNAs. Examples of the different types of RNA molecules include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7- methyl guanosine cap), a polyadenylated mRNA (poly-A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the nucleic acid molecules disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a nonspliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA).

[0081] In some embodiments, a molecule may be a denatured molecule, wherein the resulting denatured molecule has its weak linkages and / or bonds broken. The molecule may undergo denaturation by application of stress or compounds to disrupt its structures. The molecule may be denatured, for example, optionally using formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, urea, heat, or any combination thereof. In some embodiments, the molecule is not denatured for use in a method disclosed herein.

[0082] In some embodiments, a nucleic acid molecule may be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid may be denatured, for example, optionally using formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, urea, heat, or any combination thereof. In some embodiments, the nucleic acid is not denatured for use in a method disclosed herein.

[0083] In some embodiments, a polypeptide molecule may be a denatured polypeptide, wherein the resulting denatured polypeptide has its quaternary, tertiary, and / or secondary structures altered. In some embodiments, the denatured polypeptide has an intact primary structure between its amino acids. The polypeptide may be denatured, for example, optionally using formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, urea, heat, or any combination thereof. The polypeptide may be denatured through exposure to changes including but not limited to, temperature, salinity, and pressure. In some embodiments, the polypeptide is not denatured for use in a method disclosed herein.

[0084] In some embodiments, a molecule can be extracted from a cell, a virus, or a tissue sample comprising the cell or virus. Processing conditions can be adjusted to extract or release molecules (e.g., DNA, RNA, proteins, intracellular organelles, extracellular vesicles, proteinDocket No.: ESBIO0007-1WO complexes, nucleic acid complexes, or protein-nucleic acid complexes) from a cell, a virus, or a tissue sample.IV. Binders and Luminophores

[0085] The methods disclosed herein comprise using binders. A binder refers to a molecule that interacts with another molecule and results in a stable association in which the molecules are then in close proximity to each other. It may involve chemical bonding, but it is not required. A binder can be a nucleotide, a nucleic acid probe, an antibody, or an aptamer. A binder can bind to a target individually or in complex with other binders. A binder can form a non-covalent, reversible covalent, or irreversible covalent type of bonding with its target.

[0086] In some aspects, a binder is a protein that binds and forms a molecular association with a protein, a DNA, an RNA, or any of the aforementioned analytes. In some embodiments, a binder is an antibody. An antibody may bind to specific molecules. An antibody may bind to specific antigens. In some embodiments, the complementarity-determining regions, or CDRs (CDR1, CDR2, and CDR3) will contribute to the complementarity and binding specificity of an antibody to an antigen. In some embodiments, the binding of an antibody to a specific molecule is a noncovalent interaction.

[0087] In some embodiments, a binder is a native nucleotide or a nucleotide analog or modified nucleotide (e.g., labeled with one or more detectable labels). In some embodiments, a nucleotide analog comprises a nitrogenous base, five-carbon sugar, and phosphate group, wherein any component of the nucleotide may be modified and / or replaced. In some embodiments, a method disclosed herein may comprise but does not require using one or more non-incorporable nucleotides. Non-incorporable nucleotides may be modified to become incorporable at any point during the sequencing method.

[0088] Luminophores are substances that emit light. They can be used to label binders and thus allow for a sensitive approach to detect and identify molecular targets. Luminophores can be excited to a higher-energy state and as they return to their former lower-energy state, they emit light. Luminophores can be further classified as fluorophores or phosphors. Fluorophores absorb light energy at a specific wavelength and emit light at a longer wavelength. Phosphors emit light when exposed to radiant energy. In some embodiments, a method disclosed herein comprises using binders labeled with luminophores or analogs thereof. The luminophores selected to label the binders have overlapping emission spectrums and / or excitation spectrums.Docket No.: ESBIOOOQ7-1WOA. Exemplary Luminophores and Fluorophores

[0089] In some embodiments, luminophore(s) as used in methods as provided herein include, but not limited to, fluorescein derivatives; Rhodamine B derivatives; BODIPY derivatives; Pdots based on polyfluorene including polyfluorene (PFO), poly (fluorene benzothiadiazole) (PFBT), poly 11 uorcnc-^-polyf thiophene benzothiadiazole) (PFTBT5); quantum dots.

[0090] In some embodiments, fluorophore(s) as used in methods as provided herein include, but not limited to, the BODIPY series (BODIPY 493 / 503, BODIPY FL-X, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR-X, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR-X, BODIPY 630 / 650-X, BODIPY 650 / 665-X), the Alexa series (Alexa 350, Alexa 405, Alexa 488, Alexa 514, Alexa 532, Alexa 555, ATTO 550, Alexa 568, Alexa 594, Alexa 647, Alexa 680, Alexa 750), the ATTO series (ATTO 425, ATTO 430LS, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO Rho6G, ATTO 542, ATTO 565, ATTO Rho3B, ATTO 490LS, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647N, ATTO 665, ATTO 655, ATTO 680, ATTO 700, ATTO 725, ATTO 740), FAM, FITC, Cy3, Cy5, PE, Coumarin, PerCP, TRITC, Texas Red, APC, quantum dots, or a fluorescent protein (for example Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), and Red Fluorescent Protein (RFP)).

[0091] In some embodiments, fluorophore(s) as used in methods as provided herein include, but not limited to, 7-AAD (7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7-AAD), 7- Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein), BFP / GFP FRET, BOBO™-1 / BO-PRO™- 1, BOBO™-3 / BO-PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 581 / 591, BODIPY® 630 / 650-X, BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson™, Calcium Green- 1™, Calcium Orange™, Calcofhior® White,5 -Carboxy fluoroscein (5-FAM), 5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G, 5- Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Blue®,Docket No.: ESBIOOOQ7-1WOCascade Yellow™, CCF2 (GeneBLAzer™), CFP (Cyan Fluorescent Protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-DL16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP, ELF® -97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM (5- Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH), Fluoro- Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™ (high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted (rsGFP), GFP Wild Type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342 & 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6- JOE, JOJO™-1 / JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO™-1 / LO-PRO™- 1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™ Green (pH 5), LysoSensor™ Yellow / Blue (pH 4.2), LysoTracker® Green, LysoTracker® Red, LysoTracker® Yellow, Mag- Fura-2, Mag-Indo-1, Magnesium Green™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker® Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (Propidium Iodide), PKH26, PKH67, POPO™-1 / PO-PRO™-1, POPO™-3 / PO- PRO™-3, Propidium Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red) , Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green™, Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1 (low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, SpectrumGreen® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA (5- Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), Texas Red® / Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine, Thiazole Orange, TOTO®-1 / TO-PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5, Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-Rhodamine (XRITC) , Y66F, Y66H, Y66W, YFP (YellowDocket No.: ESBI00007-1WGFluorescent Protein), YOYOO-1 / YO-PRO®-1, Y0Y0®-3 / Y0-PR0®-3, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 542, ATTO 550, ATTO 565, ATTO RholOl, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5’ IRDye® 700, 5’ IRDye® 800, 5’ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3 Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHS Ester).

