Sequencing method, apparatus, computer device, and computer-readable storage medium

Abstract

Provided are a sequencing method, an apparatus, a computer device, a computer-readable storage medium and a computer program product. A nucleotide sequence cluster to be tested is subjected to a single-base extension reaction of complementary strands in the presence of four nucleotides, wherein the signal intensity distributions of signals associated with three of the nucleotides in different preset time periods are different, and the type of each complementary nucleotide of said nucleotide sequence cluster in a cycle is determined on the basis of the signal intensities in the different preset time periods.

Description

Sequencing methods, apparatus, computer equipment and computer-readable storage media Technical Field

[0001] This application relates to the field of gene sequencing, and in particular to a sequencing method, apparatus, computer equipment, computer-readable storage medium, and computer program product. Background Technology

[0002] This section is intended to provide background or context for the implementation of the embodiments of this application as set forth in the claims and detailed descriptions. The description herein is not intended to imply that it is conventional art simply because it is included in this section.

[0003] The research and application of gene sequencing technology covers almost all areas of human society, including health, agriculture, energy, and national defense and security. It has played a huge role in promoting life sciences, biomedicine and related industries, and has a more profound impact than the information economy era.

[0004] In the process of gene sequencing, fluorescence imaging of four bases is required to determine the base sequence in DNA. Related technologies distinguish the four bases by the presence or absence of signals. Summary of the Invention

[0005] This application provides a sequencing method, apparatus, computer device, computer-readable storage medium, and computer program product.

[0006] Firstly, this application provides a sequencing method, including:

[0007] The signal intensity of the nucleotide sequence cluster to be tested was obtained at different preset time periods in one cycle of nucleic acid sequencing. The nucleotide sequence cluster to be tested underwent single-base extension reaction of complementary strands in the presence of four types of nucleotides, including type I, type II, type III, and type IV. The complementary nucleotide of type I was associated with the first signal, the complementary nucleotide of type III was associated with the second signal, and the complementary nucleotide of type II was associated with the first and second signals. The signal intensity distribution of the first and second signals was different at different preset time periods. The four nucleotides carried modification groups.

[0008] The nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle is determined by the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods.

[0009] In some embodiments, determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster under test at different preset time periods includes: for each complementary nucleotide, obtaining a signal intensity vector composed of signal intensities at different preset time periods; and determining the nucleotide type of the complementary nucleotide based on the signal intensity vector of the complementary nucleotide based on the trained neural network.

[0010] In some embodiments, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods includes: for each complementary nucleotide, obtaining a data pair consisting of a first signal intensity and a second signal intensity; using the first signal intensity and the second signal intensity of the data pair as the abscissa and ordinate of a two-dimensional coordinate system, respectively, to obtain the data point of the nucleotide to be tested on the two-dimensional coordinate system; clustering multiple data points on the two-dimensional coordinate system to obtain a clustering result, the clustering result containing multiple clusters; for each complementary nucleotide, the nucleotide type corresponding to the cluster to which the data point of the complementary nucleotide belongs is taken as the nucleotide type of the complementary nucleotide.

[0011] In some embodiments, multiple nucleotide sequence clusters to be tested are arranged on the surface of a solid support. Obtaining the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods in one cycle of nucleic acid sequencing includes: obtaining fluorescence images of the solid support at different preset time periods in one cycle of nucleic acid sequencing; and obtaining the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods in one cycle of nucleic acid sequencing based on the fluorescence images.

[0012] In some embodiments, different preset time periods include a first preset time period and a second preset time period. In one cycle of nucleic acid sequencing, acquiring fluorescence images of the solid support at different preset time periods includes: acquiring fluorescence images of the solid support at fixed time intervals during one cycle of nucleic acid sequencing; using the first x frames of fluorescence images in the cycle as fluorescence images of the first preset time period; and using the last y frames of fluorescence images in the cycle as fluorescence images of the second preset time period; wherein x and y are positive integers, and the sum of x and y is less than or equal to the total number of fluorescence images acquired in one cycle of nucleic acid sequencing; the signal intensity includes the maximum signal intensity. Acquiring the signal intensity of the nucleotide sequence cluster to be tested in different preset time periods during one cycle of nucleic acid sequencing based on the fluorescence images includes: for each preset time period, traversing all fluorescence images of the preset time period to obtain the maximum pixel value of the nucleotide sequence cluster to be tested in all fluorescence images of the preset time period; and using the maximum pixel value as the maximum signal intensity of the nucleotide sequence cluster to be tested in the preset time period.

[0013] In some embodiments, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide in the cycle of the nucleotide sequence cluster to be tested based on the signal intensity at different preset time periods includes: if both the first signal intensity and the second signal intensity are within a first predetermined interval, the complementary nucleotide is identified as a first nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within the second predetermined interval, the complementary nucleotide is identified as a second nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within a third predetermined interval, the complementary nucleotide is identified as a third nucleotide; and if both the first signal intensity and the second signal intensity are within the third predetermined interval, the complementary nucleotide is identified as a fourth nucleotide.

[0014] In some embodiments, three of the four nucleotides are labeled with a marker, and the signal intensity distribution of the three nucleotide markers differs at different predetermined time periods. The marker used for nucleotide labeling (nucleotide marker) may include luciferase, which may be selected from glow-type luciferase, flash-type luciferase, mixed-light luciferase, or mixtures thereof, derivatives, etc. The aforementioned mixtures include products of luciferase and biotin or antibody or their antigen-binding fragments; the aforementioned derivatives include mutated luciferase, and also products of mutated luciferase and biotin or antibody or their antigen-binding fragments.

[0015] For example, the nucleotide carrying the modifying group is modified with biotin, and the nucleotide marker is modified with avidin. Based on the interaction between biotin and avidin, the nucleotide marker can label the nucleotide carrying the modifying group.

[0016] For example, a nucleotide carrying a modifying group is modified with an antigen, and a nucleotide marker is modified with an antibody or its antigen-binding fragment. Based on the specific recognition function of the antigen and the antibody or its antigen-binding fragment, the nucleotide marker marks the nucleotide carrying the modifying group.

[0017] For example, indirect labeling includes: dATP-biotin-streptavidin-flash luciferase, dTTP-dgoxigenin-dgoxigenin antibody or its antigen-binding fragment-flash luciferase, dCTP-biotin-streptavidin-flash luciferase, dCTP-dgoxigenin-dgoxigenin antibody or its antigen-binding fragment-flash luciferase, and dGTP is not labeled.

[0018] For example, indirect labeling includes: dTTP-anti-dTTP antibody or its antigen-binding fragment-glow luciferase, dATP-anti-dATP antibody or its antigen-binding fragment-flash luciferase, dCTP-anti-dCTP antibody or its antigen-binding fragment-flash luciferase, dCTP-anti-dCTP antibody or its antigen-binding fragment-glow luciferase, and dGTP is not labeled.

[0019] It should be noted that nucleotide labeling includes two methods: The first is labeling before the single-base extension reaction, that is, three of the four nucleotides (ribonucleotides or deoxyribonucleotides) are pre-labeled with nucleotide markers, and then the ligation product is complementary to the single-stranded nucleic acid template, and then the nucleotide marker substrate is added for signal detection; The second is labeling after the single-base extension reaction, that is, the four nucleotides introduced are first complementary to the single-stranded nucleic acid template, and then the nucleotide marker is added. After the nucleotide marker is labeled on the nucleotide, the nucleotide marker substrate is added for signal detection.

[0020] In some embodiments, at least one of the aforementioned glow-type luciferase and the aforementioned flash-type luciferase is provided in a diluted form using diluent 1 (manufacturer: MGI; catalog number: 940-000571-00).

[0021] It should be noted that the aforementioned nucleotide labeling substrate can be one or more. In this application, a preferred nucleotide labeling substrate is capable of simultaneously generating signals (e.g., optical signals, preferably fluorescent signals) from both type I and type II nucleotide labels, enabling base reading in single-channel monochrome single-substrate sequencing.

[0022] According to embodiments of this application, the nucleotide labeling substrate is selected from coelentrin, coelentrin derivatives, or isomers thereof. In some embodiments, the aforementioned nucleotide labeling substrate is provided in a diluted form using diluent 4 (manufacturer: MGI; catalog number: 940-000571-00).

[0023] In some embodiments, the marker for the first nucleotide type is a first luciferase, the marker for the third nucleotide type is a second luciferase, the marker for the second nucleotide type is a combination of the first luciferase and the second luciferase, and there is no marker for the fourth nucleotide type.

[0024] In some embodiments, the first luciferase is a glow-type luciferase and the second luciferase is a flash-type luciferase. The first luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a first signal, which is a glow-type signal. The second luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a second signal, which is a flash-type signal.

[0025] In some embodiments, the method further includes: performing a preset biochemical intervention procedure at preset time points in the cycle to change the biochemical state of each marker; obtaining the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing includes: obtaining the signal intensity of the nucleotide sequence cluster to be tested during a first preset time period before performing the biochemical intervention procedure in one cycle of nucleic acid sequencing; and obtaining the signal intensity of the nucleotide sequence cluster to be tested during a second preset time period after performing the biochemical intervention procedure.

[0026] In some embodiments, the modifying group is a reversible blocking modifying group, the signal intensity of the first signal is the same in different preset time periods, and the signal intensity of the second signal is different in different preset time periods.

[0027] In some embodiments, four nucleotide modifications are detectable markers.

[0028] In some embodiments, different preset time periods include a first preset time period and a second preset time period. Determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster in different preset time periods includes: determining an intensity difference value and a difference value interval to which the intensity difference value belongs based on a first signal intensity of the nucleotide sequence cluster in the first preset time period and a second signal intensity in the second preset time period. The intensity difference value includes an intensity ratio or an intensity difference. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0029] Secondly, this application provides a sequencing method, including:

[0030] The signal intensity of the detection position of a single-stranded nucleotide template at different preset time periods during nucleic acid sequencing is obtained. The nucleotide to be detected at the detection position binds to the complementary nucleotide for extended sequencing reaction. The signal intensity of the markers corresponding to different types of complementary nucleotides is different, and the signal intensity difference is the difference in signal intensity at different preset time periods.

[0031] Based on the difference in signal intensity at the detection site, the nucleotide type of the complementary nucleotide that binds to the detection site in the extension sequencing reaction is determined, thereby determining the nucleotide type of the nucleotide to be tested at the detection site.

[0032] In some embodiments, three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is glow-in-the-dark luciferase, the marker for the third nucleotide type is flash-in-the-dark luciferase, the marker for the second nucleotide type is a combination of glow-in-the-dark luciferase and flash-in-the-dark luciferase, and the fourth nucleotide type has no marker. The glow-in-the-dark luciferase reacts with the nucleotide marker substrate in the extension sequencing reaction to produce a glow-in-the-dark signal, and the flash-in-the-dark luciferase reacts with the nucleotide marker substrate in the extension sequencing reaction to produce a flash-in-the-dark signal.

[0033] In some embodiments, the different preset time periods include a first preset time period and a second preset time period, the signal intensity difference includes the intensity difference value of the signal intensity, and determining the nucleotide type of the complementary nucleotide bound by the extension sequencing reaction at the detection site based on the signal intensity difference at the detection site includes: determining the intensity difference value of the detection site and the difference value range to which the intensity difference value belongs based on the first signal intensity at the detection site in the first preset time period and the second signal intensity in the second preset time period, wherein the intensity difference value includes the intensity ratio or intensity difference value; and determining the nucleotide type of the complementary nucleotide bound by the extension sequencing reaction at the detection site based on the difference value range to which the intensity difference value belongs.

[0034] In some embodiments, determining the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction based on the difference value interval to which the intensity difference value belongs includes: if the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is a first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be a first nucleotide type; if the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is a second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be a second nucleotide type; if the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is a third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be a third nucleotide type; if the first signal intensity and the second signal intensity are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be a fourth nucleotide type.

[0035] In some embodiments, multiple single-stranded nucleotide templates are arranged on the surface of a solid support. Obtaining the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing includes: obtaining fluorescence images of the solid support at different preset time periods during nucleic acid sequencing; and obtaining the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing based on the fluorescence images.

[0036] In some embodiments, different preset time periods include a first preset time period and a second preset time period that do not overlap. During nucleic acid sequencing, acquiring fluorescence images of the solid support at different preset time periods includes: acquiring fluorescence images of the solid support at fixed time intervals during the first preset time period to obtain x frames of fluorescence images; acquiring fluorescence images of the solid support at fixed time intervals during the second preset time period to obtain y frames of fluorescence images; wherein x and y are positive integers; the signal intensity includes the maximum signal intensity. Acquiring the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing based on the fluorescence images includes: for each preset time period, traversing all fluorescence images of the preset time period to obtain the maximum pixel value of the detection position in all fluorescence images of the preset time period; and using the maximum pixel value of the detection position as the maximum signal intensity of the detection position in the preset time period.

[0037] Thirdly, this application provides a sequencing method, including:

[0038] The signal intensity of the nucleotide sequence cluster to be tested at the detection location at different preset time periods is obtained. The sequencing reaction of the nucleotide sequence cluster to be tested is performed in the presence of four types of nucleotides, including the first type of nucleotide, the second type of nucleotide, the third type of nucleotide, and the fourth type of nucleotide. The complementary nucleotide of the first type of nucleotide is associated with the first signal, the complementary nucleotide of the third type of nucleotide is associated with the second signal, and the complementary nucleotide of the second type of nucleotide is associated with the third signal. The signal intensity distribution of the first, second, and third signals is different at different preset time periods.

[0039] The type of complementary nucleotide that binds to the target nucleotide sequence cluster at the detection site is determined based on the signal intensity at different preset time periods.

[0040] In some embodiments, the first signal is a glow-type signal, the second signal is a flash-type signal, and the third signal is a combination of glow-type and flash-type signals. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of these markers differs across preset time periods. The marker for the first nucleotide type is glow-type luciferase, the marker for the third nucleotide type is flash-type luciferase, the marker for the second nucleotide type is a combination of glow-type and flash-type luciferase, and the fourth nucleotide type has no marker. Glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to produce a glow-type signal, and flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to produce a flash-type signal. The signal intensity distribution varies across different preset time periods. The method includes determining the nucleotide type of the complementary nucleotide bound to the test nucleotide sequence cluster at the detection position based on the signal intensity of the first signal intensity during a first preset time period and the second signal intensity during a second preset time period. This includes: if both the first and second signal intensities are within a first predetermined interval, the complementary nucleotide is identified as the first nucleotide; if both the first and second signal intensities are within the first predetermined interval and the second signal intensity is within the second predetermined interval, the complementary nucleotide is identified as the second nucleotide; if both the first and second signal intensities are within the first predetermined interval and the second signal intensity is within the third predetermined interval, the complementary nucleotide is identified as the third nucleotide; and if both the first and second signal intensities are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0041] In some embodiments, the first signal is a glow-type signal, the second signal is a first flash-type signal, and the third signal is a second flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the second nucleotide type is a first flash-type luciferase, the marker for the third nucleotide type is a second flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first flash-type signal, and the second flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second flash-type signal.

[0042] In some embodiments, the first signal is a glow-type signal, the third signal is a first flash-type signal, and the second signal is a second flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of these markers differs across preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the third nucleotide type is a first flash-type luciferase, the marker for the second nucleotide type is a second flash-type luciferase, and the fourth nucleotide type is unlabeled. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first flash-type signal, and the second flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second flash-type signal. The different preset time periods include a first preset time period and a second preset time period. The complementary nucleus binding to the target nucleotide sequence cluster at the detection position is determined based on the signal intensity of the target nucleotide sequence cluster at different preset time periods. The nucleotide type of the nucleotide includes: determining the intensity difference value of the detection location and the difference value range to which the intensity difference value belongs based on the first signal intensity of the detection location in a first preset time period and the second signal intensity in a second preset time period. The intensity difference value includes the intensity ratio or intensity difference. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value range to which the intensity difference value belongs is the first difference value range, then the nucleotide type of the complementary nucleotide is determined as the first nucleotide type. If the first signal intensity or the second signal intensity is greater than the preset intensity threshold and the difference value range to which the intensity difference value belongs is the second difference value range, then the nucleotide type of the complementary nucleotide is determined as the second nucleotide type. If the first signal intensity or the second signal intensity is greater than the preset intensity threshold and the difference value range to which the intensity difference value belongs is the third difference value range, then the nucleotide type of the complementary nucleotide is determined as the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to the preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined as the fourth nucleotide type.

[0043] Fourthly, this application also provides a sequencing device, comprising:

[0044] The data acquisition module is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods during one cycle of nucleic acid sequencing. The nucleotide sequence cluster to be tested undergoes a single-base extension reaction of complementary strands in the presence of four types of nucleotides, including a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with the first signal, the complementary nucleotide of the third nucleotide type is associated with the second signal, and the complementary nucleotide of the second nucleotide type is associated with both the first and second signals. The signal intensity distribution of the first and second signals is different at different preset time periods. The four nucleotides carry modifying groups.

[0045] The type determination module is used to determine the nucleotide type of the nucleotide to be tested in the nucleotide sequence cluster based on the signal intensity of the nucleotide sequence cluster at different preset time periods.

[0046] Fifthly, this application also provides a sequencing device, comprising:

[0047] The data acquisition module is used to acquire the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing. The nucleotide to be tested at the detection position binds to the complementary nucleotide for extended sequencing reaction. The signal intensity of the markers corresponding to different types of complementary nucleotides is different, and the signal intensity difference is the difference in signal intensity at different preset time periods.

[0048] The type determination module is used to determine the nucleotide type of the complementary nucleotide that binds to the detection site in the extension sequencing reaction based on the signal intensity difference at the detection site, thereby determining the nucleotide type of the nucleotide to be tested at the detection site.

[0049] Sixthly, this application also provides a sequencing device, comprising:

[0050] The data acquisition module is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at the detection location at different preset time periods. The nucleotide sequence cluster to be tested is sequenced in the presence of four types of nucleotides, namely, a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with the first signal, the complementary nucleotide of the third nucleotide type is associated with the second signal, and the complementary nucleotide of the second nucleotide type is associated with the third signal. The signal intensity distribution of the first, second, and third signals is different at different preset time periods.

