Enhanced optical detection for nucleic acid sequencing using heat-dependent fluorophore tags

Abstract

Disclosed herein is an improved method and system for sequencing nucleic acids using the temperature dependence of the luminescence intensity of fluorescent dyes. The temperature of the sequencing reaction is adjusted during each sequencing cycle, and the fluorescent markers of the incorporated dNTPs are detected using the emission or non-emission of light that reaches or exceeds a threshold at different temperatures or within different temperature ranges of these fluorescent dyes, thereby sequencing nucleic acids. The disclosed method enables determination of the dNTPs incorporated at any given site with a reasonable number of chemical steps without the need for the complex optics necessary for prior art systems.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Non-Provisional Application No. 16 / 569,823, filed on September 13, 2019, entitled “ENHANCED OPTICAL DETECTION FORNUCLEIC ACID SEQUENCING USING THERMALLY-DEPENDENT FLUOROPHORE TAGS,” which is hereby incorporated by reference herein for all purposes. Background Art

[0003] Deoxyribonucleic acid (DNA) is an example of a nucleic acid. DNA is a polymer or chain composed of only four possible component molecules or nucleotide bases (also referred to herein simply as "nucleotides"): adenine (A), cytosine (C), guanine (G), and thymine (T).

[0004] DNA strands can be single-stranded or double-stranded. Single-stranded DNA (ssDNA) can bind to its unique ssDNA complementary sequence to form its own double-stranded form (dsDNA). Adenine bases only bind to thymine bases, and cytosine bases only bind to guanine bases. For example, the sequence ACTGGC is complementary to and binds to TGACCG. Because bases can appear in any order, the goal of DNA sequencing is to "read" a specific ssDNA strand or template, that is, to reliably determine the sequence of its bases.

[0005] One type of nucleic acid sequencing used for DNA sequencing is called "sequencing by synthesis" (SBS). SBS involves the binding of template DNA to which primers are hybridized, the incorporation of deoxynucleoside triphosphates (dNTPs), and the detection of the incorporated dNTPs. The ssDNA (i.e., the strand to be sequenced) is sequenced by iterative construction of its complementary sequence, wherein a polymerase is used to accelerate the rate of nucleotide incorporation. In one type of SBS, a long strand of DNA to be sequenced is cut into smaller strands, and these smaller strands can be sequenced simultaneously, potentially after "amplification" (e.g., replication) using processes known in the art such as bridge amplification.

[0006] In one method of SBS, the accumulation process of the complementary sequence of the bases of the sequenced DNA chain is inferred by detecting fluorescence. The fluorescent moiety is attached to each of the four dNTPs (A, T, C and G). The incorporation of different dNTPs into the growing nucleic acid chain can then be distinguished because each of the fluorescent moieties excites and emits light of a different wavelength, which enables the determination of the target sequence. Typically, the fluorescently labeled dNTPs are excited and measured by one of the four optical filters in the fluorescent sequencing instrument, each optical filter corresponding to a different dye.

[0007] Such techniques can use fluorescence to generate images, such as using an epifluorescence microscope or a confocal microscope. The sample is illuminated with light of one or more specific wavelengths absorbed by the fluorophore, causing them to emit light of a longer wavelength (e.g., a different color than the light absorbed). Spectral emission filters separate the illumination light from the weaker emitted fluorescence. Fluorescence microscopes typically include a light source (e.g., a laser), an excitation filter, a dichroic mirror (or beam splitter), and an emission filter. The filters and dichroic mirror / beam splitter are selected to match the spectral excitation and emission characteristics of the fluorophore used to label the sample. The distribution (e.g., color, wavelength, etc.) of a single fluorophore is imaged at a time. Several monochromatic images must be combined to produce a multicolor image of several types of fluorophores.

[0008] State-of-the-art sequencing systems that rely on fluorescence signal detection can deliver throughputs of up to 20 billion reads per run. However, achieving this performance requires large-area flow cells, high-precision free-space imaging optics, and expensive high-power lasers to generate enough fluorescence signal for successful base detection.

[0009] Therefore, there is a constant need for improvements in nucleic acid sequencing technology. Summary of the invention

[0010] This summary represents non-limiting embodiments of the present invention.

[0011] Improvements to DNA sequencing are disclosed herein, in which fluorophores are used to label dNTP types. The selected fluorophore emits light in a temperature-dependent manner within a temperature range between room temperature and high temperature (e.g., 100°C). When the intensity of the emitted light is lower than the sensitivity threshold of the detector for capturing light, the fluorophore is not detected. Conversely, when the intensity of the emitted light is higher than the sensitivity threshold, the fluorophore is detected. By detecting the luminescence intensity (or the case where the luminescence intensity exceeds the threshold) at multiple different temperatures (or within multiple different and non-overlapping temperature ranges), the determination of the dNTP incorporated at any given site can be determined by a reasonable number of (e.g., less than conventional fluorophore-based systems) chemical steps, without the need for more complex optical devices required by existing systems.

[0012] In some embodiments, the method for sequencing nucleic acid uses a sequencing device including a fluid channel with multiple sites, and the multiple sites are used to attach multiple nucleic acid chains to be sequenced to the surface of the fluid channel. In some such embodiments, the method includes adding (i) multiple nucleic acid chains, (ii) multiple nucleic acid polymerase molecules, (iii) a first fluorescently labeled nucleotide precursor comprising a first fluorescent marker, and (iv) a second fluorescently labeled nucleotide precursor comprising a second fluorescent marker to the fluid channel in one or more rounds of addition. In the first temperature range and the second temperature range, the intensity of the first fluorescent marker is greater than or equal to the first threshold, the second temperature range is lower than the first temperature range, and the intensity of the second fluorescent marker is less than or equal to the second threshold in the first temperature range and greater than or equal to the second threshold in the second temperature range. The method also includes setting the temperature in the fluid channel within the first temperature range, detecting the first intensity at each site in the multiple sites when the temperature of the fluid channel is within the first temperature range, and responding to the first intensity at a specific site in the multiple sites being greater than or equal to the first value, thereby determining that the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site. The first intensity detected at the specific site can be zero or non-zero. The method also includes setting the temperature in the fluid channel within a second temperature range, detecting a second intensity at each of the plurality of sites when the temperature of the fluid channel is within the second temperature range, and determining that the second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the second intensity at the specific site being greater than or equal to the second value and the first intensity at the specific site being less than the first value. The second intensity detected at the specific site may also be zero or non-zero.

[0013] In some embodiments, the first threshold and the second threshold are approximately the same. In other embodiments, the first threshold and the second threshold are different.

[0014] In some embodiments, the first value and the second value are approximately the same. In other embodiments, the first value and the second value are different.

[0015] In some embodiments, the first value is a first threshold value, and the second value is a second threshold value. In some embodiments, the first value is based on the first threshold value, and the second value is based on the second threshold value.

[0016] In some embodiments, the method also includes adding a third fluorescently labeled nucleotide precursor containing a third fluorescent marker to the fluid channel, wherein the intensity of the third fluorescent marker is less than a third threshold value within the first temperature range and the second temperature range and is greater than or equal to the third threshold value within the third temperature range, and the third temperature range is lower than the second temperature range; setting the temperature within the fluid channel within the third temperature range; detecting a third intensity (which may be zero or non-zero) at each of the multiple sites when the temperature of the fluid channel is within the third temperature range; and determining that the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the third intensity at the specific site being greater than or equal to a third value, and the first intensity at the specific site being less than the first value, and the second intensity at the specific site being less than the second value.

[0017] In some such embodiments, the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, and the third fluorescently labeled nucleotide precursor are added to the fluidic channel substantially simultaneously.

[0018] In some embodiments, the method also includes responding to the first intensity at the specific site being less than the first value, and the second intensity at the specific site being less than the second value, and the third intensity at the specific site being less than the third value, thereby determining that the fourth unlabeled precursor has been incorporated into the extendable primer at the specific site. In some such embodiments, at least two of the first threshold, the second threshold, and the third threshold are approximately the same. In some embodiments, the first threshold and the second threshold are different.

[0019] In some embodiments, at least two of the first value, the second value, and the third value are substantially the same. In some embodiments, the first value, the second value, and the third value are different.

[0020] In some embodiments, the method also includes adding a fourth fluorescently labeled nucleotide precursor containing a fourth fluorescent marker to the fluid channel, wherein the intensity of the fourth fluorescent marker is less than a fourth threshold in each of the first temperature range, the second temperature range, and the third temperature range, and is greater than or equal to the fourth threshold in the fourth temperature range, and the fourth temperature range is lower than the third temperature range; setting the temperature within the fluid channel within the fourth temperature range; detecting a fourth intensity (which may be zero or non-zero) at each of the multiple sites when the temperature of the fluid channel is within the fourth temperature range; and determining that the fourth fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the fourth intensity at the specific site being greater than or equal to a fourth value, and the first intensity at the specific site being less than the first value, and the second intensity at the specific site being less than the second value, and the third intensity at the specific site being less than the third value.

[0021] In some such embodiments, two or more of the first threshold, the second threshold, the third threshold, and the fourth threshold are substantially the same.

[0022] In some embodiments, two or more of the first value, the second value, the third value, and the fourth value are approximately the same.

[0023] In some embodiments, the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, the third fluorescently labeled nucleotide precursor, and the fourth fluorescently labeled nucleotide precursor are added to the fluidic channel substantially simultaneously.

[0024] In some embodiments, a method for sequencing nucleic acids using a sequencing device including a fluid channel having a plurality of sites is provided, the plurality of sites being used to attach a plurality of nucleic acid chains to be sequenced to a surface of the fluid channel, the method comprising: adding to the fluid channel in one or more rounds of addition (i) a plurality of nucleic acid chains, (ii) a plurality of nucleic acid polymerase molecules, (iii) a first fluorescently labeled nucleotide precursor comprising a first fluorescent marker, wherein the intensity of the first fluorescent marker is less than a first threshold value within a first temperature range and is greater than or equal to the first threshold value within a second temperature range, the second temperature range being lower than the first temperature range, (iv) a second fluorescently labeled nucleotide precursor comprising a second fluorescent marker, wherein the intensity of the second fluorescent marker is greater than or equal to the first threshold value within a first temperature range, and a second threshold within a second temperature range, and (v) a third fluorescently labeled nucleotide precursor comprising a first fluorescent marker and a second fluorescent marker; setting the temperature within the fluid channel within the first temperature range; when the temperature of the fluid channel is within the first temperature range, detecting a first intensity (which may be zero or non-zero) at each of the multiple sites; setting the temperature within the fluid channel within a second temperature range; when the temperature of the fluid channel is within the second temperature range, detecting a second intensity (which may be zero or non-zero) at each of the multiple sites; and determining whether one of the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, or the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at a specific site among the multiple sites.

[0025] In some such embodiments, the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, and the third fluorescently labeled nucleotide precursor are added to the fluidic channel substantially simultaneously.

[0026] In some embodiments, determining whether one of a first fluorescently labeled nucleotide precursor, a second fluorescently labeled nucleotide precursor, or a third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at a specific site among the multiple sites includes: in response to a first intensity at the specific site being less than a first value, and a second intensity at the specific site being greater than or equal to a second value, thereby determining that the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site; in response to the first intensity at the specific site being greater than or equal to the first value, and the second intensity at the specific site being greater than or equal to a third value, and the third value being greater than each of the first value and the second value, thereby determining that the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site; and in response to the first intensity at the specific site being greater than or equal to the first value, and the second intensity at the specific site being greater than or equal to the second value and less than the third value, thereby determining that the second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site.

[0027] In some such embodiments, the method further comprises determining that a fourth unlabeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the first intensity at the specific site being less than a first value and the second intensity at the specific site being less than a second value.

[0028] In some embodiments, the method also includes determining that none of the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, or the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at a specific site, and in response to determining that none of the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, or the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site, inferring that a fourth unlabeled nucleotide precursor has been incorporated into the extendable primer at the specific site.

[0029] In some embodiments, the first threshold and the second threshold are approximately the same. In other embodiments, the first threshold and the second threshold are different.

[0030] In some embodiments, the first value and the second value are substantially the same. In other embodiments, the first value and the second value are different.

