Sequencing method and system, and method for dynamically adjusting concentration of sequencing reagent
By dynamically adjusting the concentrations of nucleotides and DNA polymerase, the problems of decreased sequencing quality and reagent waste in high-throughput sequencing technology have been solved, realizing a more efficient and flexible sequencing method suitable for various scenarios such as whole genome, exome, and single-cell sequencing.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- MGI TECH CO LTD
- Filing Date
- 2024-12-04
- Publication Date
- 2026-06-11
Smart Images
Figure CN2024136660_11062026_PF_FP_ABST
Abstract
Description
Sequencing methods, systems, and methods for dynamically adjusting sequencing reagent concentrations Technical Field
[0001] This application relates to the field of gene sequencing technology, specifically to sequencing methods, systems, and methods for dynamically adjusting the concentration of sequencing reagents. Background Technology
[0002] Next-generation sequencing (NGS) technology has become a mainstream tool in genomics research and clinical diagnosis due to its advantages such as high speed, low cost, and high throughput. This technology analyzes DNA or RNA sequences by sequencing while synthesizing, and is widely used in scientific research and clinical practice.
[0003] However, existing NGS sequencing technologies still have some shortcomings, including: 1) As sequencing cycles increase, the accumulated modification markers (such as fluorescent groups and blocking groups) in the synthesized base sequences in the early stages will produce a "scarring effect," leading to a decrease in the synthesis efficiency of subsequent bases, a gradual weakening of signal intensity, and a decline in the overall sequencing quality; 2) When fluorescent markers are not completely removed, background noise will be generated, interfering with the detection of subsequent bases and increasing sequencing errors; 3) Fixed-concentration reagent formulations also limit the potential for cost optimization. For example, in early sequencing cycles, even if a lower concentration of reagent is sufficient to meet the reaction requirements, existing technologies still use high-concentration reagents, resulting in reagent waste; 4) Different sequencing application scenarios, such as whole-genome sequencing, whole-exome sequencing, single-cell sequencing, and space transcriptome sequencing, often have different requirements for reagent concentrations, and fixed-concentration reagents cannot achieve optimal results in all applications.
[0004] Therefore, existing sequencing methods still need improvement. Summary of the Invention
[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a sequencing method that can flexibly adjust the nucleotide concentration according to different stages of the sequencing process, so as to improve sequencing quality, reduce costs, and enhance the universality of sequencing applications in multiple scenarios.
[0006] Specifically, this application provides the following technical solution:
[0007] In a first aspect, this application proposes a sequencing method. According to an embodiment of this application, the method includes: Step 1: incubating the nucleic acid to be tested in the presence of a nucleotide carrying a modifying group and DNA polymerase, and performing a base extension reaction; wherein the modifying group is selected from at least one of the following: a fluorescent group, an affinity group, or a reversible blocking group; Step 2: performing signal generation processing on the extension reaction product; Step 3: detecting the signal generated after processing and its intensity, determining the type of the nucleotide added to the extension reaction product, thereby determining the type of nucleotide at the extension reaction position of the nucleic acid to be tested, and determining the concentration of the nucleotide carrying the modifying group and / or DNA polymerase required for the next round of extension reaction based on the signal intensity.
[0008] In some examples of this application, the aforementioned method enhances the flexibility of the sequencing process, improves overall sequencing quality, and reduces sequencing costs by dynamically adjusting the concentration of nucleotides and / or DNA polymerases carrying modifying groups in each round of reaction. Furthermore, it can achieve optimal sequencing performance in different sequencing application scenarios (such as whole-genome sequencing, exome sequencing, single-cell sequencing, etc.).
[0009] In some examples of this application, the above method may also include at least one of the following additional technical features:
[0010] Steps 1-3 described above constitute a process for identifying the type of nucleotide added. In practical applications, the nucleic acid to be tested is composed of multiple nucleotides linked together. Therefore, after step 3, the process further includes: Step 4: Removing the modifying groups carried by the nucleotides added to the extension reaction product. Removing the modifying groups regenerates the 3' end of the DNA strand, allowing for the addition of new nucleotides in the next round of extension reactions.
[0011] Following step 4 above, the process also includes: Step 5: Repeating steps 1 to 4 to determine the nucleic acid sequence to be tested.