[0051] The type determination module is used to determine the nucleotide type of the complementary nucleotide that binds to the target nucleotide sequence cluster at the detection position based on the signal intensity of the target nucleotide sequence cluster at different preset time periods.

[0052] A computer device includes a memory and a processor, the memory storing computer-readable instructions, and the processor executing the computer-readable instructions to implement the steps of the sequencing method described above.

[0053] A computer-readable storage medium having computer-readable instructions stored thereon, which, when executed by a processor, implement the steps of the sequencing method described above.

[0054] A computer program product includes computer-readable instructions that, when executed by a processor, implement the steps of the sequencing method described above.

[0055] Details of one or more embodiments of this application are set forth in the following drawings and description. Other features, objects, and advantages of this application will become apparent from the specification, drawings, and claims. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments or related technologies of this application, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The additional details or examples used to describe the drawings should not be considered as limiting the scope of any of the disclosed invention, the currently described embodiments and / or examples, and the best mode of these inventions as currently understood.

[0057] Figure 1 is a schematic flowchart of a sequencing method in one embodiment;

[0058] Figure 2 is a schematic diagram showing the trend of signal strength over time in one embodiment;

[0059] Figure 3 is a schematic diagram of the signal strength acquisition in one embodiment;

[0060] Figure 4 is a schematic diagram of luminescence intensity and base clustering in one embodiment;

[0061] Figure 5 is a schematic diagram of luminescence intensity and base clustering in another embodiment;

[0062] Figure 6 is a schematic diagram of base clustering in another embodiment;

[0063] Figure 7 is a schematic diagram comparing the photodynamic curves of different luciferases in one embodiment;

[0064] Figure 8 is a schematic diagram of an optical signal dynamics model in one embodiment, where A is model 1, B is model 2, C is model 3, and D is model 4;

[0065] Figure 9 is a flowchart illustrating the sequencing method in another embodiment;

[0066] Figure 10 is a flowchart illustrating the sequencing method in yet another embodiment;

[0067] Figure 11 is a structural block diagram of a sequencing device in one embodiment;

[0068] Figure 12 is another structural block diagram of the sequencing device in one embodiment;

[0069] Figure 13 is a structural block diagram of the sequencing device in another embodiment;

[0070] Figure 14 is a structural block diagram of the sequencing device in yet another embodiment;

[0071] Figure 15 is an internal structure diagram of a computer device in one embodiment;

[0072] Figure 16 is an internal structural diagram of a computer device in another embodiment. Detailed Implementation

[0073] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to be used in the overall description of this application. The use of terms like "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, the number of indicated technical features, or the sequential relationship between indicated technical features.

[0074] In the description of this application, unless otherwise expressly defined, terms such as "setup," "installation," and "connection" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this application in conjunction with the specific content of the technical solution.

[0075] Over the past decade, second-generation DNA sequencing technology has gradually grown from an emerging technology to a mainstream sequencing method and has become an important detection tool in the clinical field, playing an increasingly important role in the prevention and control of infectious diseases, the diagnosis of genetic diseases, and non-invasive prenatal screening.

[0076] Microscopic imaging systems are widely used in second-generation DNA sequencing technology. During gene sequencing, fluorescence imaging of four bases on a biological sample chip is required: adenine (A), thymine (T), cytosine (C), and guanine (G). Multi-channel imaging (such as four-channel or two-channel imaging) is typically used, and then the detection images obtained from each channel are registered using algorithms to match the base positions in different images, thereby enabling the determination of the DNA base sequence. To distinguish the four bases (i.e., the four fluorescence bands), gene sequencing optical systems need to use multi-channel imaging, commonly four-channel or two-channel imaging systems. Each channel consists of a lens, filter, and camera.

[0077] In a dual-channel imaging system, two excitation wavelengths are alternately activated sequentially. For each location on the biological sample, each channel's imaging system captures an image under a different wavelength of excitation light, thus obtaining images of the four bases AT, G, C, and D. Due to camera limitations, data transmission takes tens of milliseconds after each exposure. However, each camera in a dual-channel imaging system needs to expose the same location on the sample twice, and the added time for data transmission inevitably prolongs the imaging time. In contrast, a four-channel imaging system allows both wavelengths of excitation light to be activated simultaneously and continuously. For each location on the biological sample, each channel's imaging system captures an image of the four bases AT, G, C, and D. While the four-channel imaging system can save at least half the imaging time compared to the dual-channel system, the lens and filter for each channel are more expensive, inevitably increasing the overall cost.

[0078] This application provides a sequencing method, apparatus, computer equipment, computer-readable storage medium, and computer program product that can achieve gene sequencing without the need for multiple channels, thereby reducing the cost of gene sequencing.

[0079] Please refer to Figure 1, which is a schematic flowchart of a sequencing method in one embodiment. In one embodiment, as shown in Figure 1, a sequencing method is provided. It is understood that this method can be applied to a terminal, a server, or a system including both a terminal and a server, and is implemented through interaction between the terminal and the server. In this embodiment, the method includes the following steps:

[0080] Step S102: Obtain the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing.

[0081] In this process, the nucleotide sequence clusters to be tested undergo a single-base extension reaction of complementary strands in the presence of four types of nucleotides. The four types of nucleotides include a first type, a second type, a third type, and a fourth type. The complementary nucleotide of the first type is associated with the first signal, the complementary nucleotide of the third type is associated with the second signal, and the complementary nucleotide of the second type is associated with both the first and second signals. The signal intensity distributions of the first and second signals are different at different preset time periods. The four types of nucleotides carry modifying groups.

[0082] Nucleotide sequence clusters are groups of similar or identical molecules, nucleotide sequences, or DNA chains containing multiple nucleotides to be tested. For example, a cluster can be an amplified nucleotide, oligonucleotide, or polynucleotide with the same or similar sequences. During base sequencing cycles, nucleotide sequence clusters can be immobilized onto reaction sites on a solid support surface and / or into a reaction chamber. The nucleotide sequence cluster to be tested can be a DNA nanosphere or a DNA cluster. Taking DNA nanospheres as an example, in one embodiment, before performing step S102, the DNA fragment to be tested can be circularized into a single-stranded circular DNA. Using rolling circle amplification (RCA) technology, the circular single-stranded DNA is formed into multiple copies of single-stranded DNA linked end-to-end, and then freely folded into a nanosphere structure in solution, i.e., DNA nanospheres (DNB). Due to the mutual repulsion of their own negative charges, DNBs reduce the interaction between individual DNBs, making them independent of each other. DNB-based matrix sequencing technology enables each matrix point of DNB to have at least several hundred copies. These copies aggregate together to generate a strong signal, thereby allowing identification of the nucleotide type of DNB.

[0083] To sequence nucleotide sequence clusters, the target nucleotide sequence clusters can be pumped onto a solid support surface and immobilized using an instrument's liquid chromatography system. The solid support can be a biochip. DNA reacts on the biochip, and by measuring the fluorescent groups linked to the reaction, the base sequence of the target nucleotide sequence cluster at that location can be determined. An optical system and a scientific camera can collect the DNA fluorescence signal on the biochip and convert it into a digital signal. The core principle of next-generation sequencing is sequencing-by-synthesis. Each biochemical reaction synthesizes one base, and the sequencer collects a fluorescence signal from the chip; one such cycle is called a sequence. Each cycle allows the determination of the bases contained in one target nucleotide sequence cluster on the biochip array. Through several cycles, the nucleotide sequence of the target nucleotide sequence cluster on the biochip array can be obtained.

[0084] Then, the sequencing template and sequencing reagents are pumped in. The test reagents contain four free nucleotides and DNA polymerase. Free nucleotides refer to nucleotides that are not bound to DNA or RNA chains but exist as independent molecules, such as dNTPs (deoxyribonucleoside triphosphates). The types of nucleotides referred to in this application include adenine nucleotides (A), thymine nucleotides (T), guanine nucleotides (G), and cytosine nucleotides (C). Each of the four free nucleotides contains one type of base (adenine A, thymine T, guanine G, or cytosine C). Based on the nucleotide sequence in the target nucleotide cluster, the four free nucleotides serve as substrates for the test reagents. Under the action of DNA polymerase, they undergo a complementary single-base extension reaction with the nucleotides in the target nucleotide cluster according to the base complementarity pairing principle.

[0085] In one embodiment, free nucleotides can be labeled to visualize the single-base extension reaction. The labeled free nucleotides undergo a complementary single-base extension reaction with a cluster of nucleotide sequences to be tested. Whenever a free nucleotide pairs complementaryly with a nucleotide on the nucleotide sequence cluster to be tested, the fluorescent label carried by the free nucleotide can generate a detectable fluorescent signal without the need for excitation light.

[0086] In this embodiment, the complementary nucleotide of the first nucleotide type is associated with the first signal, the complementary nucleotide of the third nucleotide type is associated with the second signal, and the complementary nucleotide of the second nucleotide type is associated with the first signal and the second signal. The signal intensity distribution of the first signal and the second signal is different in different preset time periods. Through different association methods, the signal intensity distribution of the four types of nucleotides is different in different preset time periods.

[0087] The first type of signal can be, for example, a glow signal, and the second type of signal can be, for example, a flash signal.

[0088] Optionally, different labeling methods can be used for the four free nucleotides to achieve different associations. For example, four different labels can be used to label the four nucleotides separately. The different labels react with the nucleotide labeling substrate in the single base extension reaction to produce different signals, thereby achieving association with different signals. Optionally, three of the four nucleotides can be labeled separately, and the signal intensity distribution of the labels for the three nucleotides is different at different preset time periods. For example, three different labels can be used to label three of the nucleotides, while the remaining nucleotide is not labeled (not labeling is also a labeling method). Here, "different labels" can refer to different types of labels, different amounts of labels, or labels of different types and different amounts of the corresponding types. Through labeling, the complementary nucleotide of the first nucleotide type can be associated with the first signal, the complementary nucleotide of the third nucleotide type can be associated with the second signal, and the complementary nucleotide of the second nucleotide type can be associated with both the first and second signals.

[0089] It should be noted that the first nucleotide type, second nucleotide type, third nucleotide type, and fourth nucleotide type in this application do not specifically refer to fixed nucleotide types. For example, in one sequencing scenario, the first nucleotide type may be adenine nucleotide, while in another sequencing scenario, the first nucleotide type may be cytosine nucleotide, and so on. This application does not limit this, as long as the complementary nucleotide of one of the four nucleotide types is associated with the first signal, the complementary nucleotide of one of the four nucleotide types is associated with the second signal, and the complementary nucleotide of one of the four nucleotide types is associated with both the first and second signals.

[0090] Under different association methods, the four free nucleotides exhibit different photodynamic characteristics during the single-base extension reaction of their complementary strands. This manifests as different trends in the signal intensity of the fluorescence signal generated by different types of free nucleotides during the single-base extension reaction, resulting in different signal intensity distributions for the four nucleotides at different preset time periods. In the nucleic acid sequencing cycle, the free nucleotides bound to the test nucleotide differ in each cycle. The free nucleotide bound to the test nucleotide in a cycle is the complementary nucleotide of that cycle. Depending on the type of complementary nucleotide bound, the signal intensity of the fluorescence signal collected at different preset time periods varies. By obtaining the signal intensity of the test nucleotide sequence cluster at different preset time periods within a nucleic acid sequencing cycle, the nucleotide type of the complementary nucleotide in that cycle can be determined.

[0091] Please refer to Figure 2. In the coordinate system shown in Figure 2, the vertical axis represents signal intensity I, and the horizontal axis represents time t. Base category 1 is labeled with a type 1 enzyme, such that the complementary nucleotide of base category 1 is associated with the first signal. Base category 3 is labeled with a type 2 enzyme, such that the complementary nucleotide of base category 3 is associated with the second signal. Base category 2 is labeled with both type 1 and type 2 enzymes, such that the complementary nucleotide of base category 2 is associated with both the first and second signals. Base category 4 is unlabeled. Type 1 and type 2 enzymes can be luciferases, and the first and second signals are two different fluorescent signals. Optionally, the amount of type 1 enzyme used to label base category 2 is half the amount used to label type 1, and the amount of type 2 enzyme used to label base category 2 is half the amount used to label type 3, to balance the proportions of the corresponding labels for each base category and obtain more accurate results.

[0092] Figure 2 shows the trend of signal intensity over time generated by the complementary single-base extension reaction of four free nucleotides according to the different base categories they contain. Among them, after the proportions are balanced, the signal intensity I of base category II is approximately equal to the average signal intensity of base category I and base category III at the same time.

[0093] Because different types of free nucleotides produce different signals with varying intensity trends over time during single-base extension reactions of complementary strands, by acquiring the signal intensity of the target nucleotide sequence cluster at different preset time periods in one cycle of nucleic acid sequencing, the luminescence changes of the target nucleotide sequence cluster and free nucleotides during the reaction can be monitored. This allows for the identification of the signal type generated by the target nucleotide sequence cluster in one cycle of base sequencing, thereby determining the nucleotide type of the complementary nucleotide of the target nucleotide in that cycle. Furthermore, the nucleotide type of the target nucleotide can be determined based on the base complementary pairing principle.

[0094] Currently, in next-generation sequencing, the monochrome dual-channel sequencing method uses "channel" and "signal" as distinguishing dimensions. It uses four light emission modes, namely 1 / 0, 1 / 1, 0 / 1, and 0 / 0 (1 indicates light emission, 0 indicates no light emission), to determine the four corresponding bases. Channel 1 captures the light emission signal of light-emitting label 1 and its corresponding signal activator 1, while channel 2 captures the light emission signal of light-emitting label 2 and its corresponding signal activator 2. The dual-channel method uses two data transmission paths and two signal capture devices, which inevitably increases the cost of gene sequencing.

[0095] This application captures the overall luminescence signal over time, introduces a time dimension, and focuses on the change in the overall signal intensity of the nucleotide sequence cluster to be tested over time. It does not rely on multiple channels and can achieve gene sequencing in a single channel, reducing the cost of gene sequencing.

[0096] In one embodiment, timing begins after the substrate is introduced, meaning different preset time periods are multiple preset time periods after the substrate is introduced. For example, starting from the introduction of the substrate, the signal strength is acquired every 1 second. The signal strength at the 1st second is taken as the signal strength for the preset time period 0-1s, the signal strength at the 2nd second is taken as the signal strength for the preset time period 1-2s, and so on. Optionally, the signal strength can be the fluorescence signal strength. Furthermore, the signal strength for the preset time period can be the maximum signal strength within the preset time period.

[0097] Please refer to Figure 3. As shown in Figure 3, in one embodiment, two time periods, 0 to key1 and key1 to end, are determined within one cycle (one cycle of nucleic acid sequencing). The signal intensity of the target nucleotide sequence clusters is acquired during the first preset time period 0 to key1, and the signal intensity is acquired during the second preset time period key1 to end. Optionally, the signal intensity of each preset time period is the maximum signal intensity within that preset time period, specifically the maximum value of the acquired fluorescence signal. As shown in Figure 3, the signal values ​​of each target nucleotide sequence cluster within the 0 to key1 time period are iterated, and the maximum signal value of the target nucleotide sequence cluster within the 0 to key1 time period is obtained as the signal intensity of the 0 to key1 time period. The maximum signal values ​​corresponding to the four target nucleotide sequence clusters are value1, value2, value3, and value4, respectively. The signal values ​​of each target nucleotide sequence cluster within the key1 to end time period are iterated, and the maximum signal value of the target nucleotide sequence cluster within the key1 to end time period is obtained as the signal intensity of the key1 to end time period. The maximum signal values ​​corresponding to the four target nucleotide sequence clusters are value5, value6, value7, and value8, respectively.

[0098] Because the signal intensities of different markers reacting with substrates exhibit varying trends over time, the signal intensities (value1, value5), (value2, value6), (value3, value7), and (value4, value8) of the four target nucleotide sequence clusters differ at different preset time periods. Therefore, by acquiring the signal intensities of the target nucleotide sequence clusters at different preset time periods and monitoring the luminescence changes during the reaction of markers and substrates, the markers linked to the target nucleotide sequence clusters can be identified, thereby determining the nucleotide type of the target nucleotide sequence clusters.

[0099] Step S104: Determine the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster under test at different preset time periods.

[0100] In one embodiment, multiple nucleotide samples of known nucleotide types can be prepared in advance, and each nucleotide sample can be labeled in the same way as in step S102. The signal intensity of these nucleotide samples after being introduced into the substrate is obtained at different preset time periods. The trend curve of the signal intensity of different signals generated after the reaction of each label with the substrate is plotted as a preset trend curve and stored.

[0101] Understandably, nucleotides of the same type will exhibit similar signal intensity trends over time due to the presence of the same marker. When determining the nucleotide type of a complementary nucleotide sequence cluster based on the signal intensity over different preset time periods, a signal intensity trend curve as shown in Figure 2 can be plotted. This trend curve is then matched with a preset trend curve to identify the marker linked to the complementary nucleotide of the target nucleotide sequence cluster, thereby determining the nucleotide type of the complementary nucleotide.

[0102] Understandably, nucleotides of the same type will exhibit similar or identical signal intensities over the same preset time period due to their similar signal intensity trends. When clustering nucleotide sequence clusters based on the signal intensities collected over the preset time period, the relevant signal intensity data corresponding to the same nucleotide type will be grouped together. In one embodiment, multiple nucleotide samples can be clustered based on the signal intensities of multiple nucleotide samples over different preset time periods to obtain preset clusters, where the nucleotide type of each preset cluster is known. When determining the nucleotide type of the complementary nucleotide in the cycle of the nucleotide sequence cluster based on the signal intensities of the nucleotide sequence cluster over different preset time periods, for each complementary nucleotide, a data pair or signal intensity vector composed of signal intensities over different preset time periods is obtained. The data points or signal intensity vectors corresponding to the data pairs are clustered, and the clusters obtained from the clustering are matched with the preset clusters to determine the nucleotide type of each cluster in the clustering results. The nucleotide type of the complementary nucleotide is equal to the nucleotide type of the cluster to which the data point or signal intensity vector corresponding to its data pair belongs.