[0031] In some embodiments, the third value is approximately or exactly the sum of the first value and the second value.

[0032] In some embodiments, the first value is a first threshold and the second value is a second threshold.

[0033] In some embodiments, the first value is based on a first threshold and the second value is based on a second threshold.

[0034] In some embodiments, the third value is approximately the sum of the first value and the second value.

[0035] In some embodiments, the system for performing nucleic acid sequencing includes a fluid channel, a heater connected to the fluid channel, an imaging system, and at least one processor. In some embodiments, the fluid channel has a plurality of sites, and the plurality of sites are used to attach a plurality of nucleic acid chains to be sequenced to the surface of the fluid channel. In some embodiments, the heater is configured to set the temperature of the contents of the fluid channel to any one of the non-overlapping first temperature range, the second temperature range, the third temperature range, and the fourth temperature range. In some embodiments, the imaging system is configured to detect the intensity at each site in a plurality of sites in each of the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range. In some embodiments, the second temperature range is lower than the first temperature range, the third temperature range is lower than the second temperature range, and the fourth temperature range is lower than the third temperature range.

[0036] The at least one processor is configured to perform at least one machine-readable instruction. In some embodiments, the at least one machine executable instruction, when being executed, makes the at least one processor respond to the intensity at the specific site in the multiple sites to be greater than or equal to the first threshold value in the first temperature range and the second temperature range and identify the complementary base of the first fluorescently labeled nucleotide precursor or the first fluorescently labeled nucleotide precursor. In some embodiments, the at least one machine executable instruction, when being executed, makes the at least one processor respond to the intensity at the specific site in the multiple sites to be less than the second threshold value in the first temperature range and be greater than or equal to the second threshold value in the second temperature range and identify the complementary base of the second fluorescently labeled nucleotide precursor or the second fluorescently labeled nucleotide precursor. In some embodiments, the at least one machine executable instruction, when being executed, makes the at least one processor respond to the intensity at the specific site in the multiple sites to be less than the third threshold value in the first temperature range and the second temperature range and be greater than or equal to the third threshold value in the third temperature range and identify the complementary base of the third fluorescently labeled nucleotide precursor or the third fluorescently labeled nucleotide precursor. In some embodiments, the at least one machine executable instruction, when executed, causes the at least one processor to identify a fourth fluorescently labeled nucleotide precursor or a complementary base of the fourth fluorescently labeled nucleotide precursor in response to the intensity at a particular site among the plurality of sites being less than a third threshold in each of the first temperature range, the second temperature range, and the third temperature range and greater than or equal to a fourth threshold in the fourth temperature range.

[0037] In some embodiments, a fluidic channel includes a structure (eg, a cavity, a ridge, etc.) having a plurality of sites for attaching a plurality of nucleic acid strands to be sequenced to a surface of the fluidic channel.

[0038] In some embodiments, the heater includes a thermal sensor or a microprocessor.

[0039] In some embodiments, the imaging system includes a camera, a light source (e.g., a laser), an illumination optics, a detector, an optical blocking filter, an analog-to-digital converter (ADC), or one or more sensors. The illumination optics may be configured to distribute the excitation light substantially uniformly over a plurality of sites. In some embodiments, the detector includes a lens. In some such embodiments, the imaging system includes an optical blocking filter that is located within the system so that it substantially isolates the lens from the light emitted by the light source. In some embodiments, one or more sensors of the imaging system include a charge coupled device (CCD) sensor.

[0040] In some embodiments, the at least one machine readable instruction, when executed by the at least one processor, further causes the at least one processor to control the heater, the imaging system, or both.

[0041] In some embodiments, the system for performing nucleic acid sequencing includes a fluid channel, a heater connected to the fluid channel, an imaging system, and at least one processor. In some embodiments, the fluid channel has a plurality of sites, and the plurality of sites are used to attach a plurality of nucleic acid chains to be sequenced to the surface of the fluid channel. In some embodiments, the heater is configured to set the temperature of the contents of the fluid channel in any one of the first temperature range and the second temperature range, wherein the first temperature range and the second temperature range do not overlap. In some embodiments, the imaging system is configured to detect the intensity at each site in a plurality of sites in each of the first temperature range and the second temperature range.

[0042] The at least one processor is configured to execute at least one machine-readable instruction. In some embodiments, when the at least one machine executable instruction is executed, the at least one processor instructs the heater to set the temperature in the fluid channel within a first temperature range, and obtains from the imaging system an indication of the first intensity at each of the multiple sites when the temperature of the fluid channel is within the first temperature range. In some embodiments, when the at least one machine executable instruction is executed, it also causes the at least one processor to instruct the heater to set the temperature in the fluid channel within a second temperature range, and obtains from the imaging system an indication of the second intensity at each of the multiple sites when the temperature of the fluid channel is within the second temperature range. In some embodiments, when the at least one machine executable instruction is executed, it also causes the at least one processor to determine that the first fluorescent-labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the first intensity at the specific site in the multiple sites being less than the first value and the second intensity at the specific site being greater than or equal to the second value. In some embodiments, the at least one machine executable instruction, when executed, further causes the at least one processor to determine that a second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the first intensity at the specific site being greater than or equal to the first value and the second intensity at the specific site being greater than or equal to the second value and less than a third value, the third value being greater than each of the first value and the second value. In some embodiments, the at least one machine executable instruction, when executed, further causes the at least one processor to determine that a third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site in response to the first intensity at the specific site being greater than or equal to the first value and the second intensity at the specific site being greater than or equal to the third value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The objects, features and advantages of the present disclosure will become apparent from the following description of certain embodiments in conjunction with the accompanying drawings, in which:

[0044] Figure 1A The temperature dependence of the fluorescence intensity of the fluorescent dye is shown.

[0045] Figure 1B The temperature dependence of the fluorescence intensity of the attached fluorescent dye is shown.

[0046] Figure 1C yes Figure 1B An annotated version of , showing how to exploit the temperature dependence of the emission of four fluorescent dyes for nucleic acid sequencing.

[0047] Figure 2A Shown are the use of four intensity measurement events, four temperature ranges, and four fluorescently labeled nucleotide precursors for nucleic acid sequencing according to some embodiments.

[0048] Figure 2B Conceptually illustrated is the use of three intensity measurement events, three temperature ranges, and three fluorescently labeled nucleotide precursors, each with a different fluorescent label, according to some embodiments.

[0049] Figure 2C Conceptually illustrated is the use of two intensity measurement events, two temperature ranges, and two fluorescently labeled nucleotide precursors, each with a different fluorescent label, according to some embodiments.

[0050] Figure 3A is a flow chart illustrating a method of sequencing a nucleic acid according to some embodiments.

[0051] Figure 3B shows how this method according to some embodiments can be used Figure 3A Then continue.

[0052] Figure 3C It is shown that the method according to some embodiments can be Figure 3A Another way to continue afterwards.

[0053] Figure 3D According to some embodiments, Figure 3A An alternative ordering of the elements of the method shown in .

[0054] Figure 3E According to some embodiments, Figure 3A Another alternative ordering of the elements of the method shown in .

[0055] Figure 4A and Figure 4B Methods of sequencing nucleic acids according to some embodiments are shown.

[0056] Figure 5 is a block diagram illustrating a system for nucleic acid sequencing according to some embodiments.

[0057] Figure 6 is a block diagram of components of an imaging system that may be used according to some embodiments. DETAILED DESCRIPTION

[0058] As used herein, the term "nucleotide" includes both deoxyribonucleotides and ribonucleotides, as well as modified nucleotides, such as dideoxynucleotides and other chain terminator nucleotides. It also includes nucleotide residues within a polynucleotide. The terms "nucleotide" and "base" are used interchangeably herein.

[0059] As used herein, the term "fluorescent dye" refers to a dye whose presence can be detected by fluorescence. In certain embodiments, the fluorescent dyes used herein are suitable for use in nucleic acid sequencing.

[0060] The term "nucleic acid polymerase" includes DNA polymerases and RNA polymerases. DNA polymerases can be DNA-dependent or RNA-dependent (i.e., reverse transcriptases). DNA polymerases include, for example, Taq DNA polymerase (Thermus aquaticus DNA polymerase), mutant forms of Taq DNA polymerase (such as Taq G46D F667Y DNA polymerase), and other forms known in the art.

[0061] As used herein, the term "nucleic acid substrate" refers to a nucleic acid modified by a chemical or enzymatic reaction, or a nucleic acid used as a template for a chemical or enzymatic reaction. For example, in a nucleic acid sequencing reaction, both the oligonucleotide extended by a polymerase and the template nucleic acid are the terms "nucleic acid substrate" used herein.

[0062] As used herein, the term "chain terminator nucleotide" refers to a mononucleotide that can be incorporated into a growing polynucleotide chain by a nucleic acid polymerase and terminates the chain when incorporated, because the polynucleotide end comprising a chain terminator nucleotide at its 3' position cannot be used as a substrate for adding another nucleotide by a nucleic acid polymerase. Typical terminators are those in which the nucleobase is a purine, a 7-deazapurine, a pyrimidine, a normal nucleobase or their common analogs and the sugar moiety is a pentose, and the pentose includes a 3'-substituent that blocks further synthesis, such as dideoxynucleotides (ddNTP). ddNTP is exemplified by ddGTP, ddATP, ddTTP and ddCTP. Substituents that prevent further synthesis include but are not limited to amino, deoxy, halogen, alkoxy and aryloxy. Exemplary terminators include, but are not limited to, those wherein the sugar-phosphate moiety is 3'-(C1-C6) alkylribose-5'-triphosphate, 2'-deoxy-3'-(C1-C6) alkylribose-5'-triphosphate, 2'-deoxy-3'-(C1-C6) alkoxyribose-5'-triphosphate, 2'-deoxy-3'-(C5-C6) alkoxyribose-5'-triphosphate, 14 ) aryloxyribose-5'-triphosphate, 2'-deoxy-3'-haloribose-5'-triphosphate, 2'-deoxy-3'-aminoribose-5'-triphosphate, 2',3'-dideoxyribose-5'-triphosphate or 2',3'-didehydroribose-5'-triphosphate.

[0063] Existing fluorescence-based techniques for distinguishing different bases in a sample (e.g., in fluorescence-based nucleic acid sequencing techniques) rely, for example, on the quality of the signal generated by a detection moiety associated with a particular type of nucleotide. For example, conventional fluorescence sequencing techniques utilize identifiably different fluorescent moieties that are each attached to one of the four nucleotides A, T, C, and G utilized in the sequencing reaction.

[0064] A conventional DNA sequencing method involves adapting ssDNA to attachment to a solid support of a sequencing device and using a technique such as polymerase chain reaction to amplify the amount of ssDNA to produce many DNA molecules with a short leader sequence. Oligonucleotides complementary to the short leader sequence can then be added so that a short segment of dsDNA is present at the leader sequence. The double-stranded portion of the bound molecule is a primer for a suitable DNA polymerase (such as Taq polymerase) that is effective at high temperatures.

[0065] Then one of several methods can be used to perform sequencing. For example, sequencing can use a mixture of four fluorescently labeled 3'-blocked NTPs (fluorescently labeled dideoxynucleotide terminators), where the fluorescent label is part of the 3'-blocking group. The fluorescent label is used as a "reversible terminator" for the polymerization reaction. Each NTP in the NTP is labeled with a different marker (e.g., each of the A, G, C, and T nucleotides has a different marker), and the different markers can be distinguished by fluorescence spectroscopy or by other optical means.

[0066] Four fluorescently labeled nucleotide precursors can be used to sequence millions of clusters of parallel DNA chains. DNA polymerase catalyzes the incorporation of fluorescently labeled dNTPs into the DNA template strand during the sequential cycles of DNA synthesis. In each sequencing cycle, the bound double-stranded DNA molecule is exposed to a mixture of DNA polymerase and four fluorescently labeled 3'-blocking NTPs. Polymerase adds one of the four dNTPs to the growing oligonucleotide chain (i.e., no matter which dNTP is complementary to the next unpaired base in ssDNA). Then, by washing at a temperature that prevents free dNTPs from binding to ssDNA but not high enough to dehybridize dsDNA, unincorporated dNTPs and other impurities left unreacted or generated during the reaction are separated from the vicinity of the DNA bound to the support.