[0092] The detectable label can be directly detectable by itself (e.g., fluorescent labels). The label can emit a signal or alter a signal delivered to the label so that the presence or absence of the label can be detected. In some cases, coupling may be via a linker, which may be cleavable, such as photo-cleavable (e.g., cleavable under ultra-violet light), chemically-cleavable (e.g., via a reducing agent, such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP)) or enzymatically cleavable (e.g., via an esterase, lipase, peptidase, or protease).V. Emission and Excitation Spectrums

[0093] The optical spectra include ultraviolet, visible light, and infrared wavelengths of the electromagnetic spectrum. Luminophores have spectral profiles, which can reflect the excitation intensity and the emission intensity at certain wavelengths across the optical spectrum.Spectrums are displayed in a coordinate system; nanometers are indicated on the x-axis and intensity on the y-axis. Colors are typically associated with a particular wavelength range along the optical spectra (nanometers, nM): violet (380 - 450 nM), blue (450 - 485 nM), cyan (485 - 500 nM), green (500 - 565 nM), yellow (565 - 590 nM), orange (590 - 625 nM), and red (625 - 750 nM). A light source excites the luminophores to produce a spectrum of light. The light source produces ultraviolet and / or visible light to excite the luminophores in the biological sample or in the artificial substrate. The light sources are selected by their ability to produce light of a specific wavelength or a specific wavelength range. In some embodiments, all of the luminophores used to label the binders are excited by a single wavelength, which allows all of the luminophores to emit light. In certain embodiments, this feature allows the use of a single light source to obtain spectral data on all of the luminophores used to label the binders. In some embodiments, multiple light sources are used to excite luminophores at a different wavelength or a wavelength range.

[0094] An optical system comprised of one of more light sources, excitation filters, emission filters, and one or more detectors can detect emitted luminescence intensity. The light emitted byDocket No.: ESBIO0007-1WO a luminophore can be represented by an emission spectrum, which represents the intensities emitted due to a transition from a higher energy state to a lower energy state. The collection of transitions is reflected in the collection of radiated wavelengths.

[0095] In some embodiments, binders are labeled with fluorophores. A fluorescence intensity is a measure of the light or photons emitted. A fluorophore’s emission spectrum provides a measure of intensities emitted at specific wavelengths. The excitation wavelength is fixed to obtain an emission spectrum. Conversely, a fluorophore’s excitation spectrum can be obtained by using a fixed emission wavelength and an excitation monochromator to scan the excitation intensities along the spectra. Both intensities measured for a fluorophore’s emission or excitation can be used to construct a spectral profile specific to the fluorophore.A. Intensity characteristics

[0096] In some embodiments, a method disclosed herein comprises using luminophores or analogs thereof to label binders. The luminophores selected to label binders, which target one or more molecules, have overlapping emission spectrums and / or overlapping excitation spectrums. While typically using a spectral intensity alone requires luminophores to have minimal spectral overlap, the use of intensity characteristics circumvents this requirement, provided that a unique profile can be constructed from the intensity characteristics for each luminophore used to label binders in an experiment.

[0097] After determining emission intensities or excitation intensities for a luminophore at two or more wavelengths, one or more intensity characteristics can be determined. In some embodiments, the binders are labeled with luminophores that are different but have overlapping emission spectrums. The detectors can detect the intensities at two or more different wavelengths. The emission intensities for all the luminophores will be measured at the same two or more wavelengths. For example, emission intensities for a first wavelength (XI) and a second wavelength (X2) will be determined for both luminophore-A and luminophore-B, resulting in a total of four measurements. These determined intensities can be used to determine luminophore specific intensity characteristics. In some embodiments, the binders are labeled with luminophores that are different but have overlapping excitation spectrums. Multiple light sources are then used to excite the luminophores at two or more wavelengths. A single detector is used. Similar to the example above, excitation intensities for a first wavelength (XI) and a second wavelength (X2) will be determined for both luminophore-A and luminophore-B, leadingDocket No.: ESBI00007-1WG to a total of four measurements, which can then be used to determine an intensity characteristic for luminophore-A and for luminophore-B.

[0098] In some embodiments, a first intensity characteristic (e.g., intensity ratio) is determined for a first luminophore (e.g., luminophore-A), and the determination comprises using an intensity value of the first luminophore at one of three or more wavelengths as a baseline intensity value, thereby determining a baseline corrected intensity characteristic (e.g., intensity ratio) for the first luminophore. In some embodiments, a second intensity characteristic (e.g., intensity ratio) is determined for a second luminophore (e.g., luminophore-B), and the determination comprises using an intensity value of the second luminophore at one of three or more wavelengths as a baseline intensity value, thereby determining a baseline corrected intensity characteristic (e.g., intensity ratio) for the second luminophore.

[0099] In some embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be determined using polynomial regression or fitting. Polynomial regression is a form of analysis in which the relationship between the independent variable and the dependent variable is modeled. In some embodiments, the polynomial regression or fitting can comprise using formula y = a + bx + ex2, wherein x is a wavelength andy is a measured intensity of the first or second luminophore at the wavelength x. In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be b and c which are determined using the polynomial regression or fitting. In any of the preceding embodiments, the first intensity characteristic(s) and / or the second intensity characteristic(s) can be b / c or c / b, wherein b and c are determined using the polynomial regression or fitting.

[0100] Additionally, if intensities for three or more wavelengths are measured, one intensity can provide a baseline value. For example, the difference between the first wavelength (XI) and a third wavelength (X3) is determined. The difference between the second wavelength (X2) and the third wavelength (X3) is also determined. The ratio is then obtained from these baseline corrected values ((XI -X3) / ( X2- X3)). The baseline corrected intensity characteristic is used to determine the luminophore ’s identity. This intensity characteristic can improve signal-to-noise ratio as it accounts for background luminescence and technical variations.Docket No.: ESBI00007-1WGVI. Sequencing MethodsA. Nucleotides and Nucleotide Analogs

[0101] In some embodiments, a method disclosed herein comprises using one or more nucleotides or analogs thereof as binders, including a native nucleotide or a nucleotide analog or modified nucleotide (e.g., labeled with one or more detectable labels). In some embodiments, a nucleotide analog comprises a nitrogenous base, five-carbon sugar, and phosphate group, wherein any component of the nucleotide may be modified and / or replaced. In some embodiments, a method disclosed herein may comprise but does not require using one or more non-incorporable nucleotides. Non-incorporable nucleotides may be modified to become incorporable at any point during the sequencing method.

[0102] Nucleotide analogs include, but are not limited to, alpha-phosphate modified nucleotides, alpha-beta nucleotide analogs, beta-phosphate modified nucleotides, beta-gamma nucleotide analogs, gamma-phosphate modified nucleotides, caged nucleotides, or ddNTPs. Examples of nucleotide analogs are described in U.S. Patent No. 8,071,755, which is incorporated by reference herein in its entirety.

[0103] In some embodiments, a method disclosed herein may comprise but does not require using terminators that reversibly prevent nucleotide incorporation at the 3 '-end of the primer. One type of reversible terminator is a 3'-O-blocked reversible terminator. Here the terminator moiety is linked to the oxygen atom of the 3'-OH end of the 5-carbon sugar of a nucleotide. For example, U.S. Patent Nos. 7,544,794 and 8,034,923 (the disclosures of these patents are incorporated by reference) describe reversible terminator dNTPs having the 3'-OH group replaced by a 3'-ONH2 group. Another type of reversible terminator is a 3 '-unblocked reversible terminator, wherein the terminator moiety is linked to the nitrogenous base of a nucleotide. For example, U.S. Patent No. 8,808,989 (the disclosure of which is incorporated by reference) discloses particular examples of base-modified reversible terminator nucleotides that may be used in connection with the methods described herein. Other reversible terminators that similarly can be used in connection with the methods described herein include those described in U.S.Patent Nos. 7,956,171, 8,071,755, and 9,399,798, herein incorporated by reference.