[0103] In one embodiment, when determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster in different preset time periods, for each complementary nucleotide of the nucleotide sequence cluster in the cycle, a signal intensity data pair consisting of signal intensities of different preset time periods is obtained; the nucleotide category of the complementary nucleotide is determined based on the signal intensity data pair of the complementary nucleotides according to a preset Gaussian model.

[0104] The preset Gaussian model can include a trained Gaussian mixture model, which can be trained using a sample set containing signal intensity data pairs of multiple known nucleotide types. When determining the nucleotide type of the complementary nucleotide in the cycle of the test nucleotide sequence cluster based on the trained neural network model, the preset Gaussian model can output probability values ​​of the complementary nucleotide belonging to each nucleotide type based on the input signal intensity data of the complementary nucleotide, thereby determining the nucleotide type of the complementary nucleotide through these probability values.

[0105] In one embodiment, historical data can be used to test the preset threshold range of signal intensity corresponding to various nucleotide types at different preset time periods. When determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle based on the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods, the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle can be determined by the threshold range where the signal intensity of each preset time period generated by the reaction is actually located.

[0106] For example, taking a preset time period including a first preset time period and a second preset time period as an example, historical data can be used to test the first threshold interval corresponding to each nucleotide type in the first preset time period and the second threshold interval corresponding to the second preset time period. When determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in circulation based on the signal intensity of the nucleotide sequence cluster in different preset time periods, the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in circulation is determined by combining the first threshold interval to which the first signal intensity of the first preset time period belongs and the second threshold interval to which the second signal intensity of the second preset time period belongs, with the preset threshold interval obtained from prior testing.

[0107] The above sequencing method involves a single-base extension reaction of complementary strands of the target nucleotide sequence cluster in the presence of four nucleotides: a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with both the first and second signals. The signal intensity distributions of the first and second signals differ at different preset time periods. Each of the four nucleotides carries a modifying group. The signal intensity of the target nucleotide sequence cluster at different preset time periods within one cycle of nucleic acid sequencing is obtained, and the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle is determined based on the signal intensity distribution at different preset time periods. This application achieves different trends in the signal intensity of different nucleotides over time during the single-base extension reaction by designing signals associated with four complementary nucleotide types to have different signal intensity distributions at different preset time periods. By collecting the signal intensity of the target nucleotide sequence cluster at different time points during one cycle of nucleic acid sequencing, the trend of signal intensity variation of the target nucleotide sequence cluster in that cycle can be determined, thereby identifying the nucleotide type of the complementary nucleotide in that cycle. This allows for the inference of the nucleotide type of the target nucleotide that is complementary to it, thus enabling gene sequencing without the need for multiple channels and reducing the cost of gene sequencing.

[0108] In one embodiment, determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods includes:

[0109] For each complementary nucleotide, a signal intensity vector is obtained, consisting of signal intensities at different preset time periods; based on the trained neural network, the nucleotide type of the complementary nucleotide is determined according to the signal intensity vector of the complementary nucleotide.

[0110] The trained neural network can include a trained neural network model. The neural network model can be trained using a sample set, which includes signal intensity vectors of multiple known nucleotide types. A loss function for the neural network model is designed, and the training parameters of the neural network model are progressively optimized using signal intensity vectors of known nucleotide types. A convergence condition for the loss function is designed (e.g., the value of the loss function is less than or equal to a preset threshold) until the loss function meets the convergence condition, resulting in the trained neural network model.

[0111] Furthermore, when determining the nucleotide type of a test nucleotide based on the signal intensity vector of complementary nucleotides using a trained neural network, the signal intensity vector of the complementary nucleotides can be input into the trained neural network. The neural network then outputs the probability value of each nucleotide type belonging to the complementary nucleotide based on the input signal intensity vector, thereby determining the nucleotide type of the complementary nucleotide through the probability value. For example, the nucleotide type with the highest probability value is determined as the nucleotide type of the complementary nucleotide.

[0112] In one embodiment, the neural network model may include an input layer, an output layer, convolutional layers, and a conv-LSTM layer. Optionally, the convolutional layers and the conv-LSTM layer are located between the input layer and the output layer; there may be two convolutional layers, and the conv-LSTM layer may be located between two convolutional layers.

[0113] The input layer receives the input signal intensity vector. Optionally, some data preprocessing can be performed on the signal intensity vector. The first convolutional layer convolves the signal intensity vector with a kernel, and the convolved signal intensity vector is then input into the conv-LSTM layer. The conv-LSTM layer, a variant of the Long Short-Term Memory (LSTM) network, combines the spatial feature extraction capabilities of convolutional layers with the temporal series processing capabilities of LSTM, making it suitable for handling data that varies over time. The conv-LSTM layer can handle the temporal dependencies of the convolved signal intensity vector. After processing, a second convolutional layer further extracts features from the output of the conv-LSTM layer, providing higher-level features. The features extracted by the second convolutional layer are then input into the output layer, which generates the corresponding clustering results based on the specific clustering task. For example, the output layer can use a fully connected layer to output the predicted nucleotide types corresponding to the signal intensity vector.

[0114] In the above embodiment, the neural network extracts spatial features of the signal intensity vector through two convolutional layers and processes the temporal variations of these features through an intermediate Conv-LSTM layer. This structure enables the neural network to capture complex patterns of signal intensity variations over time and space. By training the neural network with a training set, the trained neural network can determine the nucleotide type corresponding to the signal intensity vector.

[0115] In another embodiment, the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle can be determined based on the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods using a Gaussian mixture model.

[0116] In one embodiment, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods includes:

[0117] For each complementary nucleotide, a data pair consisting of a first signal intensity and a second signal intensity is obtained. The first signal intensity and the second signal intensity of the data pair are used as the abscissa and ordinate of a two-dimensional coordinate system, respectively, to obtain the data point of the complementary nucleotide in the two-dimensional coordinate system. Multiple data points are clustered in the two-dimensional coordinate system to obtain the clustering result, which contains multiple clusters. For each complementary nucleotide, the nucleotide type corresponding to the cluster to which the data point of the complementary nucleotide belongs is taken as the nucleotide type of the complementary nucleotide.

[0118] For example, by obtaining the signal intensity I1 of each complementary nucleotide during a first preset time period and the signal intensity I2 during a second preset time period, the data pair of the nucleotide sequence cluster to be tested is obtained as (I1, I2).

[0119] In one embodiment, multiple nucleotide samples of known nucleotide types can be prepared in advance, and the multiple nucleotide samples can be labeled in the same way as in step S102. The signal intensities of the multiple nucleotide samples after passing through the substrate at different preset time periods are obtained. For each nucleotide sample, a data pair composed of signal intensities at different preset time periods is obtained. The first signal intensity and the second signal intensity of the data pair are used as the abscissa and ordinate of the two-dimensional coordinate system, respectively, to obtain the data points of complementary nucleotides on the two-dimensional coordinate system. The multiple data points are clustered on the two-dimensional coordinate system to obtain multiple clusters. Each cluster is labeled according to the nucleotide type of the multiple nucleotide samples and stored as a preset cluster in the base clustering model.

[0120] When determining the nucleotide type of the complementary nucleotide in the cycle of a target nucleotide sequence cluster based on the signal intensity at different preset time periods, a data pair consisting of signal intensities at different preset time periods is obtained for each complementary nucleotide. The first and second signal intensities of the data pair are used as the x and y coordinates on a two-dimensional coordinate system, respectively, to obtain the data points of the complementary nucleotide on the two-dimensional coordinate system. Multiple data points are clustered on the two-dimensional coordinate system, and the resulting clusters are matched with preset clusters in the base clustering model to determine the nucleotide type of each cluster. Therefore, all target nucleotides belonging to the cluster after clustering are of the same nucleotide type.

[0121] Please refer to Figure 3. As shown in Figure 3, in one embodiment, two time periods, 0 to key1 and key1 to end, are determined within one cycle (one cycle of nucleic acid sequencing). The signal intensity of complementary nucleotides is obtained during the first preset time period 0 to key1, and the signal intensity of complementary nucleotides is obtained during the second preset time period key1 to end. Optionally, the signal intensity of each preset time period is the maximum signal intensity within that preset time period. As shown in Figure 3, the signal intensity of each complementary nucleotide within the 0 to key1 time period is iterated to obtain the maximum signal intensity of the complementary nucleotide within the 0 to key1 time period, which is taken as the signal intensity of the 0 to key1 time period. The signal intensities corresponding to the four complementary nucleotides are value1, value2, value3, and value4, respectively. The signal intensity of each complementary nucleotide within the key1 to end time period is iterated to obtain the maximum signal intensity of the complementary nucleotide within the key1 to end time period, which is taken as the signal intensity of the key1 to end time period. The signal intensities corresponding to the four complementary nucleotides are value5, value6, value7, and value8, respectively.

[0122] Because the signal intensity generated by different labels reacting with substrates exhibits different trends over time, the data pairs (value1, value5), (value2, value6), (value3, value7), and (value4, value8) composed of signal intensities obtained from the four complementary nucleotides at different preset time periods are all different. Therefore, by acquiring the signal intensity of the analyte nucleotide at different preset time periods and monitoring the luminescence changes during the reaction of the label and substrate, the label linked to the free nucleotide complementary to the analyte nucleotide can be identified, thereby determining the nucleotide type of the analyte nucleotide.

[0123] The cluster to which the complementary nucleotide data point belongs can be determined by calculating the Euclidean distance from the data point of the complementary nucleotide to the center point of each preset cluster.

[0124] Preferably, the preset time period for acquiring the signal intensity of the nucleotide sequence cluster to be tested in one cycle of nucleic acid sequencing is consistent with the preset time period for acquiring the signal intensity of the nucleotide sample. For example, both preset time periods are selected as the first 3 seconds and the last 3 seconds in one cycle. Optionally, the acquisition time of the aforementioned signal (e.g., fluorescence signal) can be no less than 3 seconds, for example, it can be 4 seconds, 5 seconds, 6 seconds, 7 seconds or 8 seconds, etc.

[0125] In this embodiment, the signal intensity data of multiple nucleotides to be tested are clustered together, thereby quickly classifying all nucleotides into four categories without the need for individual classification. This base clustering improves the efficiency of gene sequencing. By comparing the clusters with preset clusters in a pre-obtained base clustering model, the nucleotide type corresponding to each cluster is quickly determined, enabling high-throughput, large-scale base identification of multiple complementary nucleotides within the nucleotide sequence clusters to be tested.

[0126] In one embodiment, obtaining the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods during one cycle of nucleic acid sequencing includes:

[0127] In one cycle of nucleic acid sequencing, fluorescence images of a solid support were acquired at different preset time periods;

[0128] The signal intensity of the nucleotide sequence clusters to be tested is obtained from the fluorescence image at different preset time periods during one cycle of nucleic acid sequencing.

[0129] In this setup, multiple nucleotide sequence clusters to be sequenced are arranged on the surface of a solid support. This solid support can be a biochip or other suitable solid support for carrying the nucleotide sequence clusters. For example, multiple nucleotide sequence clusters can be carried on a biochip. A biochip, also known as a sequencing slide or sequencing chip, involves precisely and individually loading the nucleotide sequence clusters to be sequenced onto modified sites on a sequencing slide before sequencing. For example, the nucleotide sequence clusters can be placed on a flow cell with a pre-defined array pattern, and then the clusters can be bound to spots on a glass substrate using a knotting technique to obtain an array of nucleotide sequence clusters ready for sequencing.

[0130] It should be noted that the biochip can be any version. Taking version V1 as an example, the size of the biochip is approximately 75mm*25mm. The total number of fields of view (FOV, referring to the range that the objective lens of the micro-imaging optical system can observe in a single operation) included by the biochip is the total number of FOVs, for example, it can be 576 FOVs. This is for illustrative purposes only and should not be construed as a limitation of this application.

[0131] In one embodiment, the computer device can be a gene sequencer, which includes a microscope camera to directly capture fluorescence images of the solid support at different preset time periods. Alternatively, the computer device can receive fluorescence images of the solid support at different preset time periods captured by the microscope camera from an external source.

[0132] The fluorescence image refers to an image of a single field of view / view captured by a microscope camera during gene sequencing. This application does not limit the number of preset time periods or the number of fluorescence images acquired within each preset time period. There can be two or more preset time periods. One fluorescence image can be acquired in each preset time period, or multiple fluorescence images can be acquired within each preset time period, as long as fluorescence images are acquired in different preset time periods.

[0133] After obtaining fluorescence images at different preset time periods, the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods in one cycle of nucleic acid sequencing can be obtained based on the fluorescence images.

[0134] For example, signal intensity can be obtained from the grayscale value of the nucleotide sequence cluster to be tested on the fluorescence image. Since multiple nucleotide sequence clusters to be tested are arranged on the surface of the solid support, the resulting fluorescence image also contains multiple nucleotide sequence clusters to be tested. Each nucleotide sequence cluster is represented by its own pixel value on the fluorescence image according to its own luminescence. Furthermore, nucleotide sequence clusters of different nucleotide types will present different pixel values ​​on the same fluorescence image because their signal intensity changes differently over time. Based on the pixel values ​​of the nucleotide sequence clusters to be tested in the fluorescence image of one cycle of nucleic acid sequencing, the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods within one cycle of nucleic acid sequencing can be obtained, thereby identifying the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in that cycle.

[0135] In this embodiment, by arranging multiple target nucleotide sequence clusters on the surface of a solid support, gene sequencing can be performed in a stable position. The fluorescence of the multiple arrayed target nucleotide sequence clusters on the solid support surface is captured in a fluorescence image. This fluorescence image allows for the simultaneous acquisition of the fluorescence of many target nucleotide sequence clusters, improving sequencing efficiency.

[0136] In one embodiment, the different preset time periods include a first preset time period and a second preset time period. During one cycle of nucleic acid sequencing, acquiring fluorescence images of the solid support at different preset time periods includes:

[0137] In one cycle of cyclic nucleic acid sequencing, fluorescence images of the solid support are acquired at fixed time intervals; the first x frames of fluorescence images in the cycle are used as fluorescence images for the first preset time period, and the last y frames of fluorescence images in the cycle are used as fluorescence images for the second preset time period.

[0138] Where x and y are positive integers, and the sum of x and y is less than or equal to the total number of fluorescence images obtained in one loop.

[0139] For example, a fixed time interval is set to 1 / 2 second, and fluorescence images of the solid support are acquired every 0.5 seconds. 19 images are captured per cycle, meaning the signal acquisition portion takes 9.5 seconds. Signal acquisition begins simultaneously with the introduction of the substrate. The 19 images from the 50th cycle are used to process 500W nucleotide sequence clusters on the solid support. The first x frames of fluorescence images in this cycle are used as the fluorescence images for the first preset time period, and the last y frames are used as the fluorescence images for the second preset time period.

[0140] In one embodiment, the signal intensity includes the maximum signal intensity. Acquiring the signal intensity of the target nucleotide sequence cluster at different preset time periods within one cycle of nucleic acid sequencing based on fluorescence images includes:

[0141] For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum pixel value of the nucleotide sequence cluster to be tested in all fluorescence images within the preset time period; the maximum pixel value is used as the maximum signal intensity of the nucleotide sequence cluster to be tested within the preset time period.

[0142] For example, the signal intensity of the nucleotide sequence cluster to be tested can be the pixel value of the nucleotide sequence cluster to be tested in the fluorescence image. By iterating through the pixel values ​​of each nucleotide sequence cluster to be tested in the first x frames of fluorescence images, the maximum pixel value (signal value) of each nucleotide sequence cluster to be tested in these x frames of fluorescence images is obtained; by iterating through the pixel values ​​of each nucleotide sequence cluster to be tested in the next y frames of fluorescence images, the maximum pixel value (signal value) of each nucleotide sequence cluster to be tested in these y frames of fluorescence images is obtained.

[0143] Furthermore, the maximum pixel value of the nucleotide sequence cluster to be tested in the first x frames of the image can be used as the x-coordinate of the nucleotide sequence cluster to be tested, and the maximum pixel value of the nucleotide sequence cluster to be tested in the next y frames of the image can be used as the y-coordinate of the nucleotide sequence cluster to be tested. Each nucleotide sequence cluster to be tested is given a two-dimensional coordinate system composed of such x-coordinates and y-coordinates. Multiple nucleotide sequence clusters to be tested are clustered based on their two-dimensional coordinates, and the nucleotide types of complementary nucleotides of multiple nucleotide sequence clusters to be tested in the current cycle are determined based on the clustering results.

[0144] In this embodiment, for all fluorescence images acquired in a cycle, only the first few frames and the last few frames are selected to acquire signal intensity, while the intermediate fluorescence images are discarded. This is because, in the middle of a cycle, the label and substrate may still be reacting, and the signal intensity is not stable. Selecting the signal intensity acquired from the first few frames and the last few frames provides higher reliability, and due to the longer time interval, the difference in signal intensity generated by the same nucleotide sequence cluster after a longer time interval is more obvious, thereby improving the sequencing accuracy.

[0145] In one embodiment, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods includes:

[0146] If both the first signal intensity and the second signal intensity are within a first predetermined interval, the complementary nucleotide is identified as the first nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within the second predetermined interval, the complementary nucleotide is identified as the second nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within a third predetermined interval, the complementary nucleotide is identified as the third nucleotide; if both the first signal intensity and the second signal intensity are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0147] Please refer to Figure 3(a), which shows the changes in signal intensity over time for the complementary nucleotides corresponding to the four base categories. Based on the different associated signals, the first signal intensity of the complementary nucleotides corresponding to the four base categories at time point key1 and the second signal intensity at time point key2 are different in at least one of them, and these signal intensities are distributed within three predetermined intervals.