[0012] In some examples of this application, in step 3 above, the concentration of the nucleotides and / or DNA polymerase carrying the modified groups required for the next extension reaction may be the same as or different from the concentration of the nucleotides and / or DNA polymerase carrying the modified groups in the current extension reaction. The aforementioned "differentiation" includes increasing or decreasing the concentration of the nucleotides and / or DNA polymerase carrying the modified groups in the incubation system based on the difference between the signal intensity and a predetermined signal threshold. In some specific examples of this application, if the signal intensity of the current round is higher than a predetermined signal threshold, the concentration of the nucleotides and / or DNA polymerase carrying the modified groups required for the next extension reaction is decreased; if the signal intensity of the current round is lower than a predetermined signal threshold, the concentration of the nucleotides and / or DNA polymerase carrying the modified groups required for the next extension reaction is increased.
[0013] The aforementioned term "predetermined signal threshold" is determined based on the lowest signal value that can identify nucleotide types.
[0014] As the number of sequencing cycles increases, fluorescent scars gradually accumulate, leading to increased background noise, decreased synthesis efficiency, and reduced detection signal. To address this issue, in some examples of this application, based on the increase in the number of sequencing cycles, the concentration of the nucleotides and / or DNA polymerases carrying the modifying groups in the incubation system is increased to increase signal intensity and improve sequencing accuracy.
[0015] The aforementioned increase in the concentration of the nucleotides and / or DNA polymerases carrying the modified groups in the incubation system based on the increase in sequencing cycle number includes:
[0016] 1) With each additional cycle, the signal intensity is detected, and the concentration of nucleotides and / or DNA polymerase carrying the modification groups is dynamically adjusted in real time;
[0017] 2) Fixed sequencing cycles with dynamic adjustment of the concentrations of nucleotides carrying modification groups and / or DNA polymerases; for example, a first concentration is used for the first N cycles, a second concentration for the N+M cycles, and a third concentration after the N+M cycles, where the first, second, and third concentrations are the concentrations of the nucleotides carrying modification groups and / or DNA polymerases; N is an integer greater than 0, and M is an integer not less than 1. The aforementioned first concentration is lower than the second concentration, and the aforementioned second concentration is lower than the third concentration. Those skilled in the art will understand that the aforementioned fixed sequencing cycles and their corresponding concentrations can be set based on actual needs (such as the length of the nucleic acid sequence to be tested, the sequencing platform, etc.).
[0018] The determination of the aforementioned first, second, and third concentrations is based on the changes in signals during multiple sequencing cycles, and is obtained through calculation using linear fitting, nonlinear fitting, or exponential fitting methods.
[0019] The aforementioned method of adjusting the concentration of nucleotides and / or DNA polymerases carrying modifying groups based on signal strength or sequencing cycle number reduces background noise and accumulated errors in early reactions, thus extending the effective length of high-quality sequencing compared to traditional fixed-concentration sequencing protocols. Furthermore, using lower reagent concentrations in the initial reactions reduces sequencing costs.
[0020] Hot dNTPs are nucleotides carrying both fluorescent and reversible blocking groups; cold dNTPs are nucleotides carrying only reversible blocking groups. In high-throughput sequencing, hot dNTPs and cold dNTPs can be incubated simultaneously; alternatively, hot dNTPs can be added first, followed by cold dNTPs. It should be noted that the concentrations of hot dNTPs and cold dNTPs in the incubation system are different.
[0021] In some examples of this application, a single-stranded DNA template is amplified to obtain multiple DNA copies. Sequencing primers are hybridized to the DNA copies to obtain single-stranded DNA bound to the sequencing primers, which are then sequenced. This process includes: during incubation, co-incubating the aforementioned single-stranded DNA with hot dNTPs at concentration A and cold dNTPs at concentration B, causing the complementary strand of the single-stranded DNA to extend backward from the 5' end by a "hot dNTP or cold dNTP"; detecting the signal of the fluorescent group to identify the type of nucleotide synthesized in that cycle, and determining the concentration of hot dNTPs or cold dNTPs for the next cycle based on the signal intensity; and removing the fluorescent group and blocking group to regenerate the 3' end of the complementary strand for subsequent synthesis. The above steps are repeated to obtain the DNA sequence on the template.