[0104] In some embodiments, a method disclosed herein may comprise but does not require using nucleotide analogs having terminator moieties that irreversibly prevent nucleotide incorporation at the 3 '-end of the primer. Irreversible nucleotide analogs include 2', 3'-Docket No.: ESBI00007-1WG dideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'- OH group of dNTPs that is essential for polymerase-mediated synthesis.

[0105] In some embodiments, a method disclosed herein may comprise but does not require using non-incorporable nucleotides comprising a blocking moiety that inhibits or prevents the nucleotide from forming a covalent linkage to a second nucleotide (3'-OH of a primer) during the incorporation step of a nucleic acid polymerization reaction. The blocking moiety can be removed from the nucleotide, allowing for nucleotide incorporation.

[0106] In some embodiments, a method disclosed herein may comprise but does not require using 1, 2, 3, 4 or more nucleotide analogs present in the SBS reaction. In some embodiments, a nucleotide analog is replaced, diluted, or sequestered during an incorporation step. In some embodiments, a nucleotide analog is replaced with a native nucleotide. In some embodiments, a nucleotide analog is modified during an incorporation step. The modified nucleotide analog can be similar to or the same as a native nucleotide.

[0107] In some embodiments, a method disclosed herein may comprise but does not require using a nucleotide analog having a different binding affinity for a polymerase than a native nucleotide. In some embodiments, a nucleotide analog has a different interaction with a next base than a native nucleotide. Nucleotide analogs and / or non-incorporable nucleotides may basepair with a complementary base of a template nucleic acid.

[0108] In some embodiments, one or more nucleotides can be labeled with distinguishing and / or detectable luminophores. The luminophores may be distinguishable by means of their differences in fluorescence and / or luminescence intensity characteristics. The luminophore may be attached to one or more different positions on the nucleotide, so long as the fidelity of binding to the polymerase-nucleic acid complex is sufficiently maintained to enable identification of the complementary base on the template nucleic acid correctly. In some embodiments, the luminophore is attached to the nucleobase of the nucleotide. Alternatively, a luminophore is attached to the gamma phosphate position of the nucleotide.

[0109] Detectable labels can be suitable for small scale detection and / or suitable for high- throughput screening. As such, suitable detectable labels include, but are not limited to, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes. The detectable label can be quantitatively detected, and intensity characteristics can be quantified. Quantifiable detection generally includes a detection method having a quantifiable (e.g.,Docket No.: ESBI00007-1WG numerically reportable) value such as an intensity. In some embodiments, the detectable label is bound to another moiety, for example, a nucleotide or nucleotide analog, and can include a fluorescent or a chemiluminescent label.B. Polymerases

[0110] Polymerases that may be used to carry out the disclosed techniques include naturally- occurring polymerases and any modified variations thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, and analogs. Naturally occurring polymerases and modified variations thereof are not limited to polymerases that retain the ability to catalyze a polymerization reaction. In some embodiments, the naturally occurring and / or modified variations thereof retain the ability to catalyze a polymerization reaction. In some embodiments, the naturally-occurring and / or modified variations have special properties that enhance their ability to sequence DNA, including enhanced binding affinity to nucleic acids, reduced binding affinity to nucleic acids, enhanced catalysis rates, reduced catalysis rates, etc. Mutant polymerases include polymerases wherein one or more amino acids are replaced with other amino acids (naturally or non-naturally occurring), and insertions or deletions of one or more amino acids.

[0111] In some embodiments, a method disclosed herein may comprise but does not require using modified polymerases containing an external tag (e.g., an exogenous detectable label), which can be used to monitor the presence and interactions of the polymerase. In some embodiments, intrinsic signals from the polymerase can be used to monitor their presence and interactions. Thus, the provided methods can include monitoring the interaction of the polymerase, nucleotide and template nucleic acid through detection of an intrinsic signal from the polymerase. In some embodiments, the intrinsic signal is a light scattering signal. For example, intrinsic signals include native fluorescence of certain amino acids such as tryptophan.

[0112] In some embodiments, a method disclosed herein may comprise using an unlabeled polymerase, and monitoring is performed in the absence of an exogenous detectable label associated with the polymerase. Some modified polymerases or naturally occurring polymerases, under specific reaction conditions, may incorporate only single nucleotides and may remain bound to the primer-template after the incorporation of the single nucleotide.

[0113] In some embodiments, a method disclosed herein may comprise using a polymerase unlabeled with an exogenous detectable label (e.g., a fluorescent label). The label can beDocket No.: ESBI00007-1WO chemically linked to the structure of the polymerase by a covalent bond after the polymerase has been at least partially purified using protein isolation techniques. For example, the exogenous detectable label can be chemically linked to the polymerase using a free sulfhydryl or a free amine moiety of the polymerase. This can involve chemical linkage to the polymerase through the side chain of a cysteine residue, or through the free amino group of the N-terminus. In certain preferred embodiments, a fluorescent label attached to the polymerase is useful for locating the polymerase, as may be important for determining whether or not the polymerase has localized to a spot on an array corresponding to immobilized primed template nucleic acid. The fluorescent signal need not, and in some embodiments does not change absorption or emission characteristics as the result of binding any nucleotide. In some embodiments, the signal emitted by the labeled polymerase is maintained uniformly in the presence and absence of any nucleotide being investigated as a possible next correct nucleotide.

[0114] The term polymerase and its variants, as used herein, also refers to fusion proteins comprising at least two portions linked to each other, for example, where one portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand is linked to another portion that comprises a second moiety, such as, a reporter enzyme or a processivity- modifying domain. For example, T7 DNA polymerase comprises a nucleic acid polymerizing domain and a thioredoxin binding domain, wherein thioredoxin binding enhances the processivity of the polymerase. Absent the thioredoxin binding, T7 DNA polymerase is a distributive polymerase with processivity of only one to a few bases. Although DNA polymerases differ in detail, they have a similar overall shape of a hand with specific regions referred to as the fingers, the palm, and the thumb; and a similar overall structural transition, comprising the movement of the thumb and / or finger domains, during the synthesis of nucleic acids.

[0115] DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and phage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerases I, II and III, IV and V, the Klenow fragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases a, P, y, 5, e, |, , a, p, and K, as well as the Revl polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases, PZA DNADocket No.: ESBI00007-1WG polymerase, phi- 15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase. Other DNA polymerases include thermostable and / or thermophilic DNA polymerases such as DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (TH) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNA polymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase; Thermococcus fumicolans DNA polymerase; Aeropyrum pemix DNA polymerase; and the heterodimeric DNA polymerase DP1 / DP2. Engineered and modified polymerases also are useful in connection with the disclosed techniques. For example, modified versions of the extremely thermophilic marine archaea Thermococcus species 9° N (e.g., Therminator DNA polymerase from New England BioLabs Inc.; Ipswich, Mass.) can be used. Still other useful DNA polymerases, including the 3PDX polymerase are disclosed in U.S. Patent No. 8,703,461, the disclosure of which is incorporated by reference in its entirety.

[0116] RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and KI 1 polymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and Archaea RNA polymerase.