[0148] These three predetermined intervals can be referred to as the intervals of the E value (intensity value) on the vertical axis corresponding to the three horizontal lines in Figure 3(a). Assume that the signal intensities corresponding to the three horizontal lines from top to bottom are the first predetermined intensity, the second predetermined intensity, and the third predetermined intensity, respectively. Then, the first predetermined interval is the signal intensity interval containing the first predetermined intensity, the second predetermined interval is the signal intensity interval containing the second predetermined intensity, and the third predetermined interval is the signal intensity interval containing the third predetermined intensity.

[0149] If both the first and second signal intensities are within a first predetermined interval, the complementary nucleotide is identified as the first nucleotide, corresponding to the curve for base category 1 (value1, value5) in Figure 3(a). If both the first and second signal intensities are within the first predetermined interval, the complementary nucleotide is identified as the second nucleotide, corresponding to the curve for base category 2 (value2, value6) in Figure 3(a). If both the first and second signal intensities are within the first predetermined interval and the second signal intensity is within the third predetermined interval, the complementary nucleotide is identified as the third nucleotide, corresponding to the curve for base category 3 (value3, value7) in Figure 3(a). If both the first and second signal intensities are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide, corresponding to the curve for base category 4 (value4, value8) in Figure 3(a).

[0150] Figure 3(b) shows the changes in signal intensity over time for the complementary nucleotides corresponding to the four base categories in another case. The intensity distribution for each base category is different from that in Figure 3(a), but the other interpretations are the same as in Figure 3(a).

[0151] For example, the first predetermined interval can be (180, 300), the second predetermined interval can be (100, 150), and the third predetermined interval can be (0, 80).

[0152] In this embodiment, the signal intensity of different types of nucleotides is divided into predetermined intervals for different preset time periods. After obtaining the signal intensity of complementary nucleotides in different preset time periods, the category of complementary nucleotides can be directly determined according to the predetermined interval in which the signal intensity in different preset time periods is located. This is convenient and efficient, and can effectively improve sequencing efficiency.

[0153] It should be noted that this application does not distinguish between channels when acquiring fluorescence images. Regardless of whether it's a dual-channel or four-channel imaging system, each channel requires a sleeve lens, which inevitably increases the risk of manufacturing errors. This leads to slight fluctuations in the focal length of the sleeve lens, resulting in subtle differences in magnification between different imaging channels, which can easily cause errors during algorithm registration. The above embodiment, however, does not depend on the number of channels, thus allowing for single-channel sequencing. Only one camera is used to acquire images, avoiding registration errors caused by subtle differences between different imaging channels and effectively improving the accuracy of gene sequencing. The fluorescence images in this application can be acquired from a camera that does not distinguish between channels, or from a signal sensor that does not distinguish between channels (such as a CMOS sensor).

[0154] In one embodiment, three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers differs at different preset time periods.

[0155] In one embodiment, the first nucleotide type marker is a first luciferase, the third nucleotide type marker is a second luciferase, the second nucleotide type marker is a combination of the first luciferase and the second luciferase, and the fourth nucleotide type marker is unmarked.

[0156] The marker can be luciferase. Specifically, it can be selected from Gaussia luciferase, Renilla luciferase, Diplodocus luciferase, firefly luciferase, fungal luciferase, bacterial luciferase, and vargula luciferase, etc.

[0157] Different luciferases can correspond to the same or different substrates, as long as the luminescence patterns of the two enzymes reacting with the substrates are different. Similarly, different mutants of the same luciferase can correspond to the same or different substrates, as long as the luminescence patterns of the different mutants reacting with the substrates are different. For example, streptavidin-labeled luciferases can be Adivity's SA-Gluc (Streptavidin-Gaussia princeps Iuciferase); Promega's nanoKAZ; Nanolight's nanoLuc; and their corresponding Glow or Flash mutants. Digoxin antibody-labeled luciferases can be digoxin antibody-Gluc, digoxin antibody-Nluc, or digoxin antibody-nanoKAZ, and their corresponding Glow or Flash mutants. Their corresponding substrates can be coenguin; or dehydroxylated coenguin; or fluorinated coenguin; or furan ring-substituted coenguin; or other similarly modified coenguin.

[0158] Optionally, the amount of the first luciferase used to label the second nucleotide type is half the amount of the first luciferase used to label the first nucleotide type, and the amount of the second luciferase used to label the second nucleotide type is half the amount of the second luciferase used to label the third nucleotide type, in order to balance the amount of the corresponding markers for each nucleotide type and obtain more accurate results.

[0159] In one embodiment, the first luciferase can be a glow-type luciferase (MGI, catalog number: 940-000571-00), and the second luciferase is a flash-type luciferase. The first luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a first signal, which is a glow-type signal. The second luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a second signal, which is a flash-type signal. According to an embodiment of this application, the aforementioned glow-type luciferase can be selected from (MGI, catalog number: 940-000571-00). According to an embodiment of this application, the aforementioned flash-type luciferase can have the amino acid sequences shown in SEQ ID NO:2-6. The amino acid sequences shown in SEQ ID NO:2-6 are derived from 2-3, 4-1, 8-3, 6-1, and D6 shown in Table 3 of patent WO2023109981A9. The amino acid sequences of SEQ ID NO:2-6 are shown below:

[0160] In the above embodiments, four nucleotide types were labeled using four methods: a first luciferase, a second luciferase, a combination of the first and second luciferases, and no luciferase. This resulted in different trends in the signal intensity changes over time for the complementary nucleotides of the four types, thus allowing for differentiation of the complementary nucleotide types. Further controlling the consistency of the amount of each labeling agent reduces variables and ensures the accuracy of the detection results.

[0161] In one embodiment, the method further includes:

[0162] At preset time points in the cycle, a preset biochemical intervention procedure is executed to change the biochemical state of each marker.

[0163] When acquiring the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing, the signal intensity of the nucleotide sequence cluster to be tested is acquired in the first preset time period before the execution of the biochemical intervention procedure in one cycle of nucleic acid sequencing; and the signal intensity of the nucleotide sequence cluster to be tested is acquired in the second preset time period after the execution of the biochemical intervention procedure.

[0164] The pre-defined biochemical intervention program is a program that can alter the biochemical state of each labeling agent. Specifically, it can be a program for adjusting reaction temperature, pH, and ionic strength. For example, the pre-defined biochemical intervention program could be a temperature control program. When the labeling agent is luciferase, changes in temperature lead to changes in the biochemical state of the labeling agent, which can accelerate the reaction process or change the trend of signal intensity over time. In some cases, it can make the change in signal intensity over time more apparent, accelerate the experimental process, or make the clustering results of the nucleotide sequence clusters to be tested better distinguishable.

[0165] In one embodiment, the modifying group is a reversible blocking modifying group, the signal intensity of the first signal is the same in different preset time periods, and the signal intensity of the second signal is different in different preset time periods.

[0166] The same signal strength in different preset time periods can mean that the signal strength in different preset time periods is within the same predetermined range, while different signal strength in different preset time periods can mean that the signal strength in different preset time periods is within different predetermined ranges.

[0167] For example, please refer to Figures 2 and 3(a). In these figures, the complementary nucleotides corresponding to base category 1 (base T) are associated with the first type of signal, and the complementary nucleotides corresponding to base category 3 (base A) are associated with the second type of signal. The signal intensities (value1 and value5) of the first type of signal are both within the first predetermined interval at different preset time periods, and are considered the same. The signal intensities (value3 and value7) of the second type of signal are within the first predetermined interval and the third predetermined interval, respectively, at different preset time periods, and are considered different.

[0168] Furthermore, since the signal intensity of the first signal is the same in different preset time periods, while the signal intensity of the second signal is different in different preset time periods, the signal intensity of the combination of the first signal and the second signal (i.e. the signal associated with the complementary nucleotide corresponding to base category 2) is also different in different preset time periods when the two signals are superimposed, and at least one signal intensity distribution is different from the first signal and at least one signal intensity distribution is different from the second signal.

[0169] Four free nucleotides contain reversible blocking modification groups. These reversible blocking modification groups, once added to the DNA strand, temporarily prevent further elongation of the DNA polymerase, thus enabling sequencing-while-synthesizing. After each fluorescence signal detection, the reversible blocking modification groups are removed, allowing the DNA polymerase to add the next free nucleotide, and this process is repeated until the nucleotide sequence of the entire target nucleotide cluster is completely determined.

[0170] In one embodiment, four nucleotides can modify the detectable label. Specifically, the signal generating the signal intensity is a light signal; the light signal can be a chemiluminescent signal or a bioluminescent signal. The detectable label includes at least one of a bioluminescent molecule, a chemiluminescent molecule, and a fluorescent probe; preferably, the bioluminescent molecule is a luciferase. The first nucleotide type label is a first luciferase, the third nucleotide type label is a second luciferase, the second nucleotide type label is a combination of the first and second luciferases, and the fourth nucleotide type has no label. Luciferase labeling includes direct luciferase labeling or indirect luciferase labeling; wherein indirect luciferase labeling is achieved through affinity interactions; affinity interactions include antigen-antibody or antigen-binding fragment interactions, biotin-avidin interactions, etc. The aforementioned chemiluminescent molecule can be selected from acrid esters, luminol, or derivatives thereof. The aforementioned fluorescent probe can be selected from fluorescent protein-coupled probes, etc.

[0171] In one embodiment, the different preset time periods include a first preset time period and a second preset time period. Determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster in different preset time periods includes: determining an intensity difference value and the difference value interval to which the intensity difference value belongs based on the first signal intensity of the nucleotide sequence cluster in the first preset time period and the second signal intensity in the second preset time period. The intensity difference value includes an intensity ratio or an intensity difference value. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type. If the first signal intensity or the second signal intensity is greater than a preset intensity threshold and the difference value interval to which the intensity difference value belongs is the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0172] In some cases, different types of complementary nucleotides produce different signal intensities at different preset time periods. In this embodiment, the difference in signal intensity is measured using an intensity difference value. This intensity difference value can be an intensity ratio or an intensity difference. After obtaining the first signal intensity at a first preset time period and the second signal intensity at a second preset time period, the ratio or difference between the first and second signal intensities is calculated to obtain the intensity difference value. Based on the intensity difference value, the nucleotide type of the cyclic complementary nucleotide is determined.

[0173] When determining the nucleotide type of a circulating complementary nucleotide based on intensity difference values, historical data can be used to test the corresponding difference value ranges for various nucleotide types. During sequencing, the association of a complementary nucleotide with a signal is first determined based on whether at least one of its signal intensities exceeds a preset intensity threshold. If at least one of the complementary nucleotides' signal intensities exceeds the preset intensity threshold, the complementary nucleotide is considered to be associated with a signal; if all complementary nucleotides' signal intensities are less than or equal to the preset intensity threshold, the complementary nucleotide is considered not associated with a signal. Complementary nucleotides not associated with a signal can be directly identified as the fourth nucleotide type; for complementary nucleotides associated with a signal, the nucleotide type is further determined based on the difference value range within which their intensity difference values ​​fall.

[0174] Optionally, if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the first difference value interval, then the nucleotide type of the complementary nucleotide is determined as the first nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the second difference value interval, then the nucleotide type of the complementary nucleotide is determined as the second nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the third difference value interval, then the nucleotide type of the complementary nucleotide is determined as the third nucleotide type; if the intensity of the first signal or the intensity of the second signal are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined as the fourth nucleotide type.

[0175] This application also provides an application scenario where the above-described sequencing method is used for gene sequencing of E. coli DNA double-stranded PCR products. Several specific embodiments of this sequencing method in this application scenario are as follows:

[0176] Specific Example 1 (Reaction process with biochemical intervention):

[0177] Library sequences were amplified using MGI's self-produced make DNB kit, and sequencing signals were acquired using an E5 sequencer. Sequencing reagents included Hot reagents (dntps-biotin, dntps-digoxin, and BG9 polymerase), Cold reagents (dntps and BG9 polymerase), signal marker enzymes (Anti-Dig-Nluc (coupled complex product) and SA-Gluc (coupled complex product)), signal excitation substrates (CTZ buffer and CTZ, CTZ-f buffer and CTZ-f), DRB, PWB, DCB, etc. The signal marker enzyme buffer and signal excitation substrate working solution were biochemically treated (e.g., temperature was changed) to ensure that the signal intensity generated after the reaction of the four base types of markers with the substrates changed over time, as shown in Figure 4(a). A self-developed sequencing script was used to perform SE100 sequencing. After obtaining the final data, a self-developed algorithm was used for cluster analysis, resulting in the clustering results shown in Figure 4(b). After cluster analysis of 100W DNB points on the chip, downstream analysis was performed to obtain the sequencing evaluation criteria shown in Table 1.

[0178] 1. Preparation of experimental reagents:

[0179] Reagent kit: DNBSEQ One-Step DNB Preparation Reagent Kit V2.0 (OS-SB) (Manufacturer: MGI);

[0180] The luciferases used are Nluc and Gluc (i.e., G2L); their corresponding conjugation complexes are Anti-Dig-Nluc (manufacturer: MGI) and SA-Gluc (manufacturer: MGI), respectively; among them, Nluc and Gluc are glow-type luciferases.

[0181] 2. Preparation of experimental equipment:

[0182] DNBSEQ-E5RS gene sequencer (manufacturer: MGI);

[0183] DNBSEQ-E5 sequencing slide (manufacturer: MGI);

[0184] PCR amplification instrument (manufacturer: Life Pro gene amplification instrument).

[0185] 3. Experimental steps:

[0186] 1) Following the instructions of the DNBSEQ-E5RS sequencing kit, the E. coli DNA double-stranded PCR product was circularized and rolled around to prepare DNA nanospheres. The DNA rolling circle replication time was 40 min.

[0187] 2) Kit Preparation: Manually fill the reagents required for the sequencing process. The reagent components include Hot reagents (dNTPs-biotin, dNTPs-digoxin, and BG9 polymerase), Cold reagents (dNTPs and BG9 polymerase), DRB, PWB, DCB, sequencing primers, and substrates such as natural coelentrin and clonicol. It is important to note that both types of substrates are kept in the same working solution environment and are introduced into the sequencing chip together during sequencing to generate fluorescence signals simultaneously.

[0188] 3) Sequencing logic modification: The substrate interaction process during sequencing is controlled by modifying the sequencing script of DNBSEQ-E5RS: the temperature is controlled at 25°C for the first 5 seconds and 40°C for the next 4 seconds, the imaging exposure time is 1 frame per second, 1 image is captured per second, and signal acquisition starts simultaneously when the substrate is introduced.

[0189] 4) Chip loading: Select one chip from the QC (quality control) qualified batch and open the transparent vacuum packaging bag to load the chip.

[0190] 5) Set up the experimental program, correctly place the DNBSEQ-E5RS sequencing kit into the sequencer and run the reagent slot one-click loading function.

[0191] 6) Using a pipette and a wide-mouth pipette tip, add the DNB loading reaction system prepared in step 1) into the DNB well of the sequencing slide and start sequencing.

[0192] 4. Experimental Results and Analysis:

[0193] Sequencing through the above process will produce 9 images per cycle. The clustering algorithm operates as follows: Take 1 million DNB points, take the first 5 images and iterate through each DNB point to obtain the maximum gray value (signal value) of each DNB point in these 5 images, which is used as the x-axis; take the last 4 images and iterate through each DNB point to obtain the maximum gray value (signal value) of each DNB point in these 4 images, which is used as the y-axis. The plot is shown in Figure 4(b).

[0194] In terms of sequencing principles, the G base is unlabeled with any luciferase, the T base is labeled with Anti-Dig-Nluc (a glow-in-the-dark luciferase-coupled complex) (manufacturer: MGI), the A base is labeled with SA-Gluc (a glow-in-the-dark luciferase-coupled complex) (manufacturer: MGI), half of the C base is labeled with Anti-Dig-Nluc (manufacturer: MGI), and the other half of the C base is labeled with SA-Gluc (manufacturer: MGI). Therefore, these four bases will exhibit the four photodynamic characteristics shown in Figure 4(a). Cluster analysis was performed on 100W DNB points from the chip, followed by downstream analysis to obtain the sequencing evaluation criteria shown in Table 1. Over 100 cycles, the average error rate for base identification was only 2.39%.

[0195] The labeling principle is as follows: 100% of the total A bases in the system are chemically modified and labeled with biotin, 100% of the total T bases in the system are chemically modified and labeled with digoxin, 50% of the total C bases in the system are chemically modified and labeled with biotin, and 50% of the total C bases in the system are chemically modified and labeled with digoxin. Based on the affinity characteristics of SA with biotin and digoxin with anti-Digoxin, the coupling complex products Anti-Digoxin-Gluc and SA-G19 are affinity-dependent on the corresponding bases, thereby enabling the bases to be labeled with luciferase.

[0196] Table 1

[0197] Specific Implementation Example 2:

[0198] Library sequences were amplified using MGI's self-produced make DNB kit, and sequencing signals were acquired using an E25 sequencer. Sequencing reagents included Hot reagents (dntps-biotin, dntps-digoxin, and BG9 polymerase), Cold reagents (dntps and BG9 polymerase), signal marker enzymes (SA-Gluc WT, Anti-Dig-Gluc MGI), signal excitation substrates (CTZ buffer and CTZ), DRB, PWB, DCB, etc. The signal marker enzymes SA-Gluc WT and Anti-Dig-Gluc MGI have different photodynamic characteristics. The trend of signal intensity changes over time after the reaction of the four base types of markers with the substrates is shown in Figure 5(a). Sequencing was performed on the SE100 using a self-developed sequencing script. 10 million DNB points were clustered from the chip, and the clustering results are shown in Figure 5(b). Downstream analysis yielded the sequencing evaluation criteria shown in Table 2.