[0067] Because only one of the four types of dNTPs will be added to the oligonucleotide, and the four fluorescent labels are distinguishable, the identity of the incorporated dNTP can be identified by laser excitation and imaging. Specifically, each of the four filters is used to determine whether light of a specific wavelength (e.g., color) is emitted. The fluorescent label can then be enzymatically cleaved to allow the next round of incorporation. Because each base type can be paired with only one other base type, the identity of the base that just pairs in the unknown ssDNA sequence can be known from the identity of the incorporated dNTP (which is known from the wavelength of the emitted light). Therefore, the base is identified directly from the fluorescence measurement during each cycle.

[0068] One disadvantage of the above methods is that complex optical systems are required to filter out light of different wavelengths to detect the fluorescent markers of the incorporated dNTPs and distinguish between the different emission colors. Other methods have been developed to simplify the optical systems, but their sequencing is slower and requires intermediate chemical steps within each sequencing cycle. Therefore, these methods have been introduced into smaller, cheaper entry-level sequencing systems, but have not been introduced into more advanced systems that require fast throughput.

[0069] Disclosed herein is an improved method for sequencing nucleic acid.The inventors recognize that some fluorescent dyes emit light with an intensity that varies with temperature, and this temperature dependence of luminescence intensity can be used for nucleic acid sequencing applications.The method disclosed in the present invention can use an optical system simpler than the above-mentioned conventional four-dye method because they do not require wavelength filtering.On the contrary, the temperature of the system is regulated during each sequencing cycle (for example, by using an integrated heating element of a sequencing device).In addition, all four bases can be introduced in a single step.The emission of fluorescent dye light at different temperatures (or within different temperature ranges) or not emits a fluorescent marker that can be used to detect the dNTP incorporated, thereby sequencing nucleic acid.For example, and as will be described in further detail below in the context of some embodiments, in a composition comprising four chain terminator nucleotides, each chain terminator nucleotide is connected to different fluorescent dyes, and each of the four fluorescent dyes will emit light in a temperature-dependent manner so that it can be distinguished from other dyes.

[0070] The temperature response of fluorescent dyes has been used to perform microscale local temperature measurements in microfluidic and biological systems. Some such systems are described in a 2001 paper by D. Ross, M. Gaitan, and L. Locasco entitled "Temperature Measurement in Microfluidic Systems Using a Temperature-Dependent Fluorescent Dye" (Analytical Chemistry, Vol. 73, No. 17, September 1, 2001). Figure 1A The temperature dependence of the fluorescence intensity of one such fluorescent dye, Rhodamine B, is shown. Figure 1A As shown, the intensity decreases monotonically with increasing temperature. Above room temperature (assumed to be 20 degrees Celsius), the intensity of the emitted light decreases rapidly. Therefore, if the detector in the nucleic acid sequencing device has a detection threshold of, for example, 80% of the peak intensity, it will detect the dye at temperatures below about 32 degrees Celsius but not above that temperature.

[0071] As a comparison, Figure 1B The temperature dependence of the fluorescence intensity of various coumarin dyes, namely 4-methyl-7-methoxy Figure 1B substituted coumarin (labeled as "I"), 4-methyl-5,7-diethoxy (labeled as "II"), 4-methyl-5-ethoxy-7-methoxy (labeled as "III"), and 4-methyl-7,8-diethoxy (labeled as "IV"). (See, for example, R. Giri, "Temperature effect study upon the fluorescence emission of substituted coumarins", Spectrochimica Acta Part A: Molecular Spectroscopy, Vol. 48, No. 6, June 1992, pp. 843-848.) Figure 1B As shown, the intensity of 4-methyl-7,8-diethoxy (IV) is relatively insensitive to temperature. In contrast, the intensity of 4-methyl-7-methoxy (I) decreases rapidly with increasing temperature.

[0072] Therefore, the inventors realized that the intensity response can be used so that two different fluorescent labels with different responses to temperature changes can be used to distinguish two nucleotides in nucleic acid sequencing. For example, rhodamine ( Figure 1A ) and coumarin(III)( Figure 1B) shows that at 50 degrees Celsius, the intensity of rhodamine is half its value at room temperature, while the intensity of coumarin (III) is ninety percent of its value at room temperature. Assuming that the intensities of rhodamine and coumarin (III) are approximately the same at room temperature, by adjusting the photodetector to have a minimum sensitivity with the light intensity set to a value between 50% and 90% (e.g., 75%), the photodetector will detect light from both dyes at room temperature, but only light from coumarin (III) at 50 degrees Celsius. One advantage of this method over conventional methods of nucleic acid sequencing is that there is no need to filter the wavelength of emitted light or determine the color of light emitted by the dye; knowledge of the temperature dependence of the intensity of light emitted when the dye is excited to fluoresce can distinguish between different dyes. Thus, the need for the complexity and expense associated with the need to have optical filters is reduced or entirely eliminated.

[0073] It should be understood that the intensity distribution of the selected dye need not be monotonic, smooth or linear. It is contemplated that the selected dye may have predictable luminescence intensity in some temperature ranges and unpredictable (or nonlinear, random, etc.) luminescence intensity in other ranges.

[0074] Figure 1C yes Figure 1B to show how the temperature dependence of different fluorescent dyes can be used to distinguish dyes. It should be understood that Figure 1B and Figure 1C Normalized intensities for four fluorescent dyes are shown. Therefore, this explanation assumes that the peak intensity of light emitted by each of these fluorescent dyes at room temperature (approximately 273 Kelvin if 20 degrees Celsius is room temperature) is approximately the same.

[0075] like Figure 1C As shown, a threshold of approximately 85% intensity has been selected as an example. In the highest temperature range labeled "Range 1", only dye IV emits light with an intensity that reaches or exceeds the threshold. In the next highest temperature range labeled "Range 2", only dyes III and IV emit light with an intensity that reaches or exceeds the threshold. In the next highest temperature range (labeled "Range 3") and the lowest temperature range, only dyes II, III and IV emit light with an intensity that reaches or exceeds the threshold. Finally, in the lowest temperature range (labeled "Range 4"), all four fluorescent dyes emit light with an intensity that reaches or exceeds the threshold.

[0076] continue Figure 1CIn the example shown, in a DNA sequencing application, each of the four nucleotides can be labeled with a different one of the four fluorescent dyes shown. For example, A can be labeled with dye I, G can be labeled with dye II, T can be labeled with dye III, and C can be labeled with dye IV. When the temperature is in range 1, only dye IV will emit light with an intensity that reaches or exceeds the threshold. Therefore, if light is detected to be equal to or higher than the threshold in temperature range 1, the incorporated base must be cytosine (C). On the other hand, if light equal to or higher than the threshold is not detected in temperature range 1, the incorporated base is not cytosine (C). Images can be taken in each of the four temperature ranges to record the detected light (or the absence of detected light). As used herein, the term "image" refers to any record of the presence or absence of emitted light (e.g., an optical response above multiple physical locations). For example, an image can literally be an image of the fluid channel of a sequencing device, which is recorded using a camera, potentially using an optical filter to detect light of a specific wavelength at a specific physical location where a nucleic acid chain (or part of a chain) is attached or bound. As another example, an image can be a data file that records the detected intensity (e.g., in candela, lumens, or any other suitable unit) of light emitted at various physical locations where the nucleic acid is attached or bound. As another example, an image can be a data file that records whether light is detected at a specific location where the nucleic acid is attached or bound (e.g., 1 if light is detected exceeding a threshold; 0 if light is not detected exceeding a threshold). Regardless of its form, an image can be stored temporarily or permanently (e.g., so that its contents can be compared with other images), or it can be transient (e.g., created in real time and compared with previously created and stored images).

[0077] When the temperature is in range 2, only dyes III (T) and IV (C) will emit light at an intensity that reaches or exceeds the threshold. Therefore, if light is detected at or above the threshold in temperature range 2, the incorporated base is not adenine (A) or guanine (G). Based on knowing whether light above the threshold is detected when the temperature is in range 1, it can be determined whether the incorporated base is cytosine (C) (light is detected in range 1 and range 2) or thymine (T) (no light is detected in range 1, and light is detected in range 2).

[0078] When the temperature is within range 3, all dyes except dye 1 will emit light at an intensity above the threshold. Therefore, if light equal to or above the threshold is detected within temperature range 3, it can be concluded that the incorporated base is not adenine (A). Based on knowing whether light above the threshold is detected when the temperature is within range 1, within range 2, and within range 3, it can be determined whether the incorporated base is cytosine (C) (light is detected in ranges 1, 2, and 3), thymine (T) (no light is detected in range 1, but light is detected in ranges 2 and 3), or guanine (G) (no light is detected in ranges 1 and 2, but light is detected in range 3).

[0079] When the temperature is within range 4, all dyes will emit light equal to or above the threshold. Based on the evaluation of the detection of light (or absence of light) within each of the four temperature ranges, it can be determined which nucleotide has been incorporated in the sequencing step. Specifically, if light is detected in all four temperature ranges, the nucleotide is cytosine (C); if light is detected only within ranges 2, 3, and 4, the nucleotide is thymine (T); if light is detected only within ranges 3 and 4, the nucleotide is guanine (G); and if light is detected only within range 4, the nucleotide is adenine (A).

[0080] As shown in this example, by manipulating the temperature during each sequencing step (e.g., setting the temperature to different values ​​during the sequencing cycle and detecting whether there is an emission light equal to or above a threshold), different incorporated bases can be distinguished from each other. These temperature ranges can be tested in any order. It should be understood that when it is desired to determine whether and which base has been incorporated in a particular nucleic acid chain being sequenced, it may be advantageous to start with range 1, because if light is detected in range 1 during the cycle, it can be concluded that the incorporated nucleotide is cytosine (C). Therefore, if an emission light equal to or above the threshold is detected in range 1, there is no need to check ranges 2, 3, or 4. It should also be understood that it may be advantageous to test in range 3 first, because if light is not detected in range 3, it can be concluded that the incorporated nucleotide is adenine (A). Therefore, there is no need to check ranges 1, 2, or 4.

[0081] As mentioned above, in some types of SBS, long-chain DNA (or multiple long chains of DNA from a single donor organism) is divided into smaller random length fragments before sequencing.All these shorter chains from the same donor are randomized subchains of complete chains to be sequenced.For example, if the complete chain includes the sequence ATGGCTTAGCCATACCGAT, then the shorter chain may include, for example, different subchains (for example, TGGCTT and CCATACCGA), and if multiple longer chains are divided into subchains, then the shorter chain may include subchains (for example, CTTAGCCAT and ATGGCTTAGCC) that overlap with other subchains partially or completely.All shorter randomized subchains can be sequenced simultaneously, and this may be after amplification.In such applications, it should be understood that because the subchains do not represent identical subsequences, it is desirable to determine the intensity in each of these temperature ranges, because the sequencing of the subchains will not be coordinated (or synchronized) between the subchains.For example, during a single sequencing cycle, the first subchain may be mixed with cytosine, the second subchain may be mixed with thymine, and the third subchain may be mixed with adenine. In order to sequence multiple random fragments of a larger nucleic acid strand, it is desirable to determine in each sequencing cycle whether each dNTP is incorporated and at which physical position each dNTP is incorporated.

[0082] As will be understood by those skilled in the art with reference to the disclosure herein, if the peak intensities of the different dyes are different, then a peak intensity ratio such as Figure 1C The same general procedure is described in the context of . In such cases, the intensity thresholds applicable in different temperature ranges may be different, and appropriate values ​​may be determined based on knowledge of the temperature dependence properties of each fluorescent dye.

[0083] FIG. 2A to FIG. 2C It is shown how these principles can be used to identify different incorporated nucleotides according to some embodiments. Each of the four nucleotides can be labeled with a different dye, each dye having a different fluorescence intensity dependence on temperature. The color of the dye is irrelevant, as long as the temperature at which the fluorescence intensity of each dye drops below a threshold is different.