[0117] Reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from the Moloney murine leukemia virus, AMV reverse transcriptase from the avian myeloblastosis virus, and Telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes.Docket No.: ESBI00007-1WGC. Sequencing Reactions

[0118] In some embodiments of a sequencing-by-synthesis (SBS) method disclosed herein, a first labeled nucleotide that has been incorporated is not deactivated (e.g., by removal and / or photobleaching of the label) prior to the introduction and / or incorporation of the next, second labeled nucleotide. The first and second labeled nucleotides can comprise the same base or different bases. The first and second labeled nucleotides can be introduced into a sequencing reaction mix simultaneously or at different time points in any order. Further, the first and second labeled nucleotides can be introduced by itself (e.g., in a suitable solvent such as water) or in a mixture with another sequencing reagent, such as one or more other labeled nucleotides and / or one or more unlabeled nucleotides. The first and second labeled nucleotides can also comprise the same base or different bases. In some embodiments, nucleotides that have not been incorporated at a residue corresponding to a base in the template nucleic acid (e.g., because the first labeled nucleotide has been incorporated at that residue) are not removed from the sequencing reaction mix prior to the introduction and / or incorporation of the second labeled nucleotide. In some embodiments, the first and second labeled nucleotides (and optionally labeled nucleotides for interrogating subsequent bases in the template) are provided in the same sequencing reaction mix, and the first, second, and optionally any subsequent labeled nucleotide(s) are incorporated sequentially in a continuous manner.

[0119] Thus, unlike existing SBS methods, some embodiments of the method disclosed herein use continuous introduction and / or incorporation of nucleotides (e.g., fluorescently labeled A, T, C, and / or G nucleotides) without the need of label deactivation and / or wash steps in between sequential incorporation events for a given template nucleic acid molecule to be sequenced. Rather, in some embodiments, label deactivation (e.g., by cleaving and / or photobleaching the label) of a first incorporated nucleotide may occur stochastically throughout the continuous nucleotide incorporation process, for instance, prior to, during, or after the incorporation of a second, third, fourth, or a subsequent labeled nucleotide.

[0120] Nucleic acid sequencing reaction mixtures, or simply “reaction mixtures,” typically include reagents that are commonly present in polymerase based nucleic acid synthesis reactions. The reaction mixture can include other molecules including, but not limited to, enzymes. In some embodiments, the reaction mixture comprises any reagents or biomolecules generally present in a nucleic acid polymerization reaction. Reaction components may include, but are not limited to, salts, buffers, small molecules, detergents, crowding agents, metals, andDocket No.: ESBI00007-1WG ions. In some embodiments, properties of the reaction mixture may be manipulated, for example, electrically, magnetically, and / or with vibration.

[0121] The provided methods herein may further comprise but do not require one or more wash steps; a temperature change; a mechanical vibration; a pH change; or an optical stimulation that is not dye illumination or photobleaching. In some embodiments, the wash step comprises contacting the substrate and the nucleic acid molecule, the primer, and / or the polymerase with one of more buffers, detergents, protein denaturants, proteases, oxidizing agents, reducing agents, or other agents capable of crosslinking or releasing crosslinks, e.g., crosslinks within a polymerase or crosslinks between a polymerase and nucleic acid. Methods and compositions for nucleic acid sequencing are known, for example, as described in U.S. Patent Nos. 10,246,744 and 10,844,428, incorporated herein by reference in their entireties for all purposes.

[0122] Reaction mixture reagents can include, but are not limited to, enzymes (e.g., polymerase), dNTPs, template nucleic acids, primer nucleic acids, salts, buffers, small molecules, co-factors, metals, and ions. The ions may be catalytic ions, divalent catalytic ions, non-catalytic ions, non-covalent metal ions, or a combination thereof. The reaction mixture can include salts, such as NaCl, KC1, potassium acetate, ammonium acetate, potassium glutamate, or NH4C1 or the like, that ionize in aqueous solution to yield monovalent cations. The reaction mixture can include a source of ions, such as Mg2+, Mn2+, Co2+, Cd2+, and / or Ba2+ ions. The reaction mixture can include tin, Ca2+, Zn2+, Cu2+, Co2+, Fe2+, and / or Ni2+, or other divalent non-catalytic metal cations. In some embodiments, the reaction mixture can include metal cations that may inhibit formation of phosphodiester bonds between the primed template nucleic acid molecule and the cognate nucleotide. In some embodiments, the metal cations can be used (e.g., at a suitable concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0123] In some embodiments, the sequencing reaction conditions comprise contacting the nucleic acid molecule and the primer with a buffer that regulates osmotic pressure. In some embodiments, the reaction mixture comprises a buffer that regulates osmotic pressure. In some embodiments, the buffer is a high salt buffer that includes a monovalent ion, such as a monovalent metal ion (e.g., potassium ion or sodium ion) at a concentration of from about 50 to about 1,500 mM. Salt concentrations in the range of from about 100 to about 1,500 mM, or from about 200 to 1,000 mM may also be used. In some embodiments, the buffer further comprises aDocket No.: ESBI00007-1WG source of glutamate ions (e.g., potassium glutamate). In some embodiments, the buffer comprises a stabilizing agent. In some embodiments, the stabilizing agent is a non-catalytic metal ion (e.g., a divalent non-catalytic metal ion). Non-catalytic metal ions useful in this context include, but are not limited to, calcium, strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, and / or terbium. In some embodiments, the non-catalytic metal ion is strontium, tin, or nickel. In some embodiments, the sequencing reaction mixture comprises strontium chloride or nickel chloride. In some embodiments, the stabilizing agent can be used (e.g., at a suitable concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0124] The buffer can include Tris, Tricine, HEPES, MOPS, ACES, MES, phosphate-based buffers, and acetate-based buffers. The reaction mixture can include chelating agents such as EDTA, EGTA, and the like. In some embodiments, the reaction mixture includes cross-linking reagents.

[0125] In some embodiments, the interaction between the polymerase and template nucleic acid may be manipulated by modulating sequencing reaction parameters such as ionic strength, pH, temperature, or any combination thereof, or by the addition of a destabilizing agent to the reaction. In some embodiments, the destabilizing agent can be used (e.g., at a suitable concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0126] In some embodiments, high salt (e.g., 50 to 1,500 mM) and / or pH changes are utilized to destabilize a complex between the polymerase and template nucleic acid. In some embodiments, the reaction conditions favor the stabilization of a complex among the polymerase, the template nucleic acid, and a labeled nucleotide. By way of example, the pH of the reaction mixture can be adjusted from 4.0 to 10.0 to favor the stabilization of a complex among the polymerase, the template nucleic acid, and a labeled nucleotide. In some embodiments, the pH of the reaction mixture is from 4.0 to 6.0. In some embodiments, the pH of the reaction mixture is 6.0 to 10.0. In some embodiments, a suitable salt concentration and / or a suitable pH can be selected to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.Docket No.: ESBIO0007-1WO