[0199] 1. Preparation of experimental reagents:

[0200] Reagent kits: DNBSEQ One-Step DNB Preparation Kit V2.0 (OS-SB) (manufacturer: MGI), luciferase for DNBSEQ-E25RS sequencing kit (manufacturer: MGI);

[0201] Luciferase: Gluc wild-type (flash luciferase) (manufacturer: MGI), G2L (i.e., Gluc, glow luciferase) (manufacturer: MGI)

[0202] Coupling reagent: EZ-Link TM Maleimide protein labeling kit (manufacturer: Thermo Fisher Scientific), Avi-tagged protein biotin labeling kit (BirA method) (manufacturer: Beyotime), Recombinant Streptavidin (manufacturer: Beyotime), Anti-Digoxin (manufacturer: MGI)

[0203] Buffer 1: SSC (5X), Tween-20 (0.05%), cystamine (0.001–0.006M), NaSCN (0.05–0.3M), arginine (0.1–0.3M), HCl (adjust pH to 8.7–9.1);

[0204] 2. Preparation of experimental equipment:

[0205] DNBSEQ-E25RS gene sequencer (manufacturer: MGI);

[0206] DNBSEQ-E25 sequencing slide (manufacturer: MGI);

[0207] PCR amplification instrument (manufacturer: Life Pro gene amplification instrument).

[0208] 3. Experimental steps:

[0209] 1) Using EZ-Link TM The maleimide protein labeling kit conjugated Anti-Digoxin with G2L to obtain the conjugated product Anti-Digoxin-G2L. The Avi-tagged protein biotin labeling kit (BirA method) conjugated Recombinant Streptavidin with wild-type Gluc to obtain the conjugated product SA-Gluc (wild-type).

[0210] 2) Following the instructions of the DNBSEQ-E25RS sequencing kit, the E. coli DNA double-stranded PCR product was circularized and rolled around to prepare DNA nanospheres. The DNA rolling circle replication time was 25 min.

[0211] 3) Kit preparation: The reagent components include Hot reagent (dNTPs-biotin, dNTPs-digoxin and BG9 polymerase), Cold reagent (dNTPs and BG9 polymerase), DRB, PWB, DCB, sequencing primers, natural coelenterin (CTZ), etc. Except for the signal reagent, the other components and filling volumes are the same as those in the E25 SE100 kit. The signal reagents are diluted with the signal factor buffer in the E25 kit kit to dilute Anti-Digoxin-G2L and SA-Gluc (wild type) and filled into the corresponding positions in the kit.

[0212] 4) Sequencing logic modification: The sequencing script of DNBSEQ-E25RS was modified to control the substrate interaction temperature at 40℃ during the sequencing process and to maintain this process for 9 seconds. The imaging exposure time was 1 frame per second, and 1 image was acquired per second. Signal acquisition started simultaneously when the substrate was introduced.

[0213] 5) Chip loading: Select one chip from the QC (quality control) qualified batch and open the transparent vacuum packaging bag to load the chip.

[0214] 6) Set up the experimental program, correctly place the DNBSEQ-E25RS sequencing kit into the sequencer and run the reagent slot one-click loading function.

[0215] 7) Use a pipette and wide-mouth pipette tip to add the DNB loading reaction system prepared in step 1) into the DNB well of the sequencing slide and start sequencing.

[0216] 4. Experimental Results and Analysis:

[0217] Gluc (wild type) and G2L can catalyze the oxidation of natural coelenterate (CTZ) to form an unstable intermediate and release a fluorescent signal. Based on their different properties, in an environment of 40°C, the fluorescence signal collected under the catalysis of Gluc (wild type) forms a photodynamic curve of base A in Figure 5(a), while the fluorescence signal collected under the catalysis of G2L forms a photodynamic curve of base T in Figure 5(a).

[0218] Among them, the G base is not labeled with any luciferase, the T base is labeled with Anti-Digoxin-G2L (manufacturer: MGI), the A base is labeled with SA-Gluc (wild type) (manufacturer: MGI), half of the C base is labeled with Anti-Digoxin-G2L (manufacturer: MGI), and the other half of the C base is labeled with SA-Gluc (wild type). Then the four bases will have four photodynamic characteristics as shown in Figure 5(a).

[0219] Sequencing through the above process will produce 9 images per cycle. The clustering algorithm operates as follows: 10 million DNB points are taken. The first 5 images are used to iterate through each DNB point, and the maximum grayscale value (signal value) of each DNB point in these 5 images is obtained as the x-coordinate. The last 2 images are used to iterate through each DNB point, and the maximum grayscale value (signal value) of each DNB point in these 2 images is obtained as the y-coordinate. All DNB points are clustered based on their x-coordinates and y-coordinates, as shown in Figure 5(b).

[0220] Cluster analysis was performed on approximately 1 million DNB points from the chip, followed by downstream analysis to obtain the sequencing evaluation criteria shown in Table 2. Feature point extraction and bioinformatics downstream analysis of the acquired signals yielded sequencing metrics as shown in Table 2. Alignment of 1.04M reads with the reference sequence showed a match rate as high as 99.68% and an average error rate of 0.45%, demonstrating the potential and usability of the sequencing method presented in this application in the sequencing application market.

[0221] Table 2

[0222] Specific Implementation Example 3:

[0223] 1. Preparation of experimental reagents:

[0224] Same as in Specific Implementation Example 2.

[0225] 2. Preparation of experimental equipment:

[0226] Same as in Specific Implementation Example 2.

[0227] 3. Experimental steps:

[0228] The imaging exposure time was set to 0.5 seconds, with one image captured every 0.5 seconds. Starting from the introduction of the substrate cycle, the first image was acquired at the 0.5-second mark of each cycle. 19 images were captured per cycle, meaning the signal acquisition portion took 9.5 seconds. Signal acquisition began simultaneously with the introduction of the substrate. Other experimental steps were the same as in Specific Example 2.

[0229] 4. Experimental Results and Analysis:

[0230] Take 19 images from the 50th cycle and process the 5 million pixels in each image. Take the first x images and iterate through each DNB point, obtaining the maximum grayscale value (signal value) of each DNB point in these x images, and project it onto the x-axis as shown in Figure 6. Take the next y images and iterate through each DNB point, obtaining the maximum grayscale value (signal value) of each DNB point in these y images, and project it onto the y-axis as shown in Figure 6.

[0231] In Figure 6, for example, [0:2] represents the second from 0 to 2, i.e., the first, second, and third images are selected; [16:19] represents the second from 16 to 19, i.e., the 17th, 18th, and 19th images are selected. In this implementation case, an optimization experiment was conducted on the first and second preset time periods. According to the data, the preferred values ​​for the first preset time period are [0:6], [0:8], [0:10], and [0:12]. Due to the need for brevity and the objective limitations of the experimental conditions, there are other preferred values ​​such as [0:14] and [0:16], which will not be elaborated here. The preferred values ​​for the second preset time period are [14:19] and [16:19]. Due to the need for brevity and the objective limitations of the experimental conditions, there are other preferred values ​​such as [14:18] and [15:19], which will not be elaborated here. The images selected for the first preset time period cannot overlap with those selected for the second preset time period.

[0232] Specific Implementation Example 4:

[0233] The purpose of this embodiment is to evaluate the differences in photodynamic curves between commercial luciferases and other luciferases in sequencing applications. This embodiment uses a single-factor variable analysis method; except for the luciferase, all other steps can be the same.

[0234] 1. Experimental Samples and Reagents

[0235] Sequencing sample: E. coli DNA double-stranded PCR product;

[0236] Reagent kit: DNBSEQ One-Step DNB Preparation Reagent Kit V2.0 (OS-SB) (Manufacturer: MGI);

[0237] Luciferase: Luciferase used in the DNBSEQ-E25RS sequencing kit (e.g., G2L) (manufacturer: MGI), Luciferase 2, 6, 16, 18, 19 (manufacturer: MGI);

[0238] Luciferase substrate: Substrate used in the DNBSEQ-E25RS sequencing kit (manufacturer: MGI);

[0239] Diluent 1: Signal Factor Buffer (Manufacturer: MGI; Product No.: 940-000571-00);

[0240] Diluent 4: Signal reagent buffer (manufacturer: MGI; catalog number: 940-000571-00).

[0241] 2. Experimental apparatus

[0242] DNBSEQ-E5RS or DNBSEQ-E25RS gene sequencers (manufacturer: MGI);

[0243] DNBSEQ-E5 sequencing slides or DNBSEQ-E25 sequencing slides (manufacturer: MGI);

[0244] PCR amplification instrument (manufacturer: Life Pro gene amplification instrument).

[0245] 3. Experimental Procedure

[0246] 1) Following the instructions of the DNBSEQ-E5RS sequencing kit, the E. coli DNA double-stranded PCR product was circularized and rolled around to prepare DNA nanospheres. The DNA rolling circle replication time was 40 min.

[0247] 2) Kit preparation: Manually fill the reagents required for the sequencing process, prepare dilution buffer 1 for commercial luciferase and luciferase 2, 6, 16, 18, and 19, and dilution buffer 4 for substrate.

[0248] 3) Chip loading: Select one wafer from the QC (quality control) qualified batch and open the transparent vacuum packaging bag to load the wafer.

[0249] 4) Set up the experimental program, correctly place the DNBSEQ-E5RS sequencing kit into the sequencer and run the reagent slot one-click loading function.

[0250] 5) Use a pipette and wide-mouth pipette tip to add the DNB loading reaction system prepared in step 1) into the DNB well of the sequencing slide.

[0251] 6) Sequencing: Sequencing was performed based on the set program. Different luciferases diluted with dilution buffer 1 were reacted with the same substrate. A total of 66 images were taken during the reaction (one image at 1 second interval) to monitor the photodynamic curve of the luciferase.

[0252] 4. Experimental Results and Analysis

[0253] This embodiment compares the photodynamic curves of commercial luciferases and different variants of luciferase (luciferase 2, 6, 16, 18, 19) based on the signal value changes in the original micro-region image of the sequencing chip. The results are shown in Figure 7. Luciferases 2, 6, 16, 18, and 19 all exhibit flash-type photodynamic curves. Furthermore, when different luciferases diluted with dilution 1 reacted with substrates diluted with dilution 4, luciferases 2, 6, 16, 18, and 19 all showed a trend towards being more flash-type than the commercial luciferase. It can be understood that flash-type luciferases can be of various types to produce different types of flash-type signals.

[0254] In the above embodiments, four nucleotide types—A, T, C, and G—were labeled using a first luciferase, a second luciferase, 50% first luciferase + 50% second luciferase, and no luciferase, respectively. After introducing the substrate, the DNA molecules (DNBs) were clustered based on the signal intensity at different time points to determine the nucleotide type of each DNB. This enabled gene sequencing of *E. coli* DNA double-stranded PCR products in a single channel. Compared to dual-channel and four-channel gene sequencing, this method offers lower sequencing costs and faster sequencing speeds. For all fluorescence images acquired in a cycle, only the first and last few frames were selected to collect signal intensity. Due to the longer time intervals, the difference in signal intensity generated by the same DNB after a longer interval is more pronounced, thereby improving sequencing accuracy.

[0255] According to embodiments of this application, based on the characteristic differences between the aforementioned marked flash-type and glow-type luciferases, such as light intensity and half-life, four types of photodynamic models can be constructed for sequencing applications, as shown in Figures 8A, 8B, 8C, and 8D. The characteristic points of these four photodynamic application models are as follows: the peak light intensity of flash-type luciferase detection is much higher than that of glow-type luciferase (Figure 8A); the peak light intensity of flash-type luciferase detection is lower than that of glow-type luciferase (Figure 8B); the peak light intensity of flash-type luciferase detection is 1.5 to 2.5 times higher than that of glow-type luciferase (Figure 8C); and the peak light intensity of flash-type luciferase is equal to that of glow-type luciferase (Figure 8D). These four application models correspond to different clustering patterns. In some preferred examples of this application, preferred model 3 (Figure 8C) is used as a base resolution type diagram.

[0256] Please refer to Figure 9, which is a flowchart illustrating a sequencing method in one embodiment. In one embodiment, as shown in Figure 9, a sequencing method is provided. It is understood that this method can be applied to a terminal, a server, or a system including both a terminal and a server, and is implemented through interaction between the terminal and the server. In this embodiment, the method includes the following steps:

[0257] Step S202: Obtain the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing.

[0258] In this process, the nucleotide to be detected at the detection site binds to the complementary nucleotide for extended sequencing. The signal intensity of the markers corresponding to different types of complementary nucleotides varies, and the signal intensity difference is the difference in signal intensity at different preset time periods.

[0259] To sequence single-stranded nucleotide templates, the templates are pumped onto a solid support surface and immobilized using an instrument's liquid chromatography system. Then, the sequencing template and sequencing reagents are pumped in. The test reagents contain four complementary nucleotides and DNA polymerase. Based on the nucleotide sequence in the single-stranded nucleotide template, the four complementary nucleotides serve as substrates for the test reagents. Under the action of DNA polymerase, they undergo a complementary strand extension sequencing reaction with the nucleotides on the single-stranded nucleotide template according to the base pairing principle.

[0260] In one embodiment, complementary nucleotides can be labeled to visualize the extension sequencing reaction. The labeled complementary nucleotides are subjected to a complementary strand extension sequencing reaction with a single-stranded nucleotide template. Whenever a complementary nucleotide pairs complementaryly with a nucleotide on the single-stranded nucleotide template, the fluorescent label carried by the complementary nucleotide can generate a detectable fluorescent signal without the need for excitation light.

[0261] The four complementary nucleotides can be labeled using different methods. Optionally, each of the four nucleotides can be labeled separately, with the signal intensity of the labeling agents differing at different preset time periods, thus leading to different signal intensity differences at different preset time periods. For example, four different labeling agents can be used to label each of the four nucleotides separately. Alternatively, three of the four nucleotides can be labeled separately, with the signal intensity distribution of the labeling agents differing at different preset time periods, thus leading to different signal intensity differences at different preset time periods. For example, three different labeling agents can be used to label three of the nucleotides, while the remaining nucleotide is left unlabeled (leaving it unlabeled is also a labeling method). Here, "different labeling agents" can refer to different types of labeling agents, different amounts of labeling agents, or labeling agents of different types and different amounts of each type.

[0262] Under different labeling methods, the four complementary nucleotides exhibit different photodynamic characteristics during complementary strand extension sequencing reactions. This manifests as variations in the signal intensity of the fluorescence signals generated at different preset time periods during these reactions. Within the nucleic acid sequencing cycle, the detection location of the single-stranded nucleotide template varies in fluorescence signal intensity at different preset time periods depending on the type of complementary nucleotide bound. These differences can include variations in intensity ratios or intensity differences.

[0263] Since different types of complementary nucleotides produce fluorescence signals with varying intensity at different preset time periods during extended sequencing reactions, by acquiring the signal intensity of the detection site of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing, the signal intensity difference between the single-stranded nucleotide template and the complementary nucleotide during the reaction can be monitored. This allows for the identification of markers linked to the complementary nucleotides, thereby determining the nucleotide type of the complementary nucleotide bound to the detection site of the single-stranded nucleotide template in that cycle, and further determining the nucleotide type of the nucleotide to be tested at the detection site.

[0264] Since different markers produce different signal intensities when reacting with substrates at different preset time periods, by acquiring the signal intensity of single-stranded nucleotide templates at different preset time periods and monitoring the luminescence changes when markers and substrates react, markers linked by complementary nucleotides can be identified, thereby determining the nucleotide type at the detection position of the single-stranded nucleotide template.

[0265] Step S204: Based on the signal intensity difference at the detection location, determine the nucleotide type of the complementary nucleotide that binds to the detection location in the extension sequencing reaction, thereby determining the nucleotide type of the nucleotide to be tested at the detection location.

[0266] It is understandable that nucleotides of the same type will have the same or similar signal intensity when collected in the same preset time period because their signal intensity changes in the same trend over time. Therefore, the difference in signal intensity is the same in the same two preset time periods.

[0267] When determining the nucleotide type of complementary nucleotides bound at the detection site by the signal intensity difference at the detection site, historical data can be used to test the threshold ranges corresponding to various nucleotide types at different preset time periods. The nucleotide type at the detection site of the single-stranded nucleotide template can be determined by the threshold range of each signal intensity.

[0268] For example, taking a preset time period including a first preset time period and a second preset time period as an example, historical data can be used to test the threshold range corresponding to each nucleotide type in the first and second preset time periods. When determining the nucleotide type of the analyte nucleotide at the detection position of the single-stranded nucleotide template based on the signal intensity difference at the detection position in different preset time periods, the nucleotide type of the complementary nucleotide bound at the detection position by the extension sequencing reaction is determined based on the threshold range to which the signal intensity difference between the first and second preset time periods belongs. After determining the nucleotide type of the complementary nucleotide bound at the detection position by the extension sequencing reaction, the nucleotide type at the detection position of the single-stranded nucleotide template can be determined according to the base pairing principle.

[0269] The aforementioned sequencing method involves the binding of the nucleotide to be sequenced at the detection site with a complementary nucleotide for an extension sequencing reaction. Different types of complementary nucleotides correspond to different signal intensity differences in their markers, which are reflected in the signal intensity differences over different preset time periods. By acquiring the signal intensity of the detection site at the single-stranded nucleotide template during nucleic acid sequencing at different preset time periods, and based on the signal intensity differences at the detection site, the nucleotide type of the complementary nucleotide bound at the detection site in the extension sequencing reaction is determined, thereby identifying the nucleotide type of the nucleotide to be sequenced at the detection site. This application, by testing the signal intensity differences of different complementary nucleotides during the extension sequencing reaction at different preset time periods, achieves the determination of the nucleotide type of the complementary nucleotide undergoing the extension sequencing reaction based on signal intensity differences. Furthermore, based on the base pairing principle, the nucleotide type of the detection site of the single-stranded nucleotide template is determined, thus eliminating the need for multiple channels to achieve gene sequencing and reducing the cost of gene sequencing.

[0270] In one embodiment, three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is glow-in-the-dark luciferase, the marker for the third nucleotide type is flash-in-the-dark luciferase, the marker for the second nucleotide type is a combination of glow-in-the-dark luciferase and flash-in-the-dark luciferase, and the fourth nucleotide type has no marker. The glow-in-the-dark luciferase reacts with the nucleotide marker substrate in the extension sequencing reaction to produce a glow-in-the-dark signal, and the flash-in-the-dark luciferase reacts with the nucleotide marker substrate in the extension sequencing reaction to produce a flash-in-the-dark signal.