[0022] In other examples of this application, a single-stranded DNA template is amplified to obtain multiple copies of DNA. Sequencing primers are hybridized to DNA copies to obtain single-stranded DNA bound to the sequencing primers. Sequencing is then performed, including: during incubation, first adding hot dNTPs of concentration A to the aforementioned single-stranded DNA, causing the complementary strand of a portion of the single-stranded DNA to extend backward from the 5' end by a "hot dNTP"; then adding cold dNTPs of concentration B to the aforementioned single-stranded DNA, causing the complementary strand of the single-stranded DNA that did not bind to the hot dNTP at that position to extend backward from the 5' end by a "cold dNTP"; or adding a mixture of hot dNTPs and cold dNTPs of concentration A to the aforementioned single-stranded DNA, causing the complementary strand of a portion of the single-stranded DNA to extend backward from the 5' end by a "hot dNTP," and if the complementary strand of the single-stranded DNA that did not bind to the hot dNTP at that position to extend backward from the 5' end by a "cold dNTP"; detecting the signal of the fluorescent group to identify the type of nucleotide synthesized in that cycle, and determining the next cycle's hot dNTP based on the signal intensity. The concentration of dNTPs or cold dNTPs is determined; the fluorescent and blocking groups are removed, allowing the 3' end of the complementary strand to regenerate for subsequent synthesis. The above steps are repeated to determine the DNA sequence on the template.
[0023] Those skilled in the art will understand that the aforementioned hot dNTPs can be selected from nucleotides carrying fluorescent groups and reversible blocking groups; or from nucleotides carrying affinity groups (such as avidin) and reversible blocking groups.
[0024] If the aforementioned hot dNTP is selected from nucleotides carrying an affinity group (such as avidin) and a reversible blocking group, then the signal detection stage includes: first, contacting a biotin-carrying catalyst (such as luciferase) with the affinity group, linking the hot dNTP to the catalyst through affinity interaction; then adding a substrate (such as a luciferase substrate); detecting the fluorescence signal generated by the catalyst catalyzing the substrate to identify the type of nucleotide synthesized in that cycle; and determining the feed concentration of hot dNTP, cold dNTP, or biotin-based catalyst (such as luciferase) for the next cycle based on the signal intensity. The remaining steps are the same as above and will not be repeated here.
[0025] In addition, to further improve sequencing quality, in some examples of this application, at least one of the following conditions may be further adjusted based on the concentration of the nucleotides and / or DNA polymerase carrying the modified groups: 1) incubation time; 2) incubation temperature.
[0026] Secondly, this application proposes a high-throughput sequencing system. According to an embodiment of this application, the system includes: a sequencing chip, a reagent fluid system, a signal detection system, and a control system; wherein, the reagent fluid system comprises multiple reagent storage containers storing different types of sequencing reagents and buffer solutions; the control system controls the supply of various sequencing reagents and buffer solutions during the sequencing process; the control system supplies sequencing reagents of different concentrations to the sequencing chip based on the sequencing reagent concentration information provided by the detection system, or the control system supplies sequencing reagents of different concentrations to the sequencing chip based on a preset concentration calculation formula; wherein, the sequencing reagents are selected from at least one of nucleotides carrying modifying groups, polymerases, or affinity reagents (e.g., antibodies, aptamers, etc.); wherein, the provision of sequencing reagents of different concentrations is achieved by the control system supplying the sequencing chip with different volumes of sequencing reagent stock solution and different volumes of buffer solution, resulting in sequencing reagents of different concentrations.
[0027] In some examples of this application, the aforementioned system improves sequencing accuracy and efficiency by dynamically adjusting reagent concentrations according to actual reaction needs, and also reduces reagent waste in early cycles, thereby effectively reducing sequencing costs. Furthermore, this system is adaptable to diverse applications, flexibly adjusting reagents and reaction conditions according to different sequencing requirements. It is suitable for various scenarios such as whole-genome sequencing, whole-exome sequencing, or single-cell sequencing, enhancing its versatility and flexibility.
[0028] In some examples of this application, the aforementioned system may also include at least one of the following additional technical features:
[0029] The sequencing chip in the aforementioned sequencing system provides an incubation reaction carrier for the nucleic acid to be tested, the sequencing reagents, and the buffer. In this incubation reaction carrier, the four nucleotides carrying modifying groups are added simultaneously or not simultaneously. In some examples of this application, the aforementioned modifying groups are selected from at least one of the following: fluorescent groups, affinity groups, or reversible blocking groups. The aforementioned four nucleotides carrying modifying groups include hot dNTPs and cold dNTPs; wherein, hot dNTPs are nucleotides carrying both fluorescent groups and reversible blocking groups; cold dNTPs are nucleotides carrying only reversible blocking groups.
[0030] In some examples of this application, the nucleic acid to be tested is immobilized on the surface of the sequencing chip. The aforementioned sequencing chip is a closed flow cell chip or an open chip. Among them, the aforementioned open chip is suitable for reagent delivery by printing, spraying, coating or spin coating.
[0031] In some examples of this application, the signal detection system is used for sequencing signal detection.