[0127] In some embodiments, the reaction mixture comprises a competitive inhibitor, where the competitive inhibitor may reduce the occurrence of multiple incorporations events in a detection window. In one embodiment, the competitive inhibitor is a non-incorporable nucleotide. In an embodiment, the competitive inhibitor is an aminoglycoside. The competitive inhibitor is capable of replacing either the nucleotide or the catalytic metal ion in the active site, such that the competitive inhibitor occupies the active site preventing or slowing down a nucleotide incorporation. In some embodiments, both an incorporable nucleotide and a competitive inhibitor are introduced, such that the ratio of the incorporable nucleotide and the inhibitor can be adjusted to modulate the rate of incorporation of a single nucleotide at the 3 '-end of the primer. In some embodiments, the competitive inhibitor can be used (e.g., at a low concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0128] In some embodiments, the reaction mixture comprises at least one nucleotide molecule that is a non-incorporable nucleotide. In some embodiments, the reaction mixture comprises one or more nucleotide molecules incapable of incorporation into the primer of the primed template nucleic acid molecule. Such nucleotides incapable of incorporation include, for example, monophosphate nucleotides. For example, the nucleotide may contain modifications to the triphosphate group that make the nucleotide non-incorporable. Examples of non-incorporable nucleotides may be found in U.S. Pat. No. 7,482,120, which is incorporated by reference herein in its entirety. In some embodiments, the primer may not contain a free hydroxyl group at its 3'- end, thereby rendering the primer incapable of incorporating any nucleotide, and, thus, making any nucleotide non-incorporable. In some embodiments, the primer may be processed such that it contains a free hydroxyl group at its 3 '-end to allow nucleotide incorporation. In some embodiments, the non-incorporable nucleotide can be used (e.g., at a low concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0129] In some embodiments, the reaction mixture comprises at least one nucleotide molecule that is incorporable but is incorporated at a slower rate compared to a corresponding naturally- occurring nucleoside triphosphate (e.g., NTP or dNTP). Such nucleotides incorporable at a slower rate may include, for example, diphosphate nucleotides. For example, the nucleotide may contain modifications to the triphosphate group that make the nucleotide incorporable at a slower rate. In some embodiments, the nucleotide incorporable at a slower rate can be used toDocket No.: ESBIO0007-1WO slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0130] In some embodiments, the reaction mixture comprises a polymerase inhibitor. In some embodiments, the polymerase inhibitor is a pyrophosphate analog. In some embodiments, the polymerase inhibitor is an allosteric inhibitor. In some embodiments, the polymerase inhibitor is a DNA or an RNA aptamer. In some embodiments, the polymerase inhibitor competes with a catalytic-ion binding site in the polymerase. In some embodiments, the polymerase inhibitor is a reverse transcriptase inhibitor. The polymerase inhibitor may be an HIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptase inhibitor. The HIV-1 reverse transcriptase inhibitor may be a (4 / 6-halogen / MeO / EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one. In some embodiments, the polymerase inhibitor can be used (e.g., at a low concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0131] In some embodiments, the contacting step is facilitated by the use of a chamber such as a flow cell. The methods and apparatus described herein may employ next generation sequencing technology (NGS), which allows massively parallel sequencing. In some embodiments, single DNA molecules are sequenced in a massively parallel fashion within a reaction chamber. A flow cell may be used but is not necessary. Flowing liquid reagents through the flow cell, which contains an interior solid support surface (e.g., a planar surface), conveniently permits reagent exchange. Immobilized to the interior surface of the flow cell is one or more primed template nucleic acids to be sequenced or interrogated using the procedures described herein. Typical flow cells will include microfluidic valving that permits delivery of liquid reagents (e.g., components of the “reaction mixtures” discussed herein) to an entry port. Liquid reagents can be removed from the flow cell by exiting through an exit port.

[0132] In some embodiments, a reaction chamber disclosed herein can comprise a reagent wall, an imaging area, and optionally an outlet configured to remove molecules of one or more of the polymerases, the first detectably labeled nucleotide, the second detectably labeled nucleotide, and / or one or more other reagents from the imaging area. In some embodiments, the device may comprise one or more vents but no outlet or exit port for the reaction mixture. In some embodiments, a method disclosed herein does not comprise a step of removing liquid reagents through an outlet or exit port, e.g., from a reaction chamber such as a flow cell.Docket No.: ESBIG0007-1WO

[0133] The methods disclosed herein may but do not need to be used in combination with any NGS sequencing methods. The sequencing technologies of NGS include but are not limited to pyrosequencing, sequencing-by-synthesis with reversible dye terminators, sequencing by oligonucleotide probe ligation, and ion semiconductor sequencing. Nucleic acids such as DNA or RNA from individual samples can be sequenced individually (singleplex sequencing) or nucleic acids such as DNA or RNA from multiple samples can be pooled and sequenced as indexed genomic molecules (multiplex sequencing) on a single sequencing run, to generate up to several hundred million reads of sequences. Examples of sequencing technologies that can be used to obtain the sequence information according to the present method are further described here.

[0134] Some sequencing technologies are available commercially, such as the sequencing-by- synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina / Solexa (Hayward, Calif.) and Helicos Biosciences (Cambridge, Mass.).

[0135] While the automated Sanger method is considered as a ‘first generation’ technology, Sanger sequencing including the automated Sanger sequencing, can also be employed in the methods described herein. Additional suitable sequencing methods include, but are not limited to nucleic acid imaging technologies, e.g., atomic force microscopy (AFM) or transmission electron microscopy (TEM).

[0136] In some embodiments, the disclosed methods may be used in combination with massively parallel sequencing of nucleic acid molecules using Illumina's sequencing-by- synthesis and reversible terminator-based sequencing chemistry. In some implementation, a method disclosed herein can use a flow cell having a glass slide with lanes.

[0137] After sequencing of nucleic acid molecules, sequence reads of predetermined length, e.g., at least about 15 bp, are localized by mapping (alignment) to a known reference sequence or genome (e.g., viral sequences or genomes). A number of computer algorithms are available for aligning sequences, including without limitation BLAST, BLITZ, FASTA, BOWTIE, or ELAND (Illumina, Inc., San Diego, Calif., USA).

[0138] In some embodiments, the provided sequencing methods disclosed herein may regulate polymerase interaction with the nucleotides and template nucleic acid (as well as rate of nucleotide incorporation) in a manner that reveals the identity of the next base while controlling the chemical addition of a nucleotide. In some embodiments, the SBS reaction conditionDocket No.: ESBIO0007-1WO comprises a plurality of primed template nucleic acids, polymerases, nucleotides, or any combination thereof. In some embodiments, the plurality of nucleotides comprises 1, 2, 3, 4, or more types of different nucleotides, for example dATP, dTTP (or dUTP), dGTP, and dCTP.

[0139] In some embodiments, the method can further comprise contacting the nucleic acid molecule with the substrate to immobilize the nucleic acid molecule. In some embodiments, the nucleic acid molecule can be immobilized at a density of one molecule per at least about 250 nm2, at least about 200 nm2, at least about 150 nm2, at least about 100 nm2, at least about 90 nm2, at least about 80 nm2, at least about 70 nm2, at least about 60 nm2, at least about 50 nm2, at least about 40 nm2, at least about 30 nm2, at least about 20 nm2, at least about 10 nm2, at least about 5 nm2, or in between any two of the aforementioned values. Methods and compositions for arraying biomolecules on a substrate, e.g., as described in US 2005 / 0042649 (incorporated herein by reference in its entirety for all purposes), may be used in methods disclosed herein.