[0271] In one embodiment, the different preset time periods include a first preset time period and a second preset time period, the signal intensity difference includes the signal intensity difference value, and the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction is determined based on the signal intensity difference at the detection site, including:

[0272] Based on the first signal intensity at the detection location during a first preset time period and the second signal intensity during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes the intensity ratio or intensity difference value. Based on the difference value range to which the intensity difference value belongs, the nucleotide type of the complementary nucleotide that the extension sequencing reaction binds at the detection location is determined.

[0273] Taking two preset time periods as an example, the difference in signal intensity is measured using an intensity difference value. This intensity difference value can be an intensity ratio or an intensity difference. After obtaining the first signal intensity of the first preset time period and the second signal intensity of the second preset time period, the ratio or difference between the first signal intensity and the second signal intensity is calculated to obtain the intensity difference value. Based on the intensity difference value, the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction is determined.

[0274] When determining the nucleotide type of the complementary nucleotide bound at the detection site by the extension sequencing reaction based on the intensity difference value, historical data can be used to test the corresponding difference value ranges for various nucleotide types. During sequencing, the presence or absence of a signal associated with the complementary nucleotide is first determined by whether at least one of the complementary nucleotides' signal intensities is greater than a preset intensity threshold. If at least one of the complementary nucleotides' signal intensities is greater than the preset intensity threshold, the complementary nucleotide is considered to be associated with a signal; if all complementary nucleotides' signal intensities are less than or equal to the preset intensity threshold, the complementary nucleotide is considered not associated with a signal. Complementary nucleotides not associated with a signal can be directly identified as the fourth nucleotide type; for complementary nucleotides associated with a signal, the nucleotide type of the complementary nucleotide bound at the detection site by the extension sequencing reaction is further determined based on the difference value range to which the intensity difference value belongs.

[0275] In one embodiment, determining the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction, based on the difference range to which the intensity difference value belongs, includes:

[0276] If the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type; if the intensity of the first signal or the intensity of the second signal are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0277] For example, the intensity difference value can be an intensity ratio. The first difference value range can be [3.5, +∞], the second difference value range can be [0.8, 1.2], the third difference value range can be [1.5, 2.5], and the preset intensity threshold can be the non-specific adsorption signal intensity value.

[0278] For example, the intensity difference value can be the intensity difference value. Taking Figure 3(a) as an example, assuming that the values ​​of the three horizontal lines in Figure 3(a) corresponding to the E-axis are 280, 150 and 20 respectively, then with 130 as a span, the first difference value interval can be an interval close to 0, such as [0, 20], the second difference value interval can be an interval around 130, such as [120, 140], and the third difference value interval can be an interval around 280, such as [270, 290]. The preset intensity threshold can be the non-specific adsorption signal intensity value.

[0279] In one embodiment, when the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold and the intensity difference value of the complementary nucleotide is the intensity difference value, if the difference value interval to which the intensity difference value belongs is the first difference value interval, it means that both the first signal intensity and the second signal intensity are within the first predetermined interval, and the complementary nucleotide can be identified as the first nucleotide; if the difference value interval to which the intensity difference value belongs is the second difference value interval, it means that both the first signal intensity and the second signal intensity are within the first predetermined interval, and the complementary nucleotide can be identified as the second nucleotide; if the difference value interval to which the intensity difference value belongs is the third difference value interval, it means that both the first signal intensity and the second signal intensity are within the third predetermined interval, and the complementary nucleotide can be identified as the third nucleotide.

[0280] In one embodiment, multiple single-stranded nucleotide templates are arranged on the surface of a solid support, and the signal intensity at the detection location of the single-stranded nucleotide templates during different preset time periods in the nucleic acid sequencing process includes:

[0281] During nucleic acid sequencing, fluorescence images of a solid support at different preset time periods are acquired; based on the fluorescence images, the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing is obtained.

[0282] In this setup, multiple single-stranded nucleotide templates are arranged on the surface of a solid support. This solid support can be a biochip or any other suitable solid support for carrying single-stranded nucleotide templates. For example, multiple single-stranded nucleotide templates can be carried on a biochip. Biochips, also known as sequencing slides or sequencing chips, involve precisely and individually loading single-stranded nucleotide templates onto modification sites on a sequencing slide before sequencing. For instance, single-stranded nucleotide templates can be placed on a flow cell with a pre-defined array pattern, and then bound to spots on a glass substrate using a knotting technique to obtain a single-stranded nucleotide template array suitable for sequencing.

[0283] The fluorescence image refers to an image of a single field of view / view captured by a microscope camera during gene sequencing. This application does not limit the number of preset time periods or the number of fluorescence images acquired within each preset time period. There can be two or more preset time periods. One fluorescence image can be acquired in each preset time period, or multiple fluorescence images can be acquired within each preset time period, as long as fluorescence images are acquired in different preset time periods.

[0284] After obtaining fluorescence images at different preset time periods, the signal intensity of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing can be obtained based on the fluorescence images.

[0285] For example, signal intensity can be obtained from the grayscale value of the single-stranded nucleotide template in the fluorescence image. Since multiple single-stranded nucleotide templates are arranged on the surface of the solid support, the resulting fluorescence image also contains multiple single-stranded nucleotide templates. Each single-stranded nucleotide template presents its own grayscale value in the fluorescence image according to its own luminescence. Furthermore, single-stranded nucleotide templates of different nucleotide types will present different grayscale values ​​in the same fluorescence image because their signal intensity changes differently over time. Based on the grayscale value of the single-stranded nucleotide template in the fluorescence image during nucleic acid sequencing, the signal intensity of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing can be obtained, thereby identifying the nucleotide type of the complementary nucleotide bound to the detection site of the single-stranded nucleotide template, and further identifying the nucleotide type of the analyte at the detection site.

[0286] In this embodiment, gene sequencing can be performed in a stable position by arranging multiple single-stranded nucleotide templates on the surface of a solid support. The luminescence of multiple arrayed single-stranded nucleotide templates on the solid support surface is captured in a fluorescence image. Through the fluorescence image, the luminescence of many single-stranded nucleotide templates can be acquired simultaneously, improving sequencing efficiency.

[0287] In one embodiment, the different preset time periods include a first preset time period and a second preset time period that do not overlap. During nucleic acid sequencing, acquiring fluorescence images of the solid support at different preset time periods includes:

[0288] In a first preset time period, fluorescence images of the solid support are acquired at fixed time intervals to obtain x frames of fluorescence images; in a second preset time period, fluorescence images of the solid support are acquired at fixed time intervals to obtain y frames of fluorescence images.

[0289] Where x and y are positive integers.

[0290] In one embodiment, the signal intensity includes the maximum signal intensity, and the signal intensity at different preset time periods during nucleic acid sequencing, obtained from the fluorescence image at the detection location of the single-stranded nucleotide template, includes:

[0291] For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum pixel value of the detection location among all fluorescence images within the preset time period; the maximum pixel value of the detection location is taken as the maximum signal intensity of the detection location within the preset time period.

[0292] For example, the signal intensity at the detection location of a single-stranded nucleotide template can be the pixel value of the single-stranded nucleotide template in the fluorescence image. By iterating through the pixel values ​​of each single-stranded nucleotide template in the first x frames of fluorescence images, the maximum pixel value (signal value) of each single-stranded nucleotide template in these x frames of fluorescence images is obtained; by iterating through the pixel values ​​of each single-stranded nucleotide template in the next y frames of fluorescence images, the maximum pixel value (signal value) of each single-stranded nucleotide template in these y frames of fluorescence images is obtained.

[0293] Furthermore, the maximum pixel value of the single-stranded nucleotide template in the first x frames of the image can be used as the x-coordinate of the single-stranded nucleotide template, and the maximum pixel value of the single-stranded nucleotide template in the subsequent y frames of the image can be used as the y-coordinate of the single-stranded nucleotide template. Each single-stranded nucleotide template obtains a two-dimensional coordinate system composed of such x- and y-coordinates. Based on the two-dimensional coordinates of multiple single-stranded nucleotide templates, the multiple single-stranded nucleotide templates are clustered, and the nucleotide type of the nucleotide to be tested at the detection position of the multiple single-stranded nucleotide templates is determined according to the clustering results.

[0294] In this embodiment, for all fluorescence images acquired in a cycle, only the first few frames and the last few frames are selected for signal intensity acquisition, while the intermediate fluorescence images are discarded. This is because, in the middle of a cycle, the label and substrate may still be reacting, and the signal intensity is not stable. Selecting the signal intensity acquired from the first few frames and the last few frames provides higher reliability, and due to the longer time interval, the difference in signal intensity generated by the same single-stranded nucleotide template after a longer interval is more obvious, thereby improving the accuracy of sequencing.

[0295] Please refer to Figure 10, which is a schematic flowchart of a sequencing method in one embodiment. In one embodiment, as shown in Figure 8, a sequencing method is provided. It is understood that this method can be applied to a terminal, a server, or a system including both a terminal and a server, and is implemented through interaction between the terminal and the server. In this embodiment, the method includes the following steps:

[0296] S302, acquire the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods at the detection location.

[0297] In this process, the sequencing reaction of the nucleotide sequence cluster to be tested is performed in the presence of four types of nucleotides, namely, the first type, the second type, the third type, and the fourth type. The complementary nucleotide of the first type is associated with the first signal, the complementary nucleotide of the third type is associated with the second signal, and the complementary nucleotide of the second type is associated with the third signal. The signal intensity distribution of the first, second, and third signals is different at different preset time periods.

[0298] In this embodiment, three of the four nucleotides are associated with three different signals, and the signal intensity distribution of each signal is different at different preset time periods.

[0299] Among them, the first, second and third signals can be glow-type signals or flash-type signals. The signals can be distinguished by whether they are glow-type or flash-type signals, or by different types of glow-type signals or different types of flash-type signals.

[0300] For example, among the first, second, and third signals, one is a glow signal, one is a flash signal, and one is a combination of glow and flash signals.

[0301] For example, among the first, second, and third signals, one is a glow-type signal, one is a first-type flash-type signal, and one is a second-type flash-type signal.

[0302] S304, determine the nucleotide type of the complementary nucleotide that binds to the nucleotide sequence cluster at the detection position based on the signal intensity of the nucleotide sequence cluster at different preset time periods.

[0303] The aforementioned sequencing method involves sequencing a target nucleotide sequence cluster in the presence of four nucleotides: a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with a third signal. The signal intensity distributions of the first, second, and third signals differ at different preset time periods. The signal intensity of the target nucleotide sequence cluster at the detection location is obtained at different preset time periods, and the nucleotide type of the complementary nucleotide bound to the target nucleotide sequence cluster at the detection location is determined based on the signal intensity distribution of different signals at different preset time periods. This application uses three of the four complementary nucleotides associated with different signals, and by utilizing the different signal intensity distributions of different signals at different preset time periods, it achieves different trends in the signal intensity of different nucleotides over time. By collecting the signal intensity of the target nucleotide sequence cluster at the detection location at different time periods, the trend in the signal intensity of the target nucleotide sequence cluster at that detection location can be determined, thereby determining the nucleotide type of the complementary nucleotide at that detection location. Furthermore, the nucleotide type of the complementary nucleotide can be inferred, thus achieving gene sequencing without the need for multiple channels, reducing the cost of gene sequencing.

[0304] In one embodiment, the first signal is a glow-type signal, the second signal is a flash-type signal, and the third signal is a combination of glow-type and flash-type signals. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is glow-type luciferase, the marker for the third nucleotide type is flash-type luciferase, the marker for the second nucleotide type is a combination of glow-type and flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to produce a glow-type signal, and the flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to produce a flash-type signal.

[0305] In the embodiments of this application, the first nucleotide type is labeled with glow-type luciferase, the third nucleotide type is labeled with flash-type luciferase, the second nucleotide type is labeled with a combination of glow-type luciferase and flash-type luciferase, and the fourth nucleotide type is not labeled.

[0306] Assuming the signal intensity over different preset time periods includes a first signal intensity over a first preset time period and a second signal intensity over a second preset time period, in this embodiment, determining the nucleotide type of the complementary nucleotide bound to the nucleotide sequence cluster at the detection position based on the signal intensity over different preset time periods includes:

[0307] If both the first signal intensity and the second signal intensity are within a first predetermined interval, the complementary nucleotide is identified as the first nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within the second predetermined interval, the complementary nucleotide is identified as the second nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within a third predetermined interval, the complementary nucleotide is identified as the third nucleotide; if both the first signal intensity and the second signal intensity are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0308] In one embodiment, the first signal is a glow-type signal, the second signal is a first-type flash-type signal, and the third signal is a second-type flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the second nucleotide type is a first-type flash-type luciferase, the marker for the third nucleotide type is a second-type flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first-type flash-type signal, and the second-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second-type flash-type signal.

[0309] In this embodiment, a glow-type luciferase is used to label the first nucleotide type, and two different flash-type luciferases are used to label the second and third nucleotide types. Specifically, the first flash-type luciferase is used to label the second nucleotide type, and the second flash-type luciferase is used to label the third nucleotide type. The fourth nucleotide type is not labeled.

[0310] Assuming the signal intensity over different preset time periods includes a first signal intensity over a first preset time period and a second signal intensity over a second preset time period, in this embodiment, determining the nucleotide type of the complementary nucleotide bound to the nucleotide sequence cluster at the detection position based on the signal intensity over different preset time periods includes:

[0311] If both the first signal intensity and the second signal intensity are within a first predetermined interval, the complementary nucleotide is identified as the first nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within the second predetermined interval, the complementary nucleotide is identified as the second nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within a third predetermined interval, the complementary nucleotide is identified as the third nucleotide; if both the first signal intensity and the second signal intensity are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0312] In one example, the first nucleotide corresponds to base class 1 in Figure 3(a), the second nucleotide corresponds to base class 2 in Figure 3(a), the third nucleotide corresponds to base class 3 in Figure 3(a), and the fourth nucleotide corresponds to base class 4 in Figure 3(a).

[0313] Alternatively, suppose the signal intensity for different preset time periods includes a first signal intensity for a first preset time period and a second signal intensity for a second preset time period. The glow signal is shown in Figure 8B or Figure 8D, the first flash signal is shown in Figure 8D, and the second flash signal is shown in Figure 8B (i.e., the maximum value of the first flash signal is greater than the maximum value of the second flash signal). In this embodiment, determining the nucleotide type of the complementary nucleotide bound to the nucleotide sequence cluster at the detection position based on the signal intensity for different preset time periods includes: if both the first and second signal intensities are within a first predetermined interval, the complementary nucleotide is identified as a first nucleotide; if the first signal intensity is within the first predetermined interval and the second signal intensity is within a third predetermined interval, the complementary nucleotide is identified as a second nucleotide; if the first signal intensity is within the second predetermined interval and the second signal intensity is within the third predetermined interval, the complementary nucleotide is identified as a third nucleotide; if both the first and second signal intensities are within the third predetermined interval, the complementary nucleotide is identified as a fourth nucleotide. Wherein, the signal intensity in the first predetermined interval is greater than the signal intensity in the second predetermined interval, and the signal intensity in the second predetermined interval is greater than the signal intensity in the third predetermined interval. For example, the first preset time period is 0-6s, and the second preset time period is 7-12s.

[0314] In one embodiment, the first signal is a glow-type signal, the third signal is a first flash-type signal, and the second signal is a second flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the third nucleotide type is a first flash-type luciferase, the marker for the second nucleotide type is a second flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first flash-type signal, and the second flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second flash-type signal.

[0315] In this embodiment, a glow-type luciferase is used to label the first nucleotide type, and two different flash-type luciferases are used to label the second and third nucleotide types. Specifically, the first flash-type luciferase is used to label the third nucleotide type, and the second flash-type luciferase is used to label the second nucleotide type. The fourth nucleotide type is not labeled.

[0316] Assuming different preset time periods include a first preset time period and a second preset time period, in this embodiment, determining the nucleotide type of the complementary nucleotide bound to the detection position of the nucleotide sequence cluster to be tested at different preset time periods based on the signal intensity includes:

[0317] Based on the first signal intensity at the detection location during a first preset time period and the second signal intensity during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes the intensity ratio or intensity difference value.

[0318] If the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type; if the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the difference value interval to which the intensity difference value belongs is the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type; if the intensity of the first signal or the intensity of the second signal is less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type. In one example, the first nucleotide corresponds to base category 1 in Figure 3(a), the second nucleotide corresponds to base category 2 in Figure 3(a), the third nucleotide corresponds to base category 3 in Figure 3(a), and the fourth nucleotide corresponds to base category 4 in Figure 3(a).

[0319] In another example, the glow signal is shown in Figure 8B or Figure 8D, the first flash signal is shown in Figure 8D, and the second flash signal is shown in Figure 8B (i.e., the maximum value of the first flash signal is greater than the maximum value of the second flash signal); where the intensity difference value is the intensity difference value, the first difference value range is 0-19, the second difference value range is 20-40, and the third difference value range is 45-70.

[0320] According to embodiments of this application, the interval ranges in each embodiment do not overlap.

[0321] It should be understood that although the steps in the flowcharts of the above embodiments are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the above embodiments may include multiple steps or multiple stages. These steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the steps or stages of other steps.

[0322] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0323] In one embodiment, as shown in FIG11, a sequencing device 700 is provided. This device can be a software module, a hardware module, or a combination of both as part of a computer device. Specifically, the device includes: a data acquisition module 701 and a type determination module 702, wherein:

[0324] The data acquisition module 701 is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing. The nucleotide sequence cluster to be tested undergoes a single-base extension reaction of complementary strands in the presence of four types of nucleotides. The four types of nucleotides include a first type of nucleotide, a second type of nucleotide, a third type of nucleotide, and a fourth type of nucleotide. The complementary nucleotide of the first type of nucleotide is associated with the first signal, the complementary nucleotide of the third type of nucleotide is associated with the second signal, and the complementary nucleotide of the second type of nucleotide is associated with the first signal and the second signal. The signal intensity distribution of the first signal and the second signal is different at different preset time periods. The four types of nucleotides carry modification groups.