[0032] In some examples of this application, the signal detection system is used to determine the concentration information of the sequencing reagents for the next round. The concentration of the nucleotides carrying the modifying group and / or DNA polymerase or other sequencing reagents required for the next round of extension reaction may be the same as or different from the concentration in the previous round. The aforementioned "different" includes: changing the concentration of the nucleotides carrying the modifying group and / or DNA polymerase or other sequencing reagents in the incubation system based on the difference between the signal intensity and a predetermined signal threshold. In some specific examples of this application, if the signal intensity of the current round is higher than the predetermined signal threshold, the concentration of the nucleotides carrying the modifying group and / or DNA polymerase or other sequencing reagents required for the next round of extension reaction is decreased; if the signal intensity of the current round is lower than the predetermined signal threshold, the concentration of the nucleotides carrying the modifying group and / or DNA polymerase or other sequencing reagents required for the next round of extension reaction is increased.
[0033] The aforementioned term "predetermined signal threshold" is determined based on the lowest signal value that can identify nucleotides.
[0034] As the number of sequencing cycles increases, fluorescent scars gradually accumulate, leading to increased background noise, decreased synthesis efficiency, and reduced detection signal. To address this issue, in some examples of this application, based on the increase in the number of sequencing cycles, the concentration of the nucleotides and / or DNA polymerases or other sequencing reagents carrying the modifying groups in the incubation system is increased to increase signal intensity and improve sequencing accuracy.
[0035] In some examples of this application, the control system includes: based on the concentration of the nucleotide carrying the modifying group and / or DNA polymerase or other sequencing reagents, further adjusting at least one of the following conditions: 1) incubation time; 2) incubation temperature. Optimizing the incubation conditions improves sequencing quality.
[0036] Thirdly, this application proposes a method for dynamically adjusting the concentration of sequencing reagents. According to an embodiment of this application, the method includes: a) incubating the nucleic acid to be tested in the presence of a nucleotide carrying a modifying group and a DNA polymerase, and performing a base extension reaction; wherein the modifying group is selected from at least one of the following: a fluorescent group, an affinity group, or a reversible blocking group; b) detecting the signal generated by the extension reaction product and its intensity; c) determining the nucleotide carrying the modifying group and / or the DNA polymerase required for the next round of extension reaction based on the signal intensity and / or the number of sequencing cycles.
[0037] In some examples of this application, the aforementioned method can dynamically optimize the nucleotide concentration required for the next round of extension reaction based on actual reaction conditions, thereby improving the accuracy and reliability of sequencing. Furthermore, this method allows for flexible adjustment of nucleotide concentrations according to the requirements of different sequencing cycles, particularly avoiding unnecessary reagent waste in early cycles, thus reducing sequencing costs. This dynamic adjustment mechanism not only enhances adaptability to different samples and reaction conditions but also promotes efficient sequencing reactions, making the overall sequencing process simpler and more efficient.
[0038] In some examples of this application, the foregoing method may further include at least one of the following additional technical features:
[0039] In some examples of this application, determining the required concentration of the modified nucleotide and / or DNA polymerase for the next round of extension reaction based on signal intensity includes: determining the required concentration of the modified nucleotide and / or DNA polymerase or other sequencing reagents for the next round of extension reaction based on the difference between the signal intensity and a predetermined signal threshold. The predetermined signal threshold is determined based on the lowest signal value that can recognize the nucleotide.
[0040] In some examples of this application, if the signal intensity is higher than a predetermined signal threshold, the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is reduced; if the signal intensity is lower than the predetermined signal threshold, the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is increased.
[0041] In some examples of this application, if the signal intensity is X, the predetermined signal threshold is Y, and X is greater than Y, then the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is reduced to Y / X times the original concentration. For example, if the current sequencing signal intensity is 2 and the predetermined signal threshold is 1, then the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is reduced to 1 / 2 of the original concentration.
[0042] In some examples of this application, if the signal intensity is X, the predetermined signal threshold is Y, and X is less than Y, then the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is increased to Y / X times the original concentration. For example, if the current sequencing signal intensity is 1 and the predetermined signal threshold is 3, then the concentration of nucleotides and / or DNA polymerase carrying the modified group required for the next round of extension reaction is increased to 3 times the original concentration.
[0043] In some examples of this application, determining the required concentration of nucleotides and / or DNA polymerase carrying modified groups for the next extension reaction based on the sequencing cycle number includes: proportionally adjusting the required concentration of nucleotides and / or DNA polymerase carrying modified groups for the next extension reaction based on the change in signal intensity relative to a predetermined signal threshold in the previous N extension reactions, where N is an integer not less than 1. Generally, signal intensity gradually decreases with increasing sequencing cycle number. The required concentration of nucleotides and / or DNA polymerase carrying modified groups for the next extension reaction is increased accordingly based on the decrease ratio of the signal value in the previous N rounds relative to the predetermined signal threshold. However, real-time adjustment of the sequencing reagent concentration for the next round may lead to prolonged sequencing time and excessive memory consumption, increasing sequencing costs.