[0140] In some embodiments, a subset of nucleic acid molecules (e.g., nucleic acid strands to be sequenced) on the substrate may be active at one or more time points. In some embodiments, at any one time, a first subset of nucleic acid molecules on the substrate is active (e.g., allowing nucleotide incorporation into a sequencing primer using a single-stranded sequence as template) while a second subset of nucleic acid molecules on the substrate is inactive (e.g., not allowing nucleotide incorporation into a sequencing primer using a single-stranded sequence as template). In some embodiments, at one or more time points, a first subset of nucleic acid molecules on the substrate is activated (e.g., by a first set of polymerase and / or primer molecules) for nucleotide incorporation, while a second subset of nucleic acid molecules on the substrate is not activated (e.g., by the first set of polymerase and / or primer molecules), thus only signals associated with the first subset of nucleic acid molecules are detected. At one or more other time points, the second subset of nucleic acid molecules on the substrate is activated (e.g., by a second set of polymerases and / or primer molecules) for nucleotide incorporation, while the first subset of nucleic acid molecules on the substrate is not activated (e.g., by the second set of polymerases and / or primer molecules), thus only signals associated with the second subset of nucleic acid molecules are detected. In some embodiments, the first and second sets of polymerases and / or primer molecules can be introduced at different time points, e.g., in sequential cycles with optional washing steps between cycles (e.g., to remove a set of polymerases and / or primer molecules for SB S of a first subset of strands before introducing the next set of polymerases and / or primer molecules for SBS of a second subset of strands).Docket No.: ESBI00007-1WG

[0141] In some embodiments, the artificial substrate can comprise a bead, a planar substrate, a solid surface, a flow cell, a semiconductor chip, a well, a pillar, a chamber, a channel, a through hole, a nanopore, or any combination thereof. In some embodiments, the substrate can comprise a microwell, a micropillar, a microchamber, a microchannel, or any combination thereof.VII. Compositions, Kits, and Applications

[0142] Also provided herein are compositions and kits comprising one or more of the primers, nucleic acid molecules, substrates, nucleotides including detectably labeled nucleotides, polymerases, and reagents for performing the methods provided herein, for example reagents required for one or more steps comprising hybridization, ligation, amplification, detection, sequencing, and / or sample preparation as described herein, for example, in Section IV.

[0143] The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods.

[0144] In some embodiments, the kits can contain reagents and / or consumables required for performing one or more steps of the provided methods. In some embodiments, the kits contain reagents for sample processing, such as nucleic acid extraction, isolation, and / or purification, e.g., RNA extraction, isolation, and / or purification. In some embodiments, the kits contain reagents, such as enzymes and buffers for ligation and / or amplification, such as ligases and / or polymerases. In some embodiments, the kits contain reagents, such as enzymes and buffers for primer extension and / or nucleic acid sequencing, such as polymerases and / or transcriptases. In some aspects, the kit can also comprise any of the reagents described herein, e.g., buffer components for tuning the rate of nucleotide incorporation and / or for tuning the rate of signal deactivation (e.g., by photobleaching). In some embodiments, the kits contain reagents for signal detection during sequencing, such as detectable labels and detectably labeled molecules. In some embodiments, the kits optionally contain other components, for example nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, and reagents for additional assays.

[0145] In some aspects, the provided embodiments can be applied in analyzing nucleic acid sequences, such as DNA and / or RNA sequencing. In some aspects, the embodiments can be applied in an imaging or detection method for multiplexed nucleic acid analysis. In someDocket No.: ESBI00007-1WO aspects, the provided embodiments can be used to identify or detect regions of interest in target nucleic acids, such as viral DNA or RNA. In some embodiments, the region of interest comprises one or more nucleotide residues, such as a single-nucleotide polymorphism (SNP), a single-nucleotide variant (SNV), substitutions such as a single-nucleotide substitution, mutations such as a point mutation, insertions such as a single-nucleotide insertion, deletions such as a single-nucleotide deletion, translocations, inversions, duplications, and / or other sequences of interest.

[0146] In some aspects, the embodiments can be applied in investigative and / or diagnostic applications, for example, for characterization or assessment of a sample from a subject. Applications of the provided method can comprise biomedical research and clinical diagnostics. For example, in biomedical research, applications comprise, but are not limited to, genetic and genomic analysis for biological investigation or drug screening. In clinical diagnostics, applications comprise, but are not limited to, detecting gene markers such as disease, immune responses, bacterial or viral DNA / RNA for patient samples, loss of genetic heterozygosity, the presence of gene alleles indicative of a predisposition towards disease or good health, likelihood of responsiveness to therapy, or in personalized medicine or ancestry.EXAMPLESExample 1: Using fluorophores with overlapping emission wavelengths to analyze nucleotide sequences

[0147] This section provides an example of using fluorophores with different but overlapping emission wavelengths to determine the sequence of a nucleic acid template. It also provides an exemplary schematic of an optical system used for the detection of fluorophores with the aforementioned characteristics. The sequences are analyzed by contacting the nucleotides with labeled binders and measuring the emission intensities to detect the identity of the label and the binder. The binders are nucleotide bases and the fluorophore labels have overlapping emission spectrums. While typically using a spectral intensity alone requires fluorophores to have minimal spectral overlap, the use of intensity characteristics circumvents this requirement, provided that a unique profile can be constructed from the intensity characteristics for each fluorophore used to label the binders.Docket No.: ESBI00007-1WG

[0148] In the present example, four fluorophores are selected to label four binders. Binders comprise an A nucleotide, a T nucleotide, a C nucleotide, or a G nucleotide. The binders are labeled with fluorophores that have spectral overlap.

[0149] First, a sample containing one or more molecules to be analyzed is provided. For nucleotide sequencing, a nucleic acid template can be provided on an artificial substrate, and it is at an optically resolvable distance from other nucleic acid templates on the artificial substrate. In this example, the artificial substrate is a flow cell. The nucleic acid templates are then contacted with a mixture of fluorescently labeled nucleotides (dNTPs). Once one of the four dNTPs is incorporated by a polymerase into the nucleic acid template, the fluorescent molecules can be excited. In sequencing by synthesis (SBS), the fluorescently labeled nucleotides are incorporated after clonal amplification. As shown in FIG. 3B, the same light source can be used to excite all of the fluorophores and the fluorescence at different wavelengths can be split through a series of dichroic mirrors and the wavelengths can be measured with separate detectors. FIG. 3A shows three measured wavelengths (XI, X2, X3). The measured wavelengths are selected to ensure that the relative difference between the fluorescent intensities at those wavelengths is specific to a tested fluorophore.

[0150] After determining emission intensities at two or more wavelengths, in this case three wavelengths, one or more intensity ratios can be determined. In this example, emission intensities for a first wavelength (XI), a second wavelength (X2), and a third wavelength were determined for fluorophore 1, fluorophore2, fluorophore3, and fluoropho re4 (FIG. 3 A), resulting in a total of twelve measurements. These determined intensities were then used to determine fluorophore-specific intensity ratios.

[0151] Additionally, one of the three measured wavelengths provides a baseline value. For example, the difference between the first wavelength (XI) and a third wavelength (X3) was determined. The difference between the second wavelength (X2) and the third wavelength (X3) was also determined. The ratio was then obtained from these baseline corrected values ((XI- X3) / (X2- X3)), and the baseline corrected intensity ratio was used to determine the fluorophore’s identity. This intensity ratio improves signal-to-noise ratio as it accounts for background luminescence and technical variations.Docket No.: ESBI00007-1WGExample 2: Using fluorophores with overlapping excitation wavelengths to analyze nucleotide sequences

[0152] This section provides an example of using fluorophores with different but overlapping excitation wavelengths to determine the sequence of a nucleic acid template. It also provides an exemplary schematic of an optical system used for the detection of fluorophores with the aforementioned characteristics. The sequences are analyzed by contacting the nucleotides with labeled binders and measuring the excitation intensities to detect the identity of the label and the binder. The binders are nucleotide bases and the fluorophore labels have overlapping excitation spectrums.