[0084] Figure 2A Conceptually illustrated are four images, four temperature ranges, and the use of four fluorescently labeled nucleotide precursors, each with a different fluorescent label (which may be cleavable), according to some embodiments. Figure 2A is actually a logical table that can be applied separately to determine, for each nucleic acid strand or substrand, which nucleotide has been incorporated into that strand or substrand. For example, if it is assumed that Figure 1CIn the fluorescent dye, threshold value and temperature range shown in, then A is labeled with dye I, G is labeled with dye II, T is labeled with dye III, and C is labeled with dye IV. After nucleotides are incorporated into the growing nucleic acid chain, the reaction temperature is set in one of the temperature ranges. For the purpose of this explanation, it is assumed that the test is carried out from the highest temperature to the lowest temperature. As mentioned above, the order is arbitrary, and these temperature ranges can be tested in any order. In addition, according to the result obtained when the temperature is within a specific range, it may be unnecessary to test more than one temperature range in the sequencing cycle. As mentioned above, when the randomized subchain is sequenced simultaneously, it should be understood that because the subchain does not represent the same subsequence, it is desirable to determine the intensity in each of these temperature ranges, because the sequencing of the subchain will not be coordinated (or synchronized) between the subchains.

[0085] After setting the temperature to within range 1 (in this example, it is assumed that this range is the highest temperature of the four temperature ranges), the reaction is then exposed to light, and the fluorescence is observed and recorded as image 1; this constitutes a first imaging event and a first fluorescence detection pattern. The temperature is then lowered to within range 2. The reaction site is illuminated again, and the fluorescence is captured and recorded as image 2, thereby constituting a second imaging event (e.g., a second fluorescence detection pattern). The temperature is then lowered to within range 3. The reaction site is illuminated again, and the fluorescence is captured and recorded as image 3, thereby constituting a third imaging event (e.g., a third fluorescence detection pattern). The temperature is then lowered to within range 4. The reaction site is illuminated again, and any changes in fluorescence are captured and recorded, thereby constituting a fourth imaging event (e.g., a fourth fluorescence detection pattern).

[0086] like Figure 2A As shown, Image 1 is taken when the temperature is within Range 1, Image 2 is taken when the temperature is within Range 2, Image 3 is taken when the temperature is within Range 3, and Image 4 is taken when the temperature is within Range 4. Within Temperature Range 4, all four dyes emit light with intensities greater than the threshold, as shown in FIG. Figure 2AAs shown, it is therefore impossible to determine which of the four fluorescently labeled nucleotide precursors has been incorporated into the extendable primer from a single image 4. However, within the temperature range 3, only the fluorescent dyes labeled G, T, and C will emit light at an intensity greater than the threshold. Therefore, based on the presence of emission light greater than the threshold intensity in image 4 and the absence of emission light equal to or higher than the threshold in image 3, it can be determined that the incorporated nucleotide precursor is A. Similarly, only the fluorescent dyes labeled T and C will emit light at an intensity greater than the threshold within the temperature range 2. Therefore, based on the presence of emission light greater than the threshold intensity in images 4 and 3 and the absence of emission light equal to or higher than the threshold in image 2, it can be determined that the incorporated nucleotide precursor is G. Similarly, only the fluorescent dye labeled C will emit light at an intensity greater than the threshold within the temperature range 1. Therefore, based on the presence of emission light greater than the threshold intensity in images 4, 3, and 2 and the absence of emission light equal to or higher than the threshold in image 1, it can be determined that the incorporated nucleotide precursor is T. Of course, if the intensity detected exceeds the threshold in all four images, it can be determined that the incorporated nucleotide precursor is C.

[0087] Because all four fluorescently labeled nucleotide precursors emit light in temperature range 4 at intensities at or above the threshold, the procedure can be simplified by eliminating imaging in temperature range 4. Figure 2B Conceptually illustrates the use of three images, three temperature ranges, and three fluorescently labeled nucleotide precursors (each with a different fluorescent label) according to some embodiments. Figure 2A , Figure 2B In fact, it is a logic table that can be applied separately to determine which nucleotide has been incorporated into each nucleic acid chain or daughter chain. Figure 2B In this method, image 1 is taken when the temperature is within range 1, image 2 is taken when the temperature is within range 2, and image 3 is taken when the temperature is within range 3.

[0088] The fluorescent dye that labels cytosine (C) is the only dye that emits light at an intensity greater than or equal to the threshold in temperature range 1. Therefore, if an intensity greater than or equal to the threshold is detected in image 1, it can be determined that the incorporated nucleotide precursor is cytosine (C). Otherwise, image 2 can be used to determine whether the incorporated nucleotide precursor is T: because only fluorescent dyes that label thymine (T) and cytosine (C) will emit light at an intensity greater than the threshold in temperature range 2, the presence of emission light greater than the threshold intensity in image 2 and less than the threshold intensity in image 1 indicates that the incorporated nucleotide precursor is thymine (T). Similarly, the fluorescent dye that labels adenine (A) will only emit light at an intensity greater than the threshold in range 3. Therefore, based on the presence of emission light greater than the threshold intensity in image 3 but not in image 2 or image 1, it can be determined that the incorporated nucleotide precursor is adenine (A). If emission light greater than the threshold intensity is not detected in any of images 1, 2, or 3, it can be concluded (e.g., inferred) that the incorporated nucleotide is guanine (G).

[0089] Figure 2C An alternative embodiment according to some embodiments is shown in which only two images are used to determine which of the four nucleotide precursors have been incorporated. This embodiment uses a photodetector that is intensity sensitive above its intensity threshold, meaning that it detects not only the presence of emitted light, but also the intensity of the emitted light. Similar to Figure 2A and Figure 2B , Figure 2C In practice, it is a logic table that can be applied separately to determine, for each nucleic acid strand or daughter strand, which nucleotide has been incorporated into that strand or daughter strand. Figure 2C ) is labeled with a first dye, and a second nucleotide (such as Figure 2C C in FIG) is labeled with a second dye. A nucleotide (such as Figure 2C The fourth nucleotide (as shown in A in FIG. Figure 2C ) is unlabeled.

[0090] There are a variety of methods to attach fluorescent markers, and if necessary, cleave fluorescent markers. For example, fluorescent markers can be attached to bases, in which case they can be chemically cleaved. In another example, fluorescent markers can be attached to phosphates, in which case they can be cleaved by polymerases, or if attached via a linker, they can be cleaved by cleaving the linker.

[0091] In some embodiments, the fluorescent label is attached to the nitrogenous base (A, C, T, G or derivatives) of the nucleotide precursor. After incorporation of the nucleotide precursor and detection of emitted light, the fluorescent label is cleaved from the incorporated nucleotide.

[0092] In some embodiments, the fluorescent marker is attached via a cleavable linker. Cleavable linkers are known in the art and have been described, for example, in U.S. Patents 7,057,026, 7,414,116 and continuations and improvements of these patents. In some embodiments, the fluorescent marker is attached to the 5-position in pyrimidine or the 7-position in purine via a linker comprising an allyl or azido group. In other embodiments, the linker comprises a disulfide, an indole, a Sieber group, a tert-butyl Sieber group, or a dialkoxybenzyl group. The linker may also comprise a group selected from an alkyl (C 1-6 ) or alkoxy (C 1-6 ), nitro, cyano, fluorine group or one or more substituents of groups with similar properties. In short, the joint can be cracked by a water-soluble phosphine or a phosphine-based transition metal-containing catalyst. Other joints and joint cracking mechanisms are known in the art. For example, joints including trityl groups, p-alkoxybenzyl ester groups, p-alkoxybenzylamide groups, tert-butyloxycarbonyl (Boc) groups and acetal-based groups can be cracked by proton release cracking agents such as acids under acidic conditions. Sulfur-philic metals (such as nickel, silver or mercury) can be used to crack thioacetal or other sulfur-containing joints. Cleavage protecting groups can also be considered for preparing suitable joint molecules. Joints containing esters and disulfides can be cracked under reducing conditions. Joints containing triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS) can be cracked in the presence of F ions. Light-cleavable joints that are cracked at wavelengths that do not affect other components of the reaction mixture include joints containing o-nitrobenzyl groups. Joints containing benzyloxycarbonyl groups can be cracked by palladium-based catalysts.

[0093] In some embodiments, the nucleotide precursor comprises a fluorescent marker attached to a polyphosphate moiety, such as described in, for example, U.S. Patents 7,405,281 and 8,058,031. In short, the nucleotide precursor includes a chain of nucleoside moieties and 3 or more phosphate groups, wherein one or more oxygen atoms in the oxygen atom are optionally substituted with, for example, S. The marker may be attached directly or via a joint to an α, β, γ or higher phosphate group (if present). In some embodiments, the fluorescent marker is attached to the phosphate group via a non-covalent joint, such as described in, for example, U.S. Patent 8,252,910. In some embodiments, the joint is a hydrocarbon selected from the following items: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; see, for example, U.S. Patent 8,367,813. The joint may also include a nucleic acid chain; see, for example, U.S. Patent 9,464,107.

[0094] In embodiments where the fluorescent marker is attached to a phosphate group, the nucleotide precursor is incorporated into the nascent chain by a nucleic acid polymerase that also cleaves and releases the detectable fluorescent marker. In some embodiments, the fluorescent marker is removed by cleaving the linker, for example, as described in U.S. Pat. No. 9,587,275.

[0095] In some embodiments, the nucleotide precursor is a non-extendable "terminator" nucleotide, i.e., a nucleotide having a 3' end that is blocked by a "terminator" group to block the addition of the next nucleotide. The blocking group is a reversible terminator, which can be removed to continue the chain synthesis process as described herein. It is known in the art to attach a removable blocking group to a nucleotide precursor. See, for example, U.S. Patents 7,541,444, 8,071,739 and the continued application and improvement of these patents. In short, the blocking group may include an allyl group, which may be broken by reacting with a metal-allyl complex in an aqueous solution in the presence of a phosphine or nitrogen-phosphine ligand. Other examples of reversible terminator nucleotides for sequencing by synthesis include the modified nucleotides described in U.S. Provisional Patent Application Serial No. 62 / 781,638, filed on December 19, 2018 and named "3'-Protected Nucleotides", which is hereby incorporated by reference in its entirety for all purposes.

[0096] exist Figure 2C In the illustrated embodiment, image 1 is taken at a temperature within a first temperature range, and image 2 is taken at a temperature within a second, lower temperature range. If the luminescence intensity in both images is below a threshold, the incorporated nucleotide is determined to be guanine (G). If the luminescence intensity is above the threshold in image 2 but below the threshold in image 1, the incorporated nucleotide is determined to be cytosine (C). If the luminescence intensity in both images is above a threshold, adenine (A) and thymine (T) can be distinguished by comparing the intensity of the emitted light in the two images. Figure 2C In the example of , if the detected intensity is approximately the same in both images, it can be determined that the incorporated nucleotide is thymine (T) because the first dye emits light of known intensity in both temperature ranges. It will be appreciated that when thymine is incorporated (in this example), the intensity of the emitted light may be approximately the same in images 1 and 2. For example, if Figure 1C If dye IV is used to label thymine, the intensity will be approximately the same. Alternatively, the intensity associated with thymine may be different in Image 1 and Image 2. For example, if Figure 1C If dye II is used to label thymine, the measured intensity in the two images may be different. However, it will be known what the intensity is expected from the dye labeling thymine at the corresponding temperature at which images 1 and 2 were taken.

[0097] Because the third nucleotide (in Figure 2C In the example shown as adenine (A) in the figure, a nucleotide (shown as adenine (A)) has been labeled with two dyes, one of which also labels thymine (T), and the intensity of the luminescence of the dye labeling thymine is known for the two temperatures at which images 1 and 2 were acquired, it can be determined whether adenine (A) or thymine (T) was incorporated, because when the incorporated nucleotide is adenine (A), the luminescence intensity of image 2 will be higher than when it is thymine (T). Therefore, if the detected intensity is higher in image 2 than in image 1, and it is greater than the expected luminescence intensity of the dye labeling thymine (T), it can be determined that the incorporated nucleotide is adenine (A). One advantage of this embodiment is that no chemical reagents need to be added between taking the two images to change or remove any markers. The incorporated nucleotide can be determined simply by comparing the detected intensity levels in images 1 and 2. The following table provides information related to Figure 2C A decision table associated with the examples shown and described above, wherein Threshold 1 is an intensity threshold for detecting a first dye within temperature range 1; Threshold 2, which may be the same or different from Threshold 1, is an intensity threshold for detecting the first dye within temperature range 2; Threshold 3 is an intensity threshold for detecting a second dye within temperature range 2; and Threshold 4, which is greater than both Thresholds 2 and 3, is an intensity threshold for detecting the presence of both the first dye and the second dye within temperature range 2.