[0325] The type determination module 702 is used to determine the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested based on the signal intensity of the nucleotide sequence cluster at different preset time periods.

[0326] In one embodiment, when determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods, the type determination module 702 can be used to:

[0327] For each complementary nucleotide, a signal intensity vector is obtained, consisting of signal intensities at different preset time periods;

[0328] Based on the trained neural network, the nucleotide type of the complementary nucleotide is determined according to the signal intensity vector of the complementary nucleotide.

[0329] In one embodiment, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. When determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle based on the signal intensity at different preset time periods, the type determination module 702 can be used to:

[0330] For each complementary nucleotide, a data pair consisting of the first signal intensity and the second signal intensity is obtained;

[0331] By using the first and second signal intensities of the data pair as the abscissa and ordinate of the two-dimensional coordinate system, respectively, the data points of complementary nucleotides on the two-dimensional coordinate system are obtained.

[0332] Clustering multiple data points in a two-dimensional coordinate system yields clustering results, which contain multiple clusters.

[0333] For each complementary nucleotide, the nucleotide type of the cluster to which the data point of the complementary nucleotide belongs is taken as the nucleotide type of the complementary nucleotide.

[0334] In one embodiment, multiple nucleotide sequence clusters to be tested are arranged on the surface of a solid support. When acquiring the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods during one cycle of nucleic acid sequencing, the data acquisition module 701 can be used to:

[0335] In one cycle of nucleic acid sequencing, fluorescence images of a solid support were acquired at different preset time periods;

[0336] The signal intensity of the nucleotide sequence clusters to be tested is obtained from the fluorescence image at different preset time periods during one cycle of nucleic acid sequencing.

[0337] In one embodiment, the different preset time periods include a first preset time period and a second preset time period. During one cycle of nucleic acid sequencing, when acquiring fluorescence images of the solid support for different preset time periods, the data acquisition module 701 can be used for:

[0338] In one cycle of nucleic acid sequencing, fluorescence images of the solid support are acquired at fixed time intervals;

[0339] Use the first x frames of fluorescence images in the loop as the fluorescence images for the first preset time period;

[0340] Use the fluorescence image of the last y frames in the loop as the fluorescence image of the second preset time period;

[0341] Where x and y are positive integers, and the sum of x and y is less than or equal to the total number of fluorescence images obtained in one cycle of nucleic acid sequencing;

[0342] Signal intensity includes maximum signal intensity. When acquiring the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing based on fluorescence images, the data acquisition module 701 can be used for:

[0343] For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum gray value of the nucleotide sequence cluster to be tested in all fluorescence images within the preset time period.

[0344] The maximum gray value is taken as the maximum signal intensity of the nucleotide sequence cluster to be tested within a preset time period.

[0345] In one embodiment, the signal intensity at different preset time periods includes a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. When determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle based on the signal intensity at different preset time periods, the type determination module 702 can be used to:

[0346] If both the first signal intensity and the second signal intensity are within the first predetermined interval, then the complementary nucleotide is identified as the first nucleotide.

[0347] If the intensity of the first signal is within a first predetermined range and the intensity of the second signal is within a second predetermined range, then the complementary nucleotide is identified as the second nucleotide.

[0348] If the intensity of the first signal is within a first predetermined interval and the intensity of the second signal is within a third predetermined interval, then the complementary nucleotide is identified as the third nucleotide.

[0349] If both the first and second signal intensities are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0350] In one embodiment, three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers differs at different preset time periods.

[0351] In one embodiment, the marker for the first nucleotide type is a first luciferase, the marker for the third nucleotide type is a second luciferase, the marker for the second nucleotide type is a combination of the first luciferase and the second luciferase, and there is no marker for the fourth nucleotide type.

[0352] In one embodiment, the first luciferase is a glow-type luciferase, and the second luciferase is a flash-type luciferase. The first luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a first signal, which is a glow-type signal. The second luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate a second signal, which is a flash-type signal.

[0353] In one embodiment, referring to Figure 12, the sequencing device further includes a program execution module 703, used to: execute a preset biochemical intervention program at preset time points in the cycle to change the biochemical state of each marker;

[0354] When acquiring the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods during one cycle of nucleic acid sequencing, the data acquisition module 701 can be used for:

[0355] In one cycle of nucleic acid sequencing, during the first pre-set time period before performing biochemical intervention procedures, the signal intensity of the nucleotide sequence clusters to be tested is acquired; and

[0356] In the second preset time period after the biochemical intervention procedure, the signal intensity of the nucleotide sequence cluster to be tested is obtained.

[0357] In one embodiment, the modifying group is a reversible blocking modifying group, the signal intensity of the first signal is the same in different preset time periods, and the signal intensity of the second signal is different in different preset time periods.

[0358] In one embodiment, four nucleotide modifications are detectable markers.

[0359] In one embodiment, the different preset time periods include a first preset time period and a second preset time period. When determining the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster in the cycle based on the signal intensity of the nucleotide sequence cluster in different preset time periods, the type determination module 702 can be used to:

[0360] Based on the first signal intensity of the nucleotide sequence cluster to be tested in the first preset time period and the second signal intensity in the second preset time period, the intensity difference value and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes the intensity ratio or intensity difference value.

[0361] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined as the first nucleotide type.

[0362] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type.

[0363] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type.

[0364] If the intensity of the first signal and the intensity of the second signal are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0365] The aforementioned sequencing apparatus performs a single-base extension reaction of complementary strands of the target nucleotide sequence cluster in the presence of four nucleotides: a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with both the first and second signals. The signal intensity distributions of the first and second signals differ at different preset time periods. Each of the four nucleotides carries a modifying group. The signal intensity of the target nucleotide sequence cluster is obtained at different preset time periods within one cycle of nucleic acid sequencing, and the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle is determined based on the signal intensity distribution at different preset time periods. This application achieves different trends in the signal intensity of different nucleotides over time during the single-base extension reaction by designing the signals associated with the four complementary nucleotide types to have different signal intensity distributions at different preset time periods. By collecting the signal intensity of the target nucleotide sequence cluster at different time points during one cycle of nucleic acid sequencing, the trend of signal intensity variation of the target nucleotide sequence cluster in that cycle can be determined, thereby identifying the nucleotide type of the complementary nucleotide in that cycle. This allows for the inference of the nucleotide type of the target nucleotide that is complementary to it, thus enabling gene sequencing without the need for multiple channels and reducing the cost of gene sequencing.

[0366] In one embodiment, as shown in FIG13, a sequencing device 800 is provided. This device can be a software module, a hardware module, or a combination of both as part of a computer device. Specifically, the device includes: a data acquisition module 801 and a type determination module 802, wherein:

[0367] The data acquisition module 801 is used to acquire the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during the nucleic acid sequencing process. The nucleotide to be tested at the detection position binds to the complementary nucleotide to carry out the extension sequencing reaction. The signal intensity of the markers corresponding to different types of complementary nucleotides is different. The signal intensity difference is the difference of signal intensity at different preset time periods.

[0368] The type determination module 802 is used to determine the nucleotide type of the complementary nucleotide that binds to the detection site in the extension sequencing reaction based on the signal intensity difference at the detection site, thereby determining the nucleotide type of the nucleotide to be tested at the detection site.

[0369] In one embodiment, three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker of the first nucleotide type is glow-in-the-dark luciferase, the marker of the third nucleotide type is flash luciferase, the marker of the second nucleotide type is a combination of glow-in-the-dark luciferase and flash luciferase, and the fourth nucleotide type has no marker.

[0370] Glow-type luciferase reacts with nucleotide-labeled substrates in the extension sequencing reaction to produce a glow-type signal, while flash-type luciferase reacts with nucleotide-labeled substrates in the extension sequencing reaction to produce a flash-type signal.

[0371] In one embodiment, the different preset time periods include a first preset time period and a second preset time period, and the signal intensity difference includes the intensity difference value of the signal intensity. When determining the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction based on the signal intensity difference at the detection site, the type determination module 802 can be used for:

[0372] Based on the first signal intensity at the detection location during a first preset time period and the second signal intensity during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes the intensity ratio or intensity difference value.

[0373] Based on the difference range to which the intensity difference value belongs, the nucleotide type of the complementary nucleotide that the extended sequencing reaction binds at the detection site is determined.

[0374] In one embodiment, when determining the nucleotide type of the complementary nucleotide bound at the detection site by the extended sequencing reaction based on the difference range to which the intensity difference value belongs, the type determination module 802 can be used to:

[0375] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined as the first nucleotide type.

[0376] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type.

[0377] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type.

[0378] If the intensity of the first signal and the intensity of the second signal are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0379] In one embodiment, multiple single-stranded nucleotide templates are arranged on the surface of a solid support. When acquiring the signal intensity of the detection location of the single-stranded nucleotide templates at different preset time periods during nucleic acid sequencing, the data acquisition module 801 can be used to:

[0380] During nucleic acid sequencing, fluorescence images of a solid support were acquired at different preset time periods.

[0381] The signal intensity at different preset time periods during nucleic acid sequencing is obtained from the fluorescence image to determine the detection location of the single-stranded nucleotide template.

[0382] In one embodiment, the different preset time periods include a first preset time period and a second preset time period that do not overlap. During nucleic acid sequencing, when acquiring fluorescence images of the solid support at different preset time periods, the data acquisition module 801 can be used for:

[0383] During the first preset time period of the cycle, fluorescence images of the solid support are acquired at fixed time intervals to obtain x frames of fluorescence images;

[0384] During the second preset time period of the cycle, fluorescence images of the solid support are acquired at fixed time intervals to obtain y frames of fluorescence images;

[0385] Where x and y are positive integers;

[0386] Signal intensity includes maximum signal intensity. When acquiring the signal intensity of the detection position of a single-stranded nucleotide template at different preset time periods during nucleic acid sequencing based on fluorescence images, the data acquisition module 801 can be used for:

[0387] For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum pixel value of the detection location among all fluorescence images within the preset time period.

[0388] The maximum pixel value at the detection location is taken as the maximum signal strength at the detection location within a preset time period.

[0389] In the aforementioned sequencing device, the nucleotide to be tested at the detection position binds to a complementary nucleotide for an extension sequencing reaction. Different types of complementary nucleotides correspond to different signal intensity differences in their markers, which are reflected in the signal intensity differences at different preset time periods. The data acquisition module 801 acquires the signal intensity of the single-stranded nucleotide template at the detection position during nucleic acid sequencing at different preset time periods. The type determination module 802 determines the nucleotide type of the complementary nucleotide bound in the extension sequencing reaction during the cycle based on the signal intensity differences at the detection position, thereby determining the nucleotide type of the nucleotide to be tested at the detection position. This application achieves different signal intensity distributions of four types of complementary nucleotides at different preset time periods during the extension sequencing reaction by designing different signal intensity distributions of the markers for different types of complementary nucleotides. By collecting the signal intensity of the single-stranded nucleotide template at the detection position during nucleic acid sequencing at different time periods, the signal intensity difference at the detection position in that cycle can be determined, thereby determining the nucleotide type of the complementary nucleotide bound in that cycle during the extension sequencing reaction, and further determining the nucleotide type of the nucleotide to be tested at the detection position that reacts with the complementary nucleotide. This eliminates the need for multiple channels to achieve gene sequencing, reducing the cost of gene sequencing.

[0390] In one embodiment, as shown in FIG14, a sequencing device 900 is provided. This device can be a software module, a hardware module, or a combination of both as part of a computer device. Specifically, the device includes: a data acquisition module 901 and a type determination module 902, wherein:

[0391] The data acquisition module 901 is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at the detection location at different preset time periods. The nucleotide sequence cluster to be tested is sequenced in the presence of four types of nucleotides, including a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with the first signal, the complementary nucleotide of the third nucleotide type is associated with the second signal, and the complementary nucleotide of the second nucleotide type is associated with the third signal. The signal intensity distribution of the first, second, and third signals is different at different preset time periods.

[0392] The type determination module 902 is used to determine the nucleotide type of the complementary nucleotide that binds to the nucleotide sequence cluster at the detection position based on the signal intensity of the nucleotide sequence cluster at different preset time periods.

[0393] In one embodiment, the first signal is a glow-type signal, the second signal is a flash-type signal, and the third signal is a combination of glow-type and flash-type signals. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is glow-type luciferase, the marker for the third nucleotide type is flash-type luciferase, the marker for the second nucleotide type is a combination of glow-type and flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, and the flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a flash-type signal.

[0394] The signal intensities at different preset time periods include the first signal intensity of the first preset time period and the second signal intensity of the second preset time period. When determining the nucleotide type of the complementary nucleotide bound to the detection position of the nucleotide sequence cluster to be tested based on the signal intensities at different preset time periods, the type determination module 902 is further used for:

[0395] If both the first signal intensity and the second signal intensity are within the first predetermined interval, then the complementary nucleotide is identified as the first nucleotide.

[0396] If the intensity of the first signal is within a first predetermined range and the intensity of the second signal is within a second predetermined range, then the complementary nucleotide is identified as the second nucleotide.

[0397] If the intensity of the first signal is within a first predetermined interval and the intensity of the second signal is within a third predetermined interval, then the complementary nucleotide is identified as the third nucleotide.

[0398] If both the first and second signal intensities are within the third predetermined interval, the complementary nucleotide is identified as the fourth nucleotide.

[0399] In one embodiment, the first signal is a glow-type signal, the second signal is a first-type flash-type signal, and the third signal is a second-type flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the second nucleotide type is a first-type flash-type luciferase, the marker for the third nucleotide type is a second-type flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first-type flash-type signal, and the second-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second-type flash-type signal.

[0400] In one embodiment, the first signal is a glow-type signal, the third signal is a first flash-type signal, and the second signal is a second flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different at different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the third nucleotide type is a first flash-type luciferase, the marker for the second nucleotide type is a second flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a glow-type signal, the first flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a first flash-type signal, and the second flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate a second flash-type signal.

[0401] Different preset time periods include a first preset time period and a second preset time period. When determining the nucleotide type of the complementary nucleotide bound to the detection position of the nucleotide sequence cluster to be tested based on the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods, the type determination module 902 is also used for:

[0402] Based on the first signal intensity at the detection location during a first preset time period and the second signal intensity during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes the intensity ratio or intensity difference value.

[0403] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined as the first nucleotide type.

[0404] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type.

[0405] If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type.

[0406] If the intensity of the first signal and the intensity of the second signal are less than or equal to a preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

[0407] The aforementioned sequencing apparatus performs sequencing reactions on the target nucleotide sequence cluster in the presence of four types of nucleotides: a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with a third signal. The signal intensity distributions of the first, second, and third signals differ at different preset time periods. The data acquisition module 901 acquires the signal intensity of the target nucleotide sequence cluster at the detection location at different preset time periods. The type determination module 902 determines the nucleotide type of the complementary nucleotide bound to the target nucleotide sequence cluster at the detection location based on the signal intensity distribution of the target nucleotide sequence cluster at different preset time periods. This application achieves different trends in the signal intensity of different nucleotides over time by associating three of the four complementary nucleotides with different signals and by utilizing the different signal intensity distributions of different signals at different preset time periods. By collecting the signal intensity of the nucleotide sequence cluster to be tested at different time periods at the detection location, the trend of signal intensity change of the nucleotide sequence cluster to be tested at the detection location can be determined, thereby determining the nucleotide type of the complementary nucleotide at the detection location, and further inferring the nucleotide type of the complementary nucleotide to be tested. This allows gene sequencing to be achieved without the need for multiple channels, thus reducing the cost of gene sequencing.

[0408] For specific limitations regarding the sequencing device, please refer to the limitations on the sequencing method above, which will not be repeated here. Each module in the above sequencing device can be implemented entirely or partially through software, hardware, or a combination thereof. These modules can be embedded in hardware or independent of the processor in the computer device, or stored in software in the memory of the computer device, so that the processor can call and execute the operations corresponding to each module.

[0409] Terms such as “component,” “module,” and “system” are intended to refer to computer-related entities, which can be hardware, a combination of hardware and software, software, or software in execution. For example, a component can be, but is not limited to, a process running on a processor, a processor, an object, executable code, a thread of execution, a program, and / or a computer. For illustration, a running program on a server and the server itself can both be components. One or more components may reside within a process and / or a thread of execution, and components may be located within a single computer and / or distributed across two or more computers.

[0410] In an exemplary embodiment, a computer device is provided, which may be a server, and its internal structure diagram is shown in Figure 15. The computer device includes a processor, memory, input / output interfaces (I / O), and a communication interface. The processor, memory, and I / O interfaces are connected via a system bus, and the communication interface is connected to the system bus via the I / O interfaces. The processor of the computer device provides computing and control capabilities. The memory of the computer device includes a non-volatile computer-readable storage medium and internal memory. The non-volatile computer-readable storage medium stores an operating system, computer-readable instructions, and a database. The internal memory provides an environment for the operation of the operating system and computer-readable instructions in the non-volatile computer-readable storage medium. The database of the computer device stores feature data corresponding to gene loci. The input / output interfaces of the computer device are used for exchanging information between the processor and external devices. The communication interface of the computer device is used for communicating with external terminals via a network connection. When the computer-readable instructions are executed by the processor, a sequencing method is implemented.