[0153] In the present example, four fluorophores are selected to label four binders. Binders comprise an A nucleotide, a T nucleotide, a C nucleotide, or a G nucleotide. The binders are labeled with fluorophores that have spectral overlap.

[0154] First, a sample containing one or more molecules to be analyzed is provided. For nucleotide sequencing, a nucleic acid template can be provided on an artificial substrate. In this example, the nucleic acid templates are provided on a flow cell. The nucleic acid templates are then contacted with a mixture of fluorescently labeled nucleotides (dNTPs). Once one of the four dNTPs is incorporated by a polymerase into the nucleic acid template, the fluorescent molecules can be excited with separate light sources sequentially. For example, light source 1, light source 2, and light source 3 were turned on and measured sequentially as depicted in FIG. 4B. All the emission intensities were detected with the same detector and wavelength filter.FIG. 4A shows three measured wavelengths (XI, X2, X3) for excitation intensities. The measured wavelengths are selected to ensure that the relative difference between the fluorescent intensities at those wavelengths is specific to a tested fluorophore.

[0155] After determining excitation intensities at three wavelengths, an intensity ratio for each fluorophore was determined. Excitation intensities for a first wavelength (XI), a second wavelength (X2), and a third wavelength were determined for fluorophore 1, fluorophore2, fluorophore3, and fluorophore4 (FIG. 4A), resulting in a total of twelve measurements.

[0156] Additionally, one of the three measured wavelengths was used as a baseline value to obtain a baseline corrected ratio. The difference between the first wavelength (XI) and a third wavelength (X3) was determined, and the difference between the second wavelength (X2) and theDocket No.: ESBI00007-1WO third wavelength (X3) was also determined. The ratio was then obtained from these baseline corrected values ((XI -X3) / (X2- X3)), and it was used to determine the fluorophore’s identity.

[0157] It should be understood from the foregoing that, while particular implementations of the disclosed methods and systems have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents.

Claims

Docket No.: ESBIO0007-1WOCLAIMS1. A method for analyzing one or more molecules, comprising:(a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of emission wavelengths and / or different but overlapping spectrums of excitation wavelengths;(b) detecting:(i) a first intensity characteristic of emission intensities and / or of excitation intensities of the first luminophore at two or more different wavelengths, and(ii) a second intensity characteristic of emission intensities and / or of excitation intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.

2. A method for analyzing one or more molecules, comprising:(a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of emission wavelengths; and(b) detecting:(i) a first intensity characteristic of emission intensities of the first luminophore at two or more different wavelengths, and(ii) a second intensity characteristic of emission intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.Docket No.: ESBIO0007-1WO3. A method for analyzing one or more molecules, comprising:(a) contacting the one or more molecules with: (i) a first binder labeled with a first luminophore, and (ii) a second binder labeled with a second luminophore, wherein the first binder and the second binder directly or indirectly bind to different targets, and wherein the first luminophore and the second luminophore have different but overlapping spectrums of excitation wavelengths;(b) detecting:(i) a first intensity characteristic of excitation intensities of the first luminophore at two or more different wavelengths, and(ii) a second intensity characteristic of excitation intensities of the second luminophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first binder and the second binder, respectively, thereby identifying the different targets in the one or more molecules.

4. The method of any one of claims 1-3, wherein the first intensity characteristic is about 1.25-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold or more of the second intensity characteristic, or vice versa.

5. The method of any one of claims 1-4, wherein the detecting in (b) comprises:(i) detecting emission intensities or excitation intensities of the first luminophore at three or more different wavelengths; and(ii) detecting emission intensities or excitation intensities of the second luminophore at the three or more different wavelengths.

6. The method of any one of claims 1-5, wherein the detecting in (b) comprises:(i) detecting first intensity characteristics of emission intensities or of excitation intensities of the first luminophore at three or more different wavelengths; and(ii) detecting second intensity characteristics of emission intensities or of excitation intensities of the second luminophore at the three or more different wavelengths.

7. The method of any one of claims 1-6, wherein the first intensity characteristic(s) and / or the second intensity characteristic(s) are first baseline-corrected intensity ratio(s) and second baseline-corrected intensity ratio(s), respectively.Docket No.: ESBIO0007-1WO8. The method of claim 7, wherein the first baseline-corrected intensity ratio 7? I is calculated using measured intensities of the first luminophore at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, the measured intensities at XI, X2, and X3 are 71, 72 and 73, respectively, and 7? I = (72 - 71) / (73 -71).

9. The method of claim 8, wherein the second baseline-corrected intensity ratio 7?2 is calculated using measured intensities of the second luminophore at XI, X2, and X3, the measured intensities at XI, X2, and X3 are 71, 12 and 13, respectively, and 7?2 = (72 - 71) / (73 - 71).

10. The method of any one of claims 1-6, wherein the first intensity characteristic(s) and / or the second intensity characteristic(s) are determined using polynomial regression or fitting.

11. The method of claim 10, wherein the polynomial regression or fitting comprises using formula y = a + bx + ex2, wherein x is a wavelength and y is a measured intensity of the first or second luminophore at the wavelength x.

12. The method of claim 11, wherein the first intensity characteristic(s) and / or the second intensity characteristic(s) are b and c which are determined using the polynomial regression or fitting.

13. The method of claim 11, wherein the first intensity characteristic(s) and / or the second intensity characteristic(s) are b / c or c / b, wherein b and c are determined using the polynomial regression or fitting.

14. The method of any one of claims 1-13, wherein the one or more molecules comprise a polynucleotide, a polypeptide, a carbohydrate, a lipid, a small molecule, or any combination thereof.

15. The method of any one of claims 1-14, wherein each of the one or more molecules is present as a separate molecule or in complex with one or more other molecules.

16. The method of any one of claims 1-15, wherein each of the one or more molecules is present on an artificial substrate, in a biological sample, or in a solution, and wherein the first binder and the second binder are contacted with the artificial substrate, the biological sample, or the solution.

17. The method of claim 16, wherein the artificial substrate is a patterned flow cell or a nonpatterned flow cell.Docket No.: ESBI00007-1WG18. The method of claim 16, wherein the biological sample is a cell or a tissue sample.

19. The method of any one of claims 1-18, wherein each of the one or more molecules is present in and / or on a cell, an intracellular organelle, an extracellular vesicle, a virus, a viral-like particle (VLP), a lipid nanoparticle (LNP), a protein complex, a nucleic acid complex, or a protein-nucleic acid complex.

20. The method of any one of claims 1-19, wherein the one or more molecules comprise a nucleic acid template to be sequenced, optionally wherein the nucleic acid template is a DNA or an RNA.

21. The method of claim 20, wherein: the nucleic acid template is present in a clonal cluster of nucleic acid molecules each comprising a copy of a sequence to be sequenced; or the nucleic acid template is a rolling circle amplification product (RCP).

22. The method of claim 20, wherein the nucleic acid template is a single molecule located on an artificial substrate at an optically resolvable distance from other nucleic acid templates on the artificial substrate.

23. The method of any one of claims 20-22, wherein the first binder and the second binder are nucleotides that base pair with a nucleic acid residue in the nucleic acid template based on sequence complementarity, and wherein the different targets are nucleic acid residues of different bases.