[0098] Image 1 Intensity Image 2 Intensity Determination <Threshold 1 <Threshold 2 Guanine (G) <Threshold 1 ≥Threshold 3 Cytosine (C) ≥Threshold 1 ≥Threshold 2 and <Threshold 4 Thymine (T) ≥Threshold 1 ≥Threshold 4 Adenine (A)

[0099] Figure 2C One benefit of the method shown and described above is that, unlike conventional methods that use two imaging events, it does not require any additional chemical steps within the cycle.

[0100] Figure 3A 1 is a flow chart illustrating a method 100 for sequencing nucleic acids according to some embodiments. Method 100 relates to the use of a sequencing device comprising a fluid channel having a plurality of sites for binding a plurality of molecules of a nucleic acid polymerase or a plurality of nucleic acid chains (e.g., as described above and known in the art) to a surface of the fluid channel. At 102, the method begins. At 104, a material is added to the fluid channel. The material comprises at least a plurality of nucleic acid chains, a plurality of molecules of a nucleic acid polymerase, a first fluorescently labeled nucleotide precursor, and a second fluorescently labeled nucleotide precursor.

[0101] The materials added in step 104 can be added to the fluid channel simultaneously or in one or more rounds of addition. For example, in some embodiments, molecules of nucleic acid polymerase first bind to the surface of the fluid channel at these sites, and then the remaining materials are added to the fluid channel. In other embodiments, nucleic acid chains first bind to the surface of the fluid channel at these sites, and then the remaining materials are added to the fluid channel.

[0102] The first fluorescently-labeled nucleotide precursor comprises the first fluorescent marker (it can be cleavable).The intensity that the first fluorescent marker sends is greater than or equal to the first threshold value in the first temperature range and in the second temperature range that is lower than the first temperature range and does not overlap with the first temperature range.In other words, the first fluorescent marker will emit light with the intensity of at least the first threshold value in the first temperature range and the second temperature range.The first temperature range and the second temperature range can be adjacent (for example, the first temperature range is from 30 degrees Celsius to 50 degrees Celsius, and the second temperature range is from 10 degrees Celsius to 30 degrees Celsius), or they can be separated by a temperature gap (for example, the first temperature range is 40 degrees Celsius to 50 degrees Celsius, and the second temperature range is from 20 degrees Celsius to 35 degrees Celsius).

[0103] The second fluorescently labeled nucleotide precursor comprises a second fluorescent marker (which may be cleavable). The intensity emitted by the second fluorescent marker is less than the second threshold value in the first temperature range, and is greater than or equal to the second threshold value in the second temperature range. In other words, considering only the first temperature range and the second temperature range, the second fluorescent marker will only emit light with the intensity of at least the second threshold value in the second (lower) temperature range; in the first (higher) temperature range, the second fluorescent marker will not emit light with an intensity that reaches or exceeds the second threshold value. The first threshold value and the second threshold value may be the same, or they may be different. It should be understood that the second fluorescent marker can emit light with the intensity of at least the second threshold value in the temperature range outside the first temperature range and the second temperature range. For example, the second fluorescent marker can emit light with the intensity of at least the second threshold value at a temperature lower than the second temperature range. It can also be envisioned that the second fluorescent marker can emit light with the intensity of at least the second threshold value at a temperature higher than the first temperature range.

[0104] There are a variety of methods to attach fluorescent markers and, if desired, cleave fluorescent markers. For example, fluorescent markers can be attached to bases, in which case they can be chemically cleaved. As another example, fluorescent markers can be attached to phosphates, in which case they can be cleaved by a polymerase, or if attached via a linker, they can be cleaved by cleaving the linker.

[0105] In some embodiments, the fluorescent label is attached to the nitrogenous base (A, C, T, G or derivatives) of the nucleotide precursor. After incorporation of the nucleotide precursor and detection, the fluorescent label is cleaved from the incorporated nucleotide.

[0106] In some embodiments, the fluorescent marker is attached via a cleavable linker. Cleavable linkers are known in the art and have been described, for example, in U.S. Patents 7,057,026, 7,414,116 and continuations and improvements of these patents. In some embodiments, the fluorescent marker is attached to the 5-position in pyrimidine or the 7-position in purine via a linker comprising an allyl or azido group. In other embodiments, the linker comprises a disulfide, an indole, a Sieber group, a tert-butyl Sieber group, or a dialkoxybenzyl group. The linker may also comprise a group selected from an alkyl (C 1-6 ) or alkoxy (C 1-6 ), nitro, cyano, fluorine group or one or more substituents of groups with similar properties. In short, the joint can be cracked by a water-soluble phosphine or a phosphine-based transition metal-containing catalyst. Other joints and joint cracking mechanisms are known in the art. For example, joints including trityl groups, p-alkoxybenzyl ester groups, p-alkoxybenzylamide groups, tert-butyloxycarbonyl (Boc) groups and acetal-based groups can be cracked by proton release cracking agents such as acids under acidic conditions. Sulfur-philic metals (such as nickel, silver or mercury) can be used to crack thioacetal or other sulfur-containing joints. Cleavage protecting groups can also be considered for preparing suitable joint molecules. Joints containing esters and disulfides can be cracked under reducing conditions. Joints containing triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS) can be cracked in the presence of F ions. Light-cleavable joints that are cracked at wavelengths that do not affect other components of the reaction mixture include joints containing o-nitrobenzyl groups. Joints containing benzyloxycarbonyl groups can be cracked by palladium-based catalysts.

[0107] In some embodiments, the nucleotide precursor comprises a label attached to a polyphosphate portion, such as described in, for example, U.S. Patents 7,405,281 and 8,058,031. In short, the nucleotide precursor includes a chain of nucleoside moieties and 3 or more phosphate groups, wherein one or more oxygen atoms in the oxygen atom are optionally substituted with, for example, S. The label may be attached directly or via a joint to an α, β, γ or higher phosphate group (if present). In some embodiments, the label is attached to a phosphate group via a non-covalent joint, such as described in, for example, U.S. Patent 8,252,910. In some embodiments, the joint is a hydrocarbon selected from the following items: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; see, for example, U.S. Patent 8,367,813. The joint may also include a nucleic acid chain; see, for example, U.S. Patent 9,464,107.

[0108] In embodiments where the fluorescent marker is attached to a phosphate group, the nucleotide precursor is incorporated into the nascent chain by a nucleic acid polymerase that also cleaves and releases the detectable fluorescent marker. In some embodiments, the fluorescent marker is removed by cleaving the linker, for example, as described in U.S. Pat. No. 9,587,275.

[0109] In some embodiments, the nucleotide precursor is a non-extendable "terminator" nucleotide, i.e., a nucleotide having a 3' end that is blocked by a "terminator" group to block the addition of the next nucleotide. The blocking group is a reversible terminator, which can be removed to continue the chain synthesis process as described herein. It is known in the art to attach a removable blocking group to a nucleotide precursor. See, for example, U.S. Patents 7,541,444, 8,071,739 and the continued application and improvement of these patents. In short, the blocking group can include an allyl group, which can be broken by reacting with a metal-allyl complex in the presence of a phosphine or nitrogen-phosphine ligand in an aqueous solution. Other examples of reversible terminator nucleotides for sequencing by synthesis are known in the art.

[0110] See again Figure 3AAt 106, after the nucleic acid strand, the polymerase molecule, and the first fluorescently labeled nucleotide precursor and the second fluorescently labeled nucleotide precursor have been added to the fluid channel, the temperature of the contents of the fluid channel is set within a first temperature range. At 108, when the temperature is within the first temperature range, the device is illuminated (e.g., by a laser), and a first image of the contents of the fluid channel is captured, for example, when the temperature of the fluid channel is within the first temperature range, a first intensity at each of a plurality of sites is detected. The first image captures the intensity at each of the plurality of sites. As previously described, an image is a record of the presence or absence of emitted light (e.g., an optical response over a plurality of physical locations).

[0111] At 110, the image is evaluated to determine whether the intensity of the first image at a particular site among the plurality of sites is greater than or equal to a first value. If the intensity of the first image at the particular site is greater than or equal to the first value, then it is determined at 112 that the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the site. For some or all of the other sites, the same method can be used to determine whether the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at those sites.

[0112] If it is determined at 110 that the intensity of the first image at the particular site is not greater than or equal to the first value, then at 114, the temperature of the contents of the fluid channel is set within a second (lower) temperature range. At 116, when the temperature is within the second temperature range, the device is illuminated (e.g., by a laser), and a second image of the contents of the fluid channel is captured, for example, when the temperature of the fluid channel is within the second temperature range, a second intensity at each of the plurality of sites is detected. The second image captures the intensity at each of the plurality of sites. At 118, it is determined whether the intensity of the second image at a particular site in the plurality of sites is greater than or equal to the second value, and whether the intensity of the first image at the particular site is less than the first value. The first value and the second value may be substantially the same, or they may be different. In some embodiments, the first value is a first threshold value, and the second value is a second threshold value. In some embodiments, the first value is based on the first threshold value, and the second value is based on the second threshold value. For example, the first value may be a multiple of the first threshold value or a percentage of the first threshold value. Similarly, the second value may be a multiple of the second threshold value or a percentage of the second threshold value.

[0113] If the intensity of the second image at a specific site of the plurality of sites is greater than or equal to the second value and the intensity of the first image at the specific site is less than the first value, it is determined at 120 that the second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the site. For some or all of the other sites, the same method can be used to determine whether the second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at those sites.

[0114] As mentioned above, Figure 3A The method 100 shown enables determination of whether any of two fluorescently labeled nucleotide precursors has been incorporated during a cycle of a nucleotide sequencing process. Steps 104 to 120 may be performed again in one or more subsequent sequencing cycles, for example, after cleavage of the first and second fluorescent markers.

[0115] As described in more detail below, in some embodiments, method 100 includes removing Figure 3A For example, in some embodiments, method 100 includes Figure 3B The steps shown or Figure 3C Steps shown.

[0116] It should be understood that although Figure 3A Method 100 is illustrated as if the temperature of the contents of the fluid channel were initially set within the highest temperature range and then cooled, but specific implementations may start at a temperature within the second temperature range and then heat the contents of the fluid chamber to within the first (higher) temperature range. Figure 3D Such an embodiment is shown in . As shown, the steps associated with the first temperature range and the first image may occur after the steps associated with the second temperature range and the second image.

[0117] See again Figure 3A In some embodiments, method 100 terminates after 112 because it has been determined that the first fluorescently labeled nucleotide precursor has been incorporated at the site. However, it should be understood that although Figure 3A The steps are shown to occur in a particular order (e.g., step 110 occurs before step 114), but various steps in these steps may occur in an order different from that shown. Figure 3D As explained in the context of, the lower temperature range can be tested first. For another example, it is not necessary to determine whether the intensity is greater than or equal to a specified value between imaging steps. In some embodiments, the first image and the second image are captured, and then the image is analyzed to determine whether to incorporate the first fluorescently labeled nucleotide precursor or the second fluorescently labeled nucleotide precursor. Figure 3E It should be understood that although Figure 3E Steps 106 and 108 are shown occurring before steps 114 and 116 , but steps 114 and 116 may be performed before steps 106 and 108 .

[0118] See again Figure 3AIf it is determined at 118 that the intensity of the second image at the specific site is not greater than or equal to the second value or the intensity of the first image at the specific site is not less than the first value, then it is concluded that neither the first fluorescently labeled nucleotide precursor nor the second fluorescently labeled nucleotide precursor is incorporated at the specific site. In this case, method 100 may end, or further analysis may be performed. Figure 3B It shows how method 100 may continue according to some embodiments. Figure 3B The step of determining whether at least a third fluorescently labeled nucleotide precursor has been incorporated at a specific site is shown. Figure 3B Also provided is a determination of whether a fourth fluorescently labeled nucleotide precursor has been incorporated at a specific site. Figure 3A and Figure 3B The combination shows the realization of Figure 2A The method 100 of the method shown is as described above.