[0411] In an exemplary embodiment, a computer device is provided, which may be a terminal, and its internal structure diagram may be as shown in Figure 16. The computer device includes a processor, memory, input / output interface, communication interface, display unit, and input device. The processor, memory, and input / output interface are connected via a system bus, and the communication interface, display unit, and input device are connected to the system bus via the input / output interface. The processor of the computer device provides computing and control capabilities. The memory of the computer device includes a non-volatile computer-readable storage medium and internal memory. The non-volatile computer-readable storage medium stores an operating system and computer-readable instructions. The internal memory provides an environment for the operation of the operating system and computer-readable instructions in the non-volatile computer-readable storage medium. The input / output interface of the computer device is used for exchanging information between the processor and external devices. The communication interface of the computer device is used for wired or wireless communication with external terminals; wireless communication can be achieved through Wi-Fi, mobile cellular networks, NFC (Near Field Communication), or other technologies. When the computer-readable instructions are executed by the processor, they implement a sequencing method or sequencing mechanism. The display unit of the computer device is used to form a visually visible image and may be a display screen, a projection device, or a virtual reality imaging device. The display screen can be an LCD screen or an e-ink screen. The input device of the computer device can be a touch layer covering the display screen, or buttons, trackballs, or touchpads set on the casing of the computer device, or external keyboards, touchpads, or mice, etc.

[0412] Those skilled in the art will understand that the structures shown in Figures 15 and 16 are merely block diagrams of some structures related to the present application and do not constitute a limitation on the computer device to which the present application is applied. Specific computer devices may include more or fewer components than shown in the figures, or combine certain components, or have different component arrangements.

[0413] In one embodiment, a computer device is also provided, including a memory and a processor, the memory storing computer-readable instructions, the processor executing the computer-readable instructions to implement the steps in the above method embodiments.

[0414] In one embodiment, a computer-readable storage medium is provided storing computer-readable instructions that, when executed by a processor, implement the steps in the above method embodiments.

[0415] In one embodiment, a computer program product is provided, the computer program product including computer-readable instructions stored in a computer-readable storage medium. A processor of a computer device reads the computer-readable instructions from the computer-readable storage medium, and executes the computer-readable instructions, causing the computer device to perform the steps in the above-described method embodiments.

[0416] Those skilled in the art will understand that all or part of the processes in the methods of the above embodiments can be implemented by instructing related hardware through computer-readable instructions. These computer-readable instructions can be stored in a non-volatile computer-readable storage medium. When executed, these computer-readable instructions can include the processes of the embodiments of the above methods. Any references to memory, databases, or other media used in the embodiments provided in this application can include at least one of non-volatile and volatile memory. Non-volatile memory can include read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high-density embedded non-volatile memory, resistive random access memory (ReRAM), magnetic random access memory (MRAM), ferroelectric random access memory (FRAM), phase change memory (PCM), graphene memory, etc. Volatile memory can include random access memory (RAM) or external cache memory, etc. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM). The databases involved in the embodiments provided in this application may include at least one type of relational database and non-relational database. Non-relational databases may include, but are not limited to, blockchain-based distributed databases. The processors involved in the embodiments provided in this application may be general-purpose processors, central processing units, graphics processing units, digital signal processors, programmable logic devices, quantum computing-based data processing logic devices, etc., and are not limited to these.

[0417] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0418] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A sequencing method, characterized in that, include: The signal intensity of a target nucleotide sequence cluster at different preset time periods during one cycle of nucleic acid sequencing is obtained. The target nucleotide sequence cluster undergoes a single-base extension reaction of its complementary strand in the presence of four nucleotides: a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with both the first and second signals. The signal intensity distributions of the first and second signals differ at different preset time periods. Each of the four nucleotides carries a modifying group. The nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested in the cycle is determined based on the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods.

2. The method according to claim 1, characterized in that, The step of determining the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle based on the signal intensity at different preset time periods includes: For each complementary nucleotide, a signal intensity vector is obtained, consisting of signal intensities at different preset time periods; Based on the trained neural network, the nucleotide type of the complementary nucleotide is determined according to the signal intensity vector of the complementary nucleotide.

3. The method according to claim 1, characterized in that, The signal intensities at different preset time periods include a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle based on the signal intensities at different preset time periods includes: For each complementary nucleotide, a data pair consisting of the first signal intensity and the second signal intensity is obtained; Using the first and second signal intensities of the data pair as the horizontal and vertical coordinates of the two-dimensional coordinate system, respectively, the data points of the complementary nucleotides on the two-dimensional coordinate system are obtained. Multiple data points are clustered in a two-dimensional coordinate system to obtain clustering results, which contain multiple clusters; For each complementary nucleotide, the nucleotide type corresponding to the cluster to which the data point of the complementary nucleotide belongs is taken as the nucleotide type of the complementary nucleotide.

4. The method according to claim 1, characterized in that, Multiple nucleotide sequence clusters to be tested are arranged on the surface of a solid support. The step of obtaining the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods during one cycle of nucleic acid sequencing includes: During one cycle of nucleic acid sequencing, fluorescence images of the solid support were acquired at different preset time periods; The fluorescence image is used to obtain the signal intensity of the nucleotide sequence clusters to be tested at different preset time periods during one cycle of nucleic acid sequencing.

5. The method according to claim 4, characterized in that, The different preset time periods include a first preset time period and a second preset time period. Acquiring fluorescence images of the solid support at different preset time periods during one cycle of nucleic acid sequencing includes: During one cycle of nucleic acid sequencing, fluorescence images of the solid support are acquired at fixed time intervals. The fluorescence images of the first x frames in the loop are used as the fluorescence images of the first preset time period; The fluorescence image of the last y frames in the cycle is used as the fluorescence image of the second preset time period; Where x and y are positive integers, and the sum of x and y is less than or equal to the total number of fluorescence images obtained in one cycle of nucleic acid sequencing; The signal intensity includes the maximum signal intensity, and the step of obtaining the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing based on the fluorescence image includes: For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum pixel value of the nucleotide sequence cluster to be tested in all fluorescence images within the preset time period. The maximum pixel value is taken as the maximum signal intensity of the nucleotide sequence cluster to be tested during the preset time period.

6. The method according to claim 1, characterized in that, The signal intensities at different preset time periods include a first signal intensity at a first preset time period and a second signal intensity at a second preset time period. Determining the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle based on the signal intensities at different preset time periods includes: If both the first signal intensity and the second signal intensity are within a first predetermined interval, then the complementary nucleotide is identified as the first nucleotide; If the first signal intensity is within a first predetermined range and the second signal intensity is within a second predetermined range, then the complementary nucleotide is identified as the second nucleotide. If the first signal intensity is within a first predetermined interval and the second signal intensity is within a third predetermined interval, then the complementary nucleotide is identified as the third nucleotide. If both the first signal intensity and the second signal intensity are within a third predetermined range, then the complementary nucleotide is identified as a fourth nucleotide.

7. The method according to claim 1, characterized in that, Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the markers of the three nucleotides is different in different preset time periods.

8. The method according to claim 7, characterized in that, The first nucleotide type is labeled with a first luciferase, the third nucleotide type is labeled with a second luciferase, the second nucleotide type is labeled with a combination of the first luciferase and the second luciferase, and the fourth nucleotide type has no label.

9. The method according to claim 8, characterized in that, The first luciferase is a glow-type luciferase, and the second luciferase is a flash-type luciferase. The first luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate the first signal, which is a glow-type signal. The second luciferase reacts with the nucleotide-labeled substrate in the single-base extension reaction to generate the second signal, which is a flash-type signal.

10. The method according to claim 7, characterized in that, The method further includes: At preset time points in the cycle, a preset biochemical intervention procedure is executed to change the biochemical state of each marker; The acquisition of signal intensities of the target nucleotide sequence cluster at different preset time periods during one cycle of nucleic acid sequencing includes: In one cycle of nucleic acid sequencing, during the first pre-set time period before performing biochemical intervention procedures, the signal intensity of the nucleotide sequence clusters to be tested is acquired; and In the second preset time period after the biochemical intervention procedure, the signal intensity of the nucleotide sequence cluster to be tested is obtained.

11. The method according to claim 1, characterized in that, The modifying group is a reversible blocking modifying group. The first signal has the same signal intensity in different preset time periods, while the second signal has different signal intensity in different preset time periods.

12. The method according to claim 1, characterized in that, The four nucleotide modifications are detectable markers.

13. The method according to claim 1, characterized in that, The different preset time periods include a first preset time period and a second preset time period. Determining the nucleotide type of the complementary nucleotide of the target nucleotide sequence cluster in the cycle based on the signal intensity of the target nucleotide sequence cluster during different preset time periods includes: Based on the first signal intensity of the nucleotide sequence cluster to be tested in a first preset time period and the second signal intensity in a second preset time period, the intensity difference value and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes an intensity ratio or an intensity difference value. If the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to the preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

14. A sequencing method, characterized in that, include: The signal intensity of the detection position of a single-stranded nucleotide template is obtained at different preset time periods during nucleic acid sequencing. The nucleotide to be tested at the detection position binds to a complementary nucleotide for extended sequencing. The signal intensity of the markers corresponding to different types of complementary nucleotides is different. The signal intensity difference is the difference in signal intensity at different preset time periods. Based on the signal intensity difference at the detection location, the nucleotide type of the complementary nucleotide that the extended sequencing reaction binds at the detection location is determined, thereby determining the nucleotide type of the nucleotide to be tested at the detection location.

15. The method according to claim 14, characterized in that, Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the three nucleotide markers is different in different preset time periods. The marker of the first nucleotide type is glow-type luciferase, the marker of the third nucleotide type is flash-type luciferase, the marker of the second nucleotide type is a combination of glow-type luciferase and flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide-labeled substrate in the extended sequencing reaction to generate a glow-type signal, and the flash-type luciferase reacts with the nucleotide-labeled substrate in the extended sequencing reaction to generate a flash-type signal.

16. The method according to claim 14, characterized in that, The different preset time periods include a first preset time period and a second preset time period; the signal intensity difference includes the signal intensity difference value; and determining the nucleotide type of the complementary nucleotide bound at the detection location by the extended sequencing reaction based on the signal intensity difference at the detection location includes: Based on the first signal strength at the detection location during a first preset time period and the second signal strength during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes an intensity ratio or an intensity difference value. Based on the difference range to which the intensity difference value belongs, the nucleotide type of the complementary nucleotide that the extended sequencing reaction binds to at the detection site is determined.

17. The method according to claim 16, characterized in that, The step of determining the nucleotide type of the complementary nucleotide that the extended sequencing reaction binds at the detection site based on the difference range to which the intensity difference value belongs includes: If the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to the preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

18. The method according to claim 14, characterized in that, Multiple single-stranded nucleotide templates are arranged on the surface of a solid support. The signal intensity at the detection sites for obtaining the single-stranded nucleotide templates during different preset time periods in the nucleic acid sequencing process includes: During nucleic acid sequencing, fluorescence images of the solid support at different preset time periods are acquired; The signal intensity at the detection location of the single-stranded nucleotide template during different preset time periods in the nucleic acid sequencing process is obtained based on the fluorescence image.

19. The method according to claim 18, characterized in that, The different preset time periods include a first preset time period and a second preset time period that do not overlap. The step of acquiring fluorescence images of the solid support at different preset time periods during nucleic acid sequencing includes: During the first preset time period, fluorescence images of the solid support are acquired at fixed time intervals to obtain x frames of fluorescence images; During the second preset time period, fluorescence images of the solid support are acquired at fixed time intervals to obtain y frames of fluorescence images; Where x and y are positive integers; The signal intensity includes the maximum signal intensity, and the signal intensity at different preset time periods during nucleic acid sequencing, obtained from the fluorescence image at the detection location of the single-stranded nucleotide template, includes: For each preset time period, all fluorescence images within the preset time period are traversed to obtain the maximum pixel value of the detection location among all fluorescence images within the preset time period. The maximum pixel value at the detection location is taken as the maximum signal strength at the detection location during the preset time period.

20. A sequencing method, characterized in that, include: The signal intensity of the nucleotide sequence cluster to be tested at the detection location at different preset time periods is obtained. The nucleotide sequence cluster to be tested is sequenced in the presence of four types of nucleotides, including a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with a third signal. The signal intensity distributions of the first signal, the second signal, and the third signal are different at different preset time periods. The nucleotide type of the complementary nucleotide that binds to the target nucleotide sequence cluster at the detection position is determined based on the signal intensity of the target nucleotide sequence cluster at different preset time periods.

21. The method according to claim 20, characterized in that, The first signal is a glow-type signal, the second signal is a flash-type signal, and the third signal is a combination of the glow-type signal and the flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of these markers differs across different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the third nucleotide type is a flash-type luciferase, the marker for the second nucleotide type is a combination of the glow-type luciferase and the flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the glow-type signal, and the flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the flash-type signal. The signal intensities at different preset time periods include a first signal intensity during a first preset time period and a second signal intensity during a second preset time period. Determining the nucleotide type of the complementary nucleotide bound to the detection position of the nucleotide sequence cluster to be tested based on the signal intensities at different preset time periods includes: If both the first signal intensity and the second signal intensity are within a first predetermined interval, then the complementary nucleotide is identified as the first nucleotide; If the first signal intensity is within a first predetermined range and the second signal intensity is within a second predetermined range, then the complementary nucleotide is identified as the second nucleotide. If the first signal intensity is within a first predetermined interval and the second signal intensity is within a third predetermined interval, then the complementary nucleotide is identified as the third nucleotide. If both the first signal intensity and the second signal intensity are within a third predetermined range, then the complementary nucleotide is identified as a fourth nucleotide.

22. The method according to claim 20, characterized in that, The first signal is a glow-type signal, the second signal is a first-type flash-type signal, and the third signal is a second-type flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of the markers of the three nucleotides is different in different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the second nucleotide type is a first-type flash-type luciferase, the marker for the third nucleotide type is a second-type flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the glow-type signal, the first-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the first-type flash-type signal, and the second-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the second-type flash-type signal.

23. The method according to claim 20, characterized in that, The first signal is a glow-type signal, the third signal is a first-type flash-type signal, and the second signal is a second-type flash-type signal. Three of the four nucleotides are labeled with markers, and the signal intensity distribution of these markers differs across different preset time periods. The marker for the first nucleotide type is a glow-type luciferase, the marker for the third nucleotide type is a first-type flash-type luciferase, the marker for the second nucleotide type is a second-type flash-type luciferase, and the fourth nucleotide type has no marker. The glow-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the glow-type signal, the first-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the first-type flash-type signal, and the second-type flash-type luciferase reacts with the nucleotide marker substrate in the sequencing reaction to generate the second-type flash-type signal. The different preset time periods include a first preset time period and a second preset time period. Determining the nucleotide type of the complementary nucleotide bound to the detection position of the nucleotide sequence cluster under test based on the signal intensity of the nucleotide sequence cluster under test at different preset time periods includes: Based on the first signal strength at the detection location during a first preset time period and the second signal strength during a second preset time period, the intensity difference value at the detection location and the difference value range to which the intensity difference value belongs are determined. The intensity difference value includes an intensity ratio or an intensity difference value. If the intensity of the first signal or the intensity of the second signal is greater than a preset intensity threshold, and the intensity difference value belongs to the first difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the first nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the second difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the second nucleotide type. If the intensity of the first signal or the intensity of the second signal is greater than the preset intensity threshold, and the intensity difference value belongs to the third difference value interval, then the nucleotide type of the complementary nucleotide is determined to be the third nucleotide type. If the first signal intensity and the second signal intensity are less than or equal to the preset intensity threshold, then the nucleotide type of the complementary nucleotide is determined to be the fourth nucleotide type.

24. A sequencing device, characterized in that, include: The data acquisition module is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at different preset time periods in one cycle of nucleic acid sequencing. The nucleotide sequence cluster to be tested undergoes a single-base extension reaction of complementary strands in the presence of four types of nucleotides. The four types of nucleotides include a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with both the first and second signals. The signal intensity distributions of the first and second signals are different at different preset time periods. The four types of nucleotides carry modifying groups. The type determination module is used to determine the nucleotide type of the complementary nucleotide of the nucleotide sequence cluster to be tested based on the signal intensity of the nucleotide sequence cluster at different preset time periods.

25. A sequencing device, characterized in that, include: The data acquisition module is used to acquire the signal intensity of the detection position of the single-stranded nucleotide template at different preset time periods during nucleic acid sequencing. The nucleotide to be tested at the detection position binds to the complementary nucleotide for extended sequencing reaction. The signal intensity of the markers corresponding to different types of complementary nucleotides is different. The signal intensity difference is the difference in signal intensity at different preset time periods. The type determination module is used to determine the nucleotide type of the complementary nucleotide that the extended sequencing reaction binds to at the detection site based on the signal intensity difference at the detection site, thereby determining the nucleotide type of the nucleotide to be tested at the detection site.

26. A sequencing device, characterized in that, include: The data acquisition module is used to acquire the signal intensity of the nucleotide sequence cluster to be tested at the detection location at different preset time periods. The nucleotide sequence cluster to be tested is sequenced in the presence of four types of nucleotides, including a first nucleotide type, a second nucleotide type, a third nucleotide type, and a fourth nucleotide type. The complementary nucleotide of the first nucleotide type is associated with a first signal, the complementary nucleotide of the third nucleotide type is associated with a second signal, and the complementary nucleotide of the second nucleotide type is associated with a third signal. The signal intensity distributions of the first signal, the second signal, and the third signal are different at different preset time periods. The type determination module is used to determine the nucleotide type of the complementary nucleotide that binds to the target nucleotide sequence cluster at the detection position based on the signal intensity of the target nucleotide sequence cluster at different preset time periods.

27. A computer device comprising a memory and a processor, the memory storing computer-readable instructions, characterized in that, When the processor executes the computer-readable instructions, it implements the steps of the method according to any one of claims 1 to 13, or the steps of the method according to any one of claims 14 to 19, or the steps of the method according to any one of claims 20 to 23.

28. A computer-readable storage medium storing computer-readable instructions thereon, characterized in that, When executed by a processor, the computer-readable instructions implement the steps of the method according to any one of claims 1 to 13, or the steps of the method according to any one of claims 14 to 19, or the steps of the method according to any one of claims 20 to 23.

29. A computer program product comprising computer-readable instructions, characterized in that, When executed by a processor, the computer-readable instructions implement the steps of the method according to any one of claims 1 to 13, or the steps of the method according to any one of claims 14 to 19, or the steps of the method according to any one of claims 20 to 23.

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