24. The method of claim 23, wherein the nucleotides comprise one or more reversibly terminated nucleotides, one or more irreversibly terminated nucleotides, and / or one or more nucleotides that are not terminated.

25. The method of claim 23 or claim 24, wherein each of the nucleotides is independently incorporated or not incorporated into a priming strand that hybridizes to the nucleic acid template.

26. The method of any one of claims 23-25, wherein each of the nucleotides is an A nucleotide, a T / U nucleotide, a C nucleotide, or a G nucleotide.

27. The method of any one of claims 20-22, wherein the first binder and the second binder are complexes each comprising: (i) a nucleotide, and (ii) the first luminophore and the secondDocket No.: ESBIO0007-1WO luminophore, respectively, wherein the nucleotides in the complexes base pair with a nucleic acid residue in the nucleic acid template based on sequence complementarity, and wherein the different targets are nucleic acid residues of different bases.

28. The method of claim 27, wherein each of the complexes comprises: (i) a plurality of nucleotide moieties of the same base, and (ii) one or more copies of the first luminophore or the second luminophore.

29. The method of any one of claims 1-19, wherein the one or more molecules comprise different nucleic acid analytes.

30. The method of claim 29, wherein the different nucleic acid analytes comprise a genomic DNA, an RNA, a cDNA, a nucleic acid probe, a reporter oligonucleotide conjugated to a binding moiety that binds to a non-nucleic acid analyte, a rolling circle amplification product (RCP), or any combination thereof.

31. The method of claim 29 or claim 30, wherein the first binder and the second binder are nucleic acid probes, and the different targets are the different nucleic acid analytes.

32. The method of claim 31 , wherein the nucleic acid probes hybridize to the different nucleic acid analytes.

33. The method of any one of claims 1-19, wherein the one or more molecules comprise different protein analytes.

34. The method of claim 33, wherein the first binder and the second binder are antibodies or aptamers, and the different targets are the different protein analytes.

35. The method of any one of claims 1-34, wherein the first luminophore and the second luminophore are fluorophores.

36. The method of any one of claims 1-35, wherein: a single light source is used to excite the different luminophores, and a different detector is used to detect the emission intensities at each different emission wavelength; and / or a separate light source is used to excite each different luminophore at each different excitation wavelength, and a single detector is used to detect the excitation intensities of the different luminophores.Docket No.: ESBI00007-1WG37. The method of claim 36, wherein the emission intensities at the different emission wavelengths are simultaneously detected by the different detectors.

38. The method of claim 36, wherein the excitation intensities at the different excitation wavelengths are sequentially detected by the single detector.

39. The method of any one of claims 36-38, wherein the single light source or each of the separate light sources is independently a light emitting diode (LED).

40. A method for analyzing a nucleic acid template, comprising:(a) contacting the nucleic acid template with: (i) a first nucleotide which is of a first base and labeled with a first fluorophore, and (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, wherein the first base and the second base are different bases, and wherein the first fluorophore and the second fluorophore have different but overlapping spectrums of emission wavelengths and / or different but overlapping spectrums of excitation wavelengths;(b) allowing nucleotide binding to the nucleic acid template, wherein the first nucleotide or the second nucleotide binds to a complementary nucleic acid residue in the nucleic acid template, optionally wherein the bound nucleotide is incorporated into a sequencing strand hybridized to the nucleic acid template;(c) imaging the bound and optionally incorporate nucleotide, wherein (i) or (ii) is determined based on the imaging:(i) a first intensity characteristic of emission intensities and / or of excitation intensities of the first fluorophore at two or more different wavelengths,(ii) a second intensity characteristic of emission intensities and / or of excitation intensities of the second fluorophore at the two or more different wavelengths, wherein the first intensity characteristic and the second intensity characteristic correspond to the first nucleotide and the second nucleotide, respectively, thereby identifying the bound and optionally incorporate nucleotide and the complementary nucleic acid residue in the nucleic acid template.

41. The method of claim 40, wherein the imaging in (c) comprises:Docket No.: ESBI00007-1WG imaging the bound and optionally incorporate nucleotide at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, and at each of XI, X2, and X3, measuring a first, second, and third fluorescence intensity, respectively, wherein a baseline-corrected intensity ratio R is determined according to R = (value of the second fluorescence intensity - value of the first fluorescence intensity) / (value of the third fluorescence intensity - value of the first fluorescence intensity), and wherein the baseline-corrected intensity ratio R is the first intensity characteristic or the second intensity characteristic, thereby identifying the bound and optionally incorporate nucleotide as the first nucleotide or the second nucleotide.

42. The method of claim 41, wherein the baseline-corrected intensity ratio R is a first baseline-corrected intensity ratio R 1 corresponding to the first nucleotide, wherein the measured fluorescence intensities of the first nucleotide at XI, X2, and X3 are 71, 72 and 73, respectively, and Tfl = (72 -71 ) / (73 - 71).

43. The method of claim 41, wherein the baseline-corrected intensity ratio 7? is a second baseline-corrected intensity ratio 7?2 corresponding to the second nucleotide, wherein the measured fluorescence intensities of the first nucleotide at XI, X2, and X3 are il, 12 and 13, respectively, and 7?2 = (12 - Z1 ) / (Z3 - i 1 ).

44. The method of claim 40, wherein the imaging in (c) comprises: imaging the bound and optionally incorporate nucleotide at a first wavelength XI, a second wavelength X2, and a third wavelength X3, wherein XI, X2, and X3 are different, and at each of XI, X2, and X3, measuring a first, second, and third fluorescence intensity, respectively, subjecting the measured fluorescence intensities and XI, X2, and X3 to polynomial regression or fitting, thereby generating formula y = a + bx + ex2, wherein x is a wavelength and y is a fluorescence intensity at the wavelength x, and wherein (i) b and c and / or (ii) b / c or c / b is the first intensity characteristic or the second intensity characteristic, thereby identifying the bound and optionally incorporate nucleotide as the first nucleotide or the second nucleotide.

45. The method of any one of claims 40-44, wherein the contacting in (a) comprises contacting a plurality of different nucleic acid templates with: (i) a first nucleotide which is of aDocket No.: ESBI00007-1WO first base and labeled with a first fluorophore, (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, (iii) a third nucleotide which is of a third base and labeled with a third fluorophore, and (iv) a fourth nucleotide which is of a fourth base and labeled with a fourth fluorophore, wherein the plurality of different nucleic acid templates comprise the nucleic acid template, and the first base, the second base, the third base, and the fourth base are different bases.

46. The method of claim 45, wherein the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore have different but overlapping spectrums of emission wavelengths.

47. The method of claim 46, wherein the imaging in (c) comprising using a single light source to excite the fluorophores and using multiple detectors each for detecting an emission intensity at a different wavelength.

48. The method of claim 45, wherein the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore have different but overlapping spectrums of excitation wavelengths.

49. The method of claim 48, wherein the imaging in (c) comprising using a single detector and using multiple light sources each for exciting the fluorophores at a different wavelength.

50. A kit or composition, comprising: (i) a first nucleotide which is of a first base and labeled with a first fluorophore, (ii) a second nucleotide which is of a second base and labeled with a second fluorophore, (iii) a third nucleotide which is of a third base and labeled with a third fluorophore, and (iv) a fourth nucleotide which is of a fourth base and labeled with a fourth fluorophore, wherein the first base, the second base, the third base, and the fourth base are different bases, and the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore have different but overlapping emission spectrums and / or different but overlapping excitation spectrums.

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