[0119] See also Figure 3B At 130, a third fluorescently labeled nucleotide precursor is added to the fluid channel. Step 130 can be combined with Figure 3A The step 104 shown is combined (for example, the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor and the third fluorescently labeled nucleotide precursor can be added to the fluid chamber substantially at the same time), or can be a separate step. The third fluorescently labeled nucleotide precursor comprises a third fluorescent marker (which can be cleavable). The intensity emitted by the third fluorescent marker is less than the third threshold value in the first temperature range and the second temperature range, and is greater than or equal to the third threshold value in the third temperature range lower than both the first temperature range and the second temperature range. In other words, only considering the first temperature range, the second temperature range and the third temperature range, the third fluorescent marker will only emit light with the intensity of at least the third threshold value in the third temperature range; in the higher first temperature range and the second temperature range, the third fluorescent marker will not emit light with an intensity that reaches or exceeds the third threshold value. The first temperature range, the second temperature range and the third temperature range are non-overlapping, and they may be adjacent to each other, or there may be a temperature gap between the first temperature range and the second temperature range and / or between the second temperature range and the third temperature range. It should be understood that the third fluorescent marker can emit light with the intensity of at least the third threshold value in the temperature range outside the first temperature range, the second temperature range and the third temperature range. For example, the third fluorescent marker can emit light at an intensity of at least a third threshold at a temperature below the third temperature range. It is also conceivable that the third fluorescent marker can emit light at an intensity of at least a third threshold at a temperature above the first temperature range. The third threshold may be substantially the same as (i.e., equal to) the first threshold and / or the second threshold. Alternatively, the third threshold may be different from the first threshold and / or the second threshold.

[0120] At 132, the temperature of the contents of the fluid channel is set within a third temperature range. At 134, the device is illuminated while the temperature is within the third temperature range, and a third image of the contents of the fluid channel is captured, e.g., a third intensity is detected at each of the plurality of sites while the temperature of the fluid channel is within the third temperature range. The third image captures the intensity at each of the plurality of sites.

[0121] At 136, the third image is evaluated to determine whether the intensity of the third image at a specific site in the plurality of sites is greater than or equal to a third value. The third value may be substantially the same as (or equal to) the first value and / or the second value. If the intensity of the third image at the specific site is greater than or equal to the third value, and the intensity of the first image at the specific site is less than the first value, and the intensity of the second image at the specific site is less than the second value, then at 138, it is determined that the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the site. For some or all of the other sites, the same method can be used to determine whether the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at those sites.

[0122] If it is determined at 136 that the intensity of the third image at the specific site is not greater than or equal to the third value, then at 140, a fourth fluorescently labeled nucleotide precursor is added to the fluid chamber. Step 140 may be combined with step 130 and / or step 104 (at Figure 3A(shown in) combination. The fourth fluorescently labeled nucleotide precursor comprises a fourth fluorescent marker (which may be cleavable). The intensity emitted by the fourth fluorescent marker is less than the fourth threshold value in the first temperature range, the second temperature range and the third temperature range, and is greater than or equal to the fourth threshold value in the fourth temperature range lower than all the first temperature ranges, the second temperature range and the third temperature range. In other words, only considering the first temperature range, the second temperature range, the third temperature range and the fourth temperature range, the fourth fluorescent marker will only emit light with the intensity of at least the fourth threshold value in the fourth temperature range; in the higher first temperature range, the second temperature range and the third temperature range, the fourth fluorescent marker will not emit light with intensity reaching or exceeding the fourth threshold value. It should be understood that the fourth fluorescent marker can emit light with the intensity of at least the fourth threshold value in the temperature range outside the first temperature range, the second temperature range, the third temperature range and the fourth temperature range. For example, the fourth fluorescent marker can emit light with the intensity of at least the fourth threshold value at a temperature lower than the fourth temperature range. It can also be imagined that the fourth fluorescent marker can emit light with the intensity of at least the fourth threshold value at a temperature higher than the first temperature range. The first temperature range, the second temperature range, the third temperature range, and the fourth temperature range are non-overlapping, and they may be adjacent to each other, or there may be a temperature gap between the first temperature range and the second temperature range, between the second temperature range and the third temperature range, and / or between the third temperature range and the fourth temperature range. The fourth threshold value may be substantially the same as the first threshold value, the second threshold value, and / or the third threshold value (i.e., equal to the first threshold value, the second threshold value, and / or the third threshold value), or it may be different from the first threshold value, the second threshold value, and / or the third threshold value.

[0123] At 142, the temperature of the contents of the fluid channel is set within a fourth temperature range. At 144, when the temperature is within the fourth temperature range, the irradiation device is used, and a fourth image of the contents of the fluid channel is captured, for example, when the temperature of the fluid channel is within the fourth temperature range, a fourth intensity at each of the plurality of sites is detected. The fourth image captures the intensity at each of the plurality of sites. At 146, it is determined whether the intensity of the fourth image at a specific site in the plurality of sites is greater than or equal to a fourth value. The fourth value may be substantially the same (i.e., equal to) as the first value, the second value, and / or the third value. If so, and if the intensity of the first image at the specific site is less than the first value, and the intensity of the second image at the specific site is less than the second value, and the intensity of the third image at the specific site is less than the third value, then at 148, it is determined that the nucleotide precursor of the fourth fluorescent label has been incorporated into the extendable primer at the site. For some or all of the other sites, the same method can be used to determine whether the nucleotide precursor of the fourth fluorescent label has been incorporated into the extendable primer at those sites.

[0124] It should be understood that Figure 3D or Figure 3E Alternative Figure 3A Likewise, it should be understood that Figure 3A and Figure 3B The combination is similar to the above Figure 3D and Figure 3E In some embodiments, the temperature ranges are tested in the order of the temperature ranges described in the context of the present invention. Specifically, the order of the test temperature ranges need not be from highest to lowest, or even monotonous (e.g., it is conceivable that the temperature ranges can be tested in any order). Similarly, it is not necessary to analyze each image after taking each image and before changing the temperature of the contents of the fluid channel (e.g., two or more images can be taken before any analysis). In addition, and as described above, when two or more nucleotide precursors are to be added to the fluid chamber, they can be added in one or more rounds of addition (e.g., all materials can be added in step 104, or two or more of steps 104, 130, and 140 can be combined).

[0125] As mentioned above Figure 2B As explained in the context of , some embodiments use only three images to determine which of the four nucleotide precursors has been incorporated at a particular site. Figure 3A and Figure 3C The combination of shows method 100 according to some such embodiments. In step 118 ( Figure 3A ), method 100 continues with step 160, where a third fluorescently labeled nucleotide precursor and a fourth unlabeled nucleotide precursor are added to the fluid channel. Step 160 may be combined with step 104 ( Figure 3A ) combination, or it can be a separate step. The third fluorescently labeled nucleotide precursor comprises a third fluorescent marker, as described above. The fourth nucleotide precursor is unlabeled.

[0126] At 162, the temperature of the contents of the fluid channel is set within a third temperature range. At 164, the device is illuminated while the temperature is within the third temperature range, and a third image of the contents of the fluid channel is captured, e.g., a third intensity is detected at each of the plurality of sites while the temperature of the fluid channel is within the third temperature range. The third image captures the intensity at each of the plurality of sites.

[0127] At 166, the third image is evaluated to determine whether the intensity of the third image at a specific site in the plurality of sites is greater than or equal to a third value. The third value may be substantially the same (i.e., equal to) as the first value and / or the second value, or it may be different. If the intensity of the third image at the specific site is greater than or equal to the third value, and the intensity of the first image at the specific site is less than the first value, and the intensity of the second image at the specific site is less than the second value, then at 168, it is determined that the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the site. For some or all of the other sites, the same method can be used to determine whether the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at those sites.

[0128] If it is determined at 166 that the intensity of the third image at the specific site is not greater than or equal to the third value, it is inferred at 170 that a fourth unlabeled nucleotide precursor has been incorporated at the specific site.

[0129] As mentioned above Figure 2C As explained in the context of , some embodiments use only two images to determine which of four nucleotide precursors has been incorporated at a particular site. Figure 4A and Figure 4B Method 200 is shown according to some such embodiments.

[0130] At 202, in one or more rounds of addition, materials are added to a fluid channel of a sequencing device. The materials include at least a plurality of nucleic acid chains, a plurality of nucleic acid polymerase molecules, a first fluorescently labeled nucleotide precursor, a second fluorescently labeled nucleotide precursor, a third fluorescently labeled nucleotide precursor, and a fourth unlabeled nucleotide precursor. Figure 3A Nucleic acid chains and nucleic acid polymerases have been described in the context of, and these descriptions also apply here. In addition, it has been previously explained how nucleic acid polymerases or multiple nucleic acid chain molecules can be bound to the surface of the fluid channel of the sequencing device. These explanations also apply here.

[0131] The first fluorescently-labeled nucleotide precursor comprises the first fluorescent marker.The intensity that the first fluorescent marker sends is less than the first threshold value in the first temperature range, is greater than or equal to the first threshold value in the second temperature range lower than the first temperature range.In other words, the first fluorescent marker will emit light with the intensity of at least the first threshold value in the second temperature range but not in the first temperature range.The marker does not need to be cleavable.It should be understood that the first fluorescent marker can emit light with the intensity of at least the first threshold value in the temperature range outside the first temperature range and the second temperature range.For example, the first fluorescent marker can emit light with the intensity of at least the first threshold value at a temperature lower than the second temperature range.It can also be imagined that the first fluorescent marker can emit light with the intensity of at least the first threshold value at a temperature higher than the first temperature range.

[0132] The second fluorescently-labeled nucleotide precursor comprises the second fluorescent marker (it does not need to be cleavable).The intensity that the second fluorescent marker sends is greater than or equal to the second threshold value in the first temperature range and the second temperature range.In other words, only consider the first temperature range and the second temperature range, the second fluorescent marker will emit light with the intensity of at least the second threshold value in the first temperature range and the second temperature range.The second threshold value can be roughly the same as the first threshold value (that is, equal to), or two threshold values ​​can be different.

[0133] The third fluorescently labeled nucleotide precursor comprises both the first fluorescent label and the second fluorescent label.

[0134] The fourth nucleotide precursor does not include a fluorescent label.

[0135] There are a variety of methods for attaching fluorescent labels to the first fluorescently labeled nucleotide precursor and the second fluorescently labeled nucleotide precursor, as described above in Figure 3A described in the context of .

[0136] See again Figure 4A At 204, the temperature of the contents of the fluid channel is set within a first temperature range. At 206, the device is illuminated while the temperature is within the first temperature range, and a first image of the contents of the fluid channel is captured, e.g., a first intensity is detected at each of a plurality of sites while the temperature of the fluid channel is within the first temperature range. The first image captures the intensity at each of the plurality of sites. At 208, the temperature of the contents of the fluid channel is set within a second temperature range. At 210, the device is illuminated while the temperature is within the second temperature range, and a second image of the contents of the fluid channel is captured, e.g., a second intensity is detected at each of the plurality of sites while the temperature of the fluid channel is within the second temperature range. The second image captures the intensity at each of the plurality of sites.

[0137] At 212, using the first image and the second image, it is determined which of a first nucleotide precursor, a second nucleotide precursor, a third nucleotide precursor, or a fourth nucleotide precursor has been incorporated at a particular site in the plurality of sites. Figure 4BStep 212 according to some embodiments is shown. At 220, determine whether the intensity of the first image at the specific site is less than the first value, and whether the intensity of the second image at the specific site is greater than or equal to the second value. The first value and the second value may be approximately the same (i.e., equal), or they may be different. The first value and the second value may be equal to the first threshold and the second threshold, respectively. Alternatively, the first value and the second value may be based on the first threshold and the second threshold, respectively (e.g., they may be multiples or percentages of the first threshold and the second threshold). If the intensity of the first image at the specific site is less than the first value and the intensity of the second image at the specific site is greater than or equal to the second value, then determine at 222 that the nucleotide precursor of the first fluorescent label has been incorporated at the specific site. If not, then at 224, determine whether the intensity of the first image at the specific site is greater than or equal to the first value, and whether the intensity of the second image at the specific site is greater than or equal to the third value, the third value is greater than each of the first value and the second value. The third value may be, for example, the sum of the first value and the second value. If the intensity of the first image at the specific site is greater than or equal to the first value and the intensity of the second image at the specific site is greater than or equal to the third value, then it is determined at 226 that the third fluorescently labeled nucleotide precursor has been incorporated at the specific site. If not, then at 228, it is determined whether the intensity of the first image at the specific site is greater than or equal to the first value, and whether the intensity of the second image at the specific site is greater than or equal to the second value but less than the third value. If so, then at 230, it is determined that the second fluorescently labeled nucleotide precursor has been incorporated at the specific site.

[0138] If it is not determined at 228 that the intensity of the first image at the specific site is greater than or equal to the first value, and the intensity of the second image at the specific site is greater than or equal to the second value but less than the third value, then it can be inferred at 234 that the fourth unlabeled nucleotide precursor has been incorporated at the specific site. Alternatively, and optionally, it can be determined affirmatively at step 232 whether the intensity of the first image at the specific site is less than the first value, and the intensity of the second image at the specific site is less than the second value, in which case it is then determined at 234 that the fourth unlabeled nucleotide precursor has been incorporated at the specific site.

[0139] FIG. 3A to FIG. 3E as well as Figure 4A and Figure 4B The interpretation assumes that the emission intensity of the fluorescent markers is the same as elsewhere in this document (e.g., 1A to Figure 1C as well as FIG. 2A to FIG. 2C It will be appreciated that if the intensity of the fluorescent marker has different characteristics, then the intensity distribution may be modified appropriately. FIG. 3A to FIG. 3E (e.g. 110, 118, 136, 146, 166) and / or Figure 4A and Figure 4B (For example, certain elements of 220, 224, 228, 232). According to the disclosure of this article, ordinary technicians in this field will understand that modifications are appropriate.

[0140] Figure 5 A system 500 for sequencing nucleic acid is shown. As previously described, system 500 includes fluid channel 515 having multiple sites for attaching multiple nucleic acid chains to be sequenced to the surface of fluid channel 515. For example, fluid channel 515 may include a structure (e.g., a cavity or ridge) configured to anchor nucleic acid or nucleic acid polymerase to a proximal wall. Heater 510 is coupled to fluid channel 515. Heater 510 sets the temperature of the contents of fluid channel 515. It should be understood that heater 510 may include cooling elements and or replace heating elements. The main characteristic of heater 510 is that it can control the temperature of the contents of fluid channel 515. For example, heater 510 can set the temperature of the contents of fluid channel 515 in at least any one of a non-overlapping first temperature range, a second temperature range, a third temperature range, and a fourth temperature range. The second temperature range may be lower than the first temperature range, the third temperature range may be lower than the second temperature range, and the fourth temperature range may be lower than the third temperature range. Heater 510 may include, for example, a thermal sensor, a microprocessor, or software.

[0141] The system 500 also includes an imaging system 520. The imaging system 520 is configured to detect intensity at each of a plurality of sites of the fluid channel 515. For example, the imaging system 520 can detect intensity at each of a plurality of sites within each of a first temperature range, a second temperature range, a third temperature range, and a fourth temperature range.

[0142] like Figure 6 As shown, the imaging system 520 may include, for example, some or all of the following: a camera 521, an excitation light source 522 (e.g., a laser), an illumination optical device 523 (e.g., configured to evenly distribute the excitation light and reduce stray light), a detector 524 (e.g., a lens with a large aperture to increase light collection efficiency), an optical blocking filter 525 (e.g., for isolating the detector 524 from the excitation light), an analog-to-digital converter (ADC), and / or one or more sensors 526 that convert the detected intensity into a digitized signal or value (e.g., a charge coupled device (CCD) sensor, which itself may include other components, such as an ADC). It should be understood that the components and elements listed above are merely exemplary and are not intended to be limiting. The imaging system 520 may be coupled to a storage device (not shown). In such an embodiment, the storage device may store intensity data collected by the imaging system 520.

[0143] The system 500 also includes at least one processor 505 coupled to the imaging system 520 and the heater 510. The processor 505 executes machine-readable instructions. The processor 505 can direct or control other elements of the system 500. For example, the processor 505 can direct the heater 510 to set the temperature of the contents of the fluid channel 515 within a specified range. Similarly, the processor 505 can direct the imaging system 520 to detect the intensity at all or a subset of the multiple sites of the fluid channel 515. In addition or alternatively, the processor 505 can obtain an indication of the detected intensity at all or a subset of the multiple sites of the fluid channel 515 from the imaging system.

[0144] For example, in response to the intensity at a specific site among the plurality of sites being greater than or equal to a first threshold value within the first temperature range and the second temperature range, the processor 505 may identify a first fluorescently labeled nucleotide precursor or a complementary base of the first fluorescently labeled nucleotide precursor. For example, if the processor 505 determines that the intensity at the specific site corresponds to a fluorescent marker attached to a guanine precursor, the processor 505 may identify guanine as the detected marker, or identify cytosine as the next base in the sequenced nucleic acid fragment.

[0145] Similarly, in response to the intensity at this specific site in the multiple sites being less than the second threshold value in the first temperature range and being greater than or equal to the second threshold value in the second temperature range, the processor 505 can identify the complementary base of the second fluorescently labeled nucleotide precursor or the second fluorescently labeled nucleotide precursor. Similarly, in response to the intensity at this specific site in the multiple sites being less than the third threshold value in the first temperature range and the second temperature range and being greater than or equal to the third threshold value in the third temperature range, the processor 505 can identify the complementary base of the third fluorescently labeled nucleotide precursor or the third fluorescently labeled nucleotide precursor. And finally, in response to the intensity at this specific site in the multiple sites being less than the fourth threshold value in each of the first temperature range, the second temperature range and the third temperature range and being greater than or equal to the fourth threshold value in the fourth temperature range, the processor 505 can identify the complementary base of the fourth fluorescently labeled nucleotide precursor or the fourth fluorescently labeled nucleotide precursor.

[0146] The processor 505 may instruct the heater 510 to set the temperature of the contents in the fluid channel within a first temperature range. Then, when the temperature of the contents of the fluid channel 520 is within the first temperature range, the processor 505 may obtain an indication of the first intensity at some or all of the multiple sites from the imaging system 520. The processor 505 may instruct the heater 510 to set the temperature in the fluid channel within a second temperature range. Then, when the temperature of the fluid channel 515 is within the second temperature range, the processor 505 may obtain an indication of the second intensity at some or all of the multiple sites from the imaging system 520. In response to the first intensity at a specific site in the multiple sites being less than the first value, and the second intensity at the specific site being greater than or equal to the second value, the processor 505 may determine that the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site. For example, if the processor 505 determines that the intensity at the specific site corresponds to a fluorescent marker attached to an adenine precursor, the processor 505 may identify adenine as a detected marker, or identify thymine as the next base in the sequenced nucleic acid fragment. Alternatively, in response to the first intensity at the specific site being greater than or equal to the first value, and the second intensity at the specific site being greater than or equal to the second value and less than a third value, the third value being greater than each of the first value and the second value, the processor 505 can determine that the second fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site. Finally, in response to the first intensity at the specific site being greater than or equal to the first value, and the second intensity at the specific site being greater than or equal to the third value, the processor 505 can determine that the third fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site.

[0147] In the foregoing description and drawings, specific terminology is set forth to provide a thorough understanding of the embodiments disclosed herein. In some cases, the terminology or drawings may suggest specific details that are not necessary to practice the invention.

[0148] In order to avoid unnecessarily obscuring the present disclosure, well-known components are shown in block diagram form and / or have not been discussed in detail, or in some cases not discussed at all.

[0149] Unless otherwise specifically defined herein, all terms should be given their broadest possible interpretation, including the meaning implied by the specification and drawings and the meaning understood by those skilled in the art and / or as defined in dictionaries, books, etc. As expressly set forth herein, some terms may not be conformed to their ordinary or customary meanings.

[0150] As used in this specification and the appended claims, the singular forms "a", "an", and "the" do not exclude plural references unless otherwise indicated. Unless otherwise indicated, the word "or" should be interpreted as inclusive. Thus, the phrase "A or B" should be interpreted as all of the following meanings: "both A and B", "A but not B", and "B but not A". Any use of "and / or" herein does not mean that the word "or" alone is exclusive.

[0151] As used in this specification and the appended claims, phrases of the form "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, or C," and "one or more of A, B, and C" are interchangeable and each encompasses all of the following meanings: "only A," "only B," "only C," "A and B but not C," "A and C but not B," "B and C but not A," and "all of A, B, and C."

[0152] To the extent that the terms "including," "having," and variations thereof are used in the detailed description or claims, such terms are intended to be inclusive in a manner similar to the term "comprising," i.e., meaning "including, but not limited to." The terms "exemplary" and "embodiment" are used to indicate examples, not preferences or requirements.

[0153] The drawings are not necessarily drawn to scale, and dimensions, shapes, and sizes of features may vary significantly from how they are depicted in the drawings.

[0154] Although specific embodiments have been disclosed, it will be apparent that various modifications and changes may be made thereto without departing from the broader spirit and scope of the present disclosure. For example, at least where practicable, features or aspects of any one of the embodiments may be applied in combination with any other embodiment of the embodiments, or in place of corresponding features or aspects thereof. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

Claims

1. A method for sequencing nucleic acid using a sequencing device, the sequencing device comprising a fluid channel having a plurality of sites for attaching a plurality of nucleic acid chains to be sequenced to a surface of the fluid channel, the method comprising: In one or more rounds of addition, (i) the plurality of nucleic acid chains, (ii) a plurality of nucleic acid polymerase molecules, (iii) a first fluorescently labeled nucleotide precursor comprising a first fluorescent marker, wherein when excited to fluoresce, the normalized intensity of light emitted by the first fluorescent marker is greater than or equal to a threshold value within a first temperature range and a second temperature range, the second temperature range being lower than the first temperature range, wherein the first temperature range and the second temperature range do not overlap, and (iv) a second fluorescently labeled nucleotide precursor comprising a second fluorescent marker, wherein when excited to fluoresce, the normalized intensity of light emitted by the second fluorescent marker is less than or equal to the threshold value within the first temperature range and greater than or equal to the threshold value within the second temperature range, wherein the first fluorescent marker and the second fluorescent marker are chemically different, and wherein the first temperature range and the second temperature range are determined according to the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, the normalized intensity of light emitted by the second fluorescent marker, and the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker. (v) a third fluorescently labeled nucleotide precursor comprising a third fluorescent marker, wherein when excited into fluorescence, the normalized intensity of light emitted by the third fluorescent marker is less than the threshold value in the first temperature range and the second temperature range and is greater than or equal to the threshold value in the third temperature range, the third temperature range being lower than the second temperature range, wherein the first temperature range, the second temperature range and the third temperature range do not overlap, and wherein the first temperature range, the second temperature range and the third temperature range are divided according to the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the second fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the third fluorescent marker and the threshold value, and wherein the third fluorescent marker is chemically different from each of the first fluorescent marker and the second fluorescent marker; Using a heater, setting the temperature in the fluid channel to be within the first temperature range; using an imaging system, detecting a first intensity at each of the plurality of sites when the temperature of the fluid channel is within the first temperature range; In response to the first intensity at a specific site among the plurality of sites being greater than or equal to the threshold, determining that the first fluorescently labeled nucleotide precursor has been incorporated into the extendable primer at the specific site; using the heater, setting the temperature in the fluid channel within the second temperature range; using the imaging system, detecting a second intensity at each of the plurality of sites when the temperature of the fluid channel is within the second temperature range; In response to the second intensity at the specific site being greater than or equal to the threshold and the first intensity at the specific site being less than the threshold, determining that the second fluorescent-labeled nucleotide precursor has been incorporated into the extendable primer at the specific site; Using the heater, setting the temperature in the fluid channel within the third temperature range; using the imaging system, detecting a third intensity at each of the plurality of sites when the temperature of the fluid channel is within the third temperature range; as well as In response to the third intensity at the specific site being greater than or equal to the threshold, the first intensity at the specific site being less than the threshold, and the second intensity at the specific site being less than the threshold, it is determined that the third fluorescent-labeled nucleotide precursor has been incorporated into the extendable primer at the specific site. 2 . The method of claim 1 , wherein the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, and the third fluorescently labeled nucleotide precursor are added to the fluid channel simultaneously.

3. The method according to claim 1, further comprising: In response to the first intensity at the specific site being less than the threshold, the second intensity at the specific site being less than the threshold, and the third intensity at the specific site being less than the threshold, it is determined that the fourth unlabeled precursor has been incorporated into the extendable primer at the specific site.

4. The method according to claim 1, further comprising: adding a fourth fluorescently labeled nucleotide precursor comprising a fourth fluorescent marker to the fluid channel, wherein when excited to fluorescence, a normalized intensity of light emitted by the fourth fluorescent marker is less than the threshold value within each of the first temperature range, the second temperature range, and the third temperature range and is greater than or equal to the threshold value within a fourth temperature range, the fourth temperature range being lower than the third temperature range, wherein the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range do not overlap, and wherein the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range are divided according to a temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, a temperature dependence of the normalized intensity of light emitted by the second fluorescent marker, a temperature dependence of the normalized intensity of light emitted by the third fluorescent marker, a temperature dependence of the normalized intensity of light emitted by the fourth fluorescent marker, and the threshold value, and wherein the fourth fluorescent marker is chemically different from each of the first fluorescent marker, the second fluorescent marker, and the third fluorescent marker; using the heater, setting the temperature in the fluid channel within the fourth temperature range; using the imaging system, detecting a fourth intensity at each of the plurality of sites when the temperature of the fluid channel is within the fourth temperature range; as well as In response to the fourth intensity at the specific site being greater than or equal to the threshold, and the first intensity at the specific site being less than the threshold, and the second intensity at the specific site being less than the threshold, and the third intensity at the specific site being less than the threshold, it is determined that the fourth fluorescent-labeled nucleotide precursor has been incorporated into the extendable primer at the specific site. 5 . The method of claim 4 , wherein the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, the third fluorescently labeled nucleotide precursor, and the fourth fluorescently labeled nucleotide precursor are added to the fluid channel simultaneously.

6. A method for sequencing nucleic acid using a sequencing device, the sequencing device comprising a fluid channel having a plurality of sites for attaching a plurality of nucleic acid chains to be sequenced to a surface of the fluid channel, the method comprising: In one or more rounds of addition, (i) the plurality of nucleic acid chains, (ii) a plurality of nucleic acid polymerase molecules, (iii) a first fluorescently labeled nucleotide precursor comprising a first fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the first fluorescent marker is less than a threshold value in a first temperature range and greater than or equal to the threshold value in a second temperature range, the second temperature range being lower than the first temperature range, wherein the first temperature range and the second temperature range do not overlap, (iv) a second fluorescently labeled nucleotide precursor comprising a second fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the second fluorescent marker is greater than or equal to the threshold value in the first temperature range and the second temperature range, wherein the first temperature range and the second temperature range are divided according to the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the second fluorescent marker, and the threshold value, and (v) a third fluorescently labeled nucleotide precursor comprising the first fluorescent marker and the second fluorescent marker, wherein the first fluorescent marker and the second fluorescent marker are chemically different from each other; Using a heater, setting the temperature in the fluid channel to be within the first temperature range; using an imaging system, detecting a first intensity at each of the plurality of sites when the temperature of the fluid channel is within the first temperature range; using the heater, setting the temperature in the fluid channel within the second temperature range; using the imaging system, detecting a second intensity at each of the plurality of sites when the temperature of the fluid channel is within the second temperature range; In response to the first intensity being less than the threshold value, and the second intensity being greater than or equal to the threshold value and less than two times the threshold value, determining that the first fluorescently labeled nucleotide precursor has been incorporated; In response to the first intensity being greater than or equal to the threshold, and the second intensity being greater than or equal to the threshold and less than two times the threshold, determining that the second fluorescent-labeled nucleotide precursor has been incorporated; as well as In response to the first intensity being greater than or equal to the threshold and the second intensity being greater than or equal to twice the threshold, it is determined that the third fluorescent-labeled nucleotide precursor has been incorporated. 7 . The method of claim 6 , wherein the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, and the third fluorescently labeled nucleotide precursor are added to the fluid channel simultaneously.

8. The method according to claim 6, further comprising: In response to the first intensity being less than the threshold value, and the second intensity being less than the threshold value, it is determined that a fourth unlabeled nucleotide precursor has been incorporated.

9. The method according to claim 6, further comprising: determining that none of the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, or the third fluorescently labeled nucleotide precursor has been incorporated; as well as In response to determining that none of the first fluorescently labeled nucleotide precursor, the second fluorescently labeled nucleotide precursor, or the third fluorescently labeled nucleotide precursor has been incorporated, it is inferred that a fourth unlabeled nucleotide precursor has been incorporated.

10. A system for sequencing nucleic acids, comprising: A fluid channel having a plurality of sites for attaching a plurality of nucleic acid strands to be sequenced to a surface of the fluid channel, at least some of the plurality of nucleic acid strands comprising a fluorescent marker, the fluorescent marker comprising: (a) a first fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the first fluorescent marker is greater than or equal to a threshold value within a first temperature range, a second temperature range, a third temperature range, and a fourth temperature range, wherein the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range do not overlap, and wherein the second temperature range is lower than the first temperature range, the third temperature range is lower than the second temperature range, and the fourth temperature range is lower than the third temperature range, (b) a second fluorescent marker, wherein when excited to fluoresce, the normalized intensity of light emitted by the second fluorescent marker is less than or equal to the threshold value within the first temperature range, and greater than or equal to the threshold value within the second temperature range, the third temperature range, and the fourth temperature range, and (c) a third fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the third fluorescent marker is less than or equal to the threshold value in the first temperature range and the second temperature range, and is greater than or equal to the threshold value in the third temperature range and the fourth temperature range, wherein the first fluorescent marker, the second fluorescent marker and the third fluorescent marker are chemically different, and wherein the first temperature range, the second temperature range, the third temperature range and the fourth temperature range are divided according to the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the second fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the third fluorescent marker and the threshold value; a heater coupled to the fluid channel for setting a temperature of the contents of the fluid channel within any one of the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range; an imaging system configured to detect an intensity of light emitted at each of the plurality of sites within each of the first temperature range, the second temperature range, the third temperature range, and the fourth temperature range; and at least one processor coupled to the imaging system and the heater and configured to execute at least one machine-readable instruction that, when executed, causes the at least one processor to: identifying a first fluorescently labeled nucleotide precursor comprising the first fluorescent marker or a complementary base of the first fluorescently labeled nucleotide precursor in response to the intensity of light emitted at a specific site among the plurality of sites being greater than or equal to the threshold value within the first temperature range and the second temperature range, identifying a second fluorescently labeled nucleotide precursor comprising the second fluorescent marker or a complementary base of the second fluorescently labeled nucleotide precursor in response to the intensity of light emitted at the specific site of the plurality of sites being less than the threshold within the first temperature range and greater than or equal to the threshold within the second temperature range, In response to the intensity of the light emitted at the specific site of the plurality of sites being less than the threshold value within the first temperature range and the second temperature range and being greater than or equal to the threshold value within the third temperature range, identifying a region containing the third fluorescent marker a third fluorescently labeled nucleotide precursor of the marker or a complementary base of the third fluorescently labeled nucleotide precursor, and In response to the intensity of light emitted at the specific site among the multiple sites being less than the threshold within each of the first temperature range, the second temperature range and the third temperature range and greater than or equal to the threshold within the fourth temperature range, a fourth fluorescently labeled nucleotide precursor containing a fourth fluorescent marker or a complementary base of the fourth fluorescently labeled nucleotide precursor is identified.

11. The system of claim 10, wherein the fluid channel comprises a structure, wherein the structure comprises the plurality of sites for attaching the plurality of nucleic acid strands to be sequenced to the surface of the fluid channel.

12. The system of claim 11, wherein the structure comprises a cavity or a ridge.

13. The system of claim 10, wherein the heater comprises a thermal sensor or a microprocessor.

14. The system of claim 10, wherein the imaging system comprises one or more of: a camera, a light source, illumination optics, a detector, an optical blocking filter, an analog-to-digital converter, or one or more sensors.

15. The system of claim 14, wherein the imaging system comprises the light source, and wherein the light source comprises a laser.

16. The system of claim 14, wherein the imaging system includes the illumination optics, and wherein the illumination optics are configured to evenly distribute excitation light over the plurality of sites.

17. The system of claim 14, wherein the imaging system comprises the detector, and wherein the detector comprises a lens.

18. The system of claim 17, wherein the imaging system includes the optical blocking filter, and wherein the optical blocking filter is located within the system to isolate the lens from light emitted by the light source.

19. The system of claim 14, wherein the imaging system comprises the one or more sensors, and wherein the one or more sensors comprise charge coupled device sensors.

20. The system of claim 10, wherein the at least one machine-readable instruction, when executed by the at least one processor, further causes the at least one processor to control the heater.

21. The system of claim 10, wherein the at least one machine-readable instruction, when executed by the at least one processor, further causes the at least one processor to control the imaging system.

22. A system for sequencing nucleic acids, comprising: A fluid channel having a plurality of sites for attaching a plurality of nucleic acid strands to be sequenced to a surface of the fluid channel, at least some of the plurality of nucleic acid strands comprising at least one of two fluorescent markers, the two fluorescent markers comprising: (a) a first fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the first fluorescent marker is greater than or equal to a threshold value within a first temperature range and a second temperature range, wherein the first temperature range and the second temperature range do not overlap, and wherein the second temperature range is lower than the first temperature range, and (b) a second fluorescent marker, wherein when excited to fluoresce, a normalized intensity of light emitted by the second fluorescent marker is less than or equal to the threshold value within the first temperature range, and is greater than or equal to the threshold value within the second temperature range, wherein the first fluorescent marker and the second fluorescent marker are chemically different from each other, wherein the first temperature range and the second temperature range are divided according to the temperature dependence of the normalized intensity of light emitted by the first fluorescent marker, the temperature dependence of the normalized intensity of light emitted by the second fluorescent marker, and the threshold value; a heater for setting the temperature of the contents of the fluid channel within any one of the first temperature range and the second temperature range; an imaging system configured to detect an intensity of light emitted at each of the plurality of sites within each of the first temperature range and the second temperature range; and at least one processor coupled to the imaging system and configured to execute at least one machine-readable instruction that, when executed, causes the at least one processor to: instructing the heater to set the temperature in the fluid channel to be within the first temperature range; acquiring from the imaging system an indication of a first intensity of light emitted at each of the plurality of sites when the temperature of the fluid channel is within the first temperature range; instructing the heater to set the temperature within the fluid channel within the second temperature range; acquiring from the imaging system an indication of a second intensity of light emitted at each of the plurality of sites when the temperature of the fluid channel is within the second temperature range; determining that a first fluorescently labeled nucleotide precursor comprising the first fluorescent marker has been incorporated into the extendable primer at the specific site in response to the first intensity of light emitted at the specific site being less than the threshold and the second intensity of light emitted at the specific site being greater than or equal to the threshold and less than two times the threshold, determining that a second fluorescently labeled nucleotide precursor comprising the second fluorescent marker has been incorporated into the extendable primer at the specific site in response to the first intensity of light emitted at the specific site being greater than or equal to the threshold and the second intensity of light emitted at the specific site being greater than or equal to the threshold and less than two times the threshold, and In response to the first intensity of light emitted at the specific site being greater than or equal to the threshold, and the second intensity of light emitted at the specific site being greater than or equal to twice the threshold, it is determined that a third fluorescently labeled nucleotide precursor comprising both the first fluorescent marker and the second fluorescent marker has been incorporated into the extendable primer at the specific site.

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