[0044] Therefore, in some preferred embodiments of this application, determining the required concentration of nucleotides and / or DNA polymerase carrying the modified group for the next extension reaction based on the sequencing cycle number includes: proportionally adjusting the required concentration of nucleotides and / or DNA polymerase carrying the modified group for the next extension reaction based on a predetermined linear or exponential concentration variation formula. Generally, the signal intensity gradually decreases as the sequencing cycle number increases. Based on the attenuation ratio of the signal value in the first N rounds relative to a predetermined signal threshold, the required concentration of nucleotides and / or DNA polymerase carrying the modified group for the next extension reaction is increased accordingly.
[0045] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description
[0046] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:
[0047] Figure 1 is a schematic diagram of the input conditions for the concentration change of the test case provided in the embodiment of this application;
[0048] Figure 2 is a schematic diagram comparing the signal value results of the test examples provided in the embodiments of this application;
[0049] Figure 3 is a schematic diagram comparing the test case Q30 results provided in the embodiments of this application. Detailed Implementation
[0050] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.
[0051] In this application, unless otherwise stated, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.
[0052] The present application is described below with reference to examples. It should be noted that these embodiments are merely descriptive and do not limit the present application in any way. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.
[0053] Example 1: The impact of different sequencing protocols on sequencing performance
[0054] The general conditions for all test cases are as follows:
[0055] Sequencing workflow: Throughout the sequencing process, the incubation order of hot dNTPs and cold dNTPs is as follows: hot dNTPs are incubated first, followed by cold dNTPs;
[0056] Incubation time: The default incubation time uses a fixed incubation schedule;
[0057] Incubation temperature: The same temperature conditions were used for all incubation steps.
[0058] Test Example 1: Sequencing at a Fixed Concentration in the Control Group
[0059] Throughout the sequencing process, the concentrations of hot dNTPs, cold dNTPs, and DNA polymerase in the sequencing reagents remained constant. Specifically:
[0060] Hot reagent: dNTP concentration is fixed at A1, DNA polymerase concentration is fixed at A2;
[0061] Cold reagent: dNTP concentration is fixed at B1, DNA polymerase concentration is fixed at B2.
[0062] For the measured verification data of Test Example 1, the concentration change scheme is as follows:
[0063] Hot reagent: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%;
[0064] Cold reagent: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%.
[0065] Test Example 2: SE and PE were tested using linear concentration increments.
[0066] Independent concentration settings: During sequencing, the concentration strategies for SE (single-end read) and PE (two-end read) are independent of each other.
[0067] For the measured verification data of Test Example 2, the concentration change scheme is as follows:
[0068] Hot reagent:
[0069] SE chain: dNTP concentration was linearly increased from 50% to 150%; DNA polymerase concentration remained constant at 100%.
[0070] PE chain: dNTP concentration increased linearly from 50% to 150%; DNA polymerase concentration remained constant at 100%.
[0071] Cold reagent:
[0072] SE strand: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%.
[0073] PE chain: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%.
[0074] Test Example 3: SE and PE were tested using exponentially increasing concentrations.
[0075] Independent concentration settings:
[0076] During sequencing, the concentration strategies for SE (single-end read) and PE (two-end read) are independent of each other.
[0077] For the measured verification data of Test Example 3, the concentration change scheme is as follows:
[0078] Hot reagent:
[0079] SE chain: dNTP concentration increased exponentially from approximately 60% to approximately 150%; DNA polymerase concentration remained constant at 100%.
[0080] PE chain: dNTP concentration increased exponentially from approximately 60% to approximately 150%; DNA polymerase concentration remained constant at 100%.
[0081] Cold reagent:
[0082] SE strand: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%.
[0083] PE chain: dNTP concentration is fixed at 100%, DNA polymerase concentration is fixed at 100%.
[0084] Test Example 4: Concentration Adjustment Based on Real-Time Signal
[0085] Signal adaptive adjustment:
[0086] During the sequencing process, the concentrations of dNTPs and DNA polymerase in subsequent sequencing processes are adaptively adjusted based on parameters such as signal intensity and background noise from the previous sequencing results to optimize signal quality.
[0087] Adaptive adjustment scheme:
[0088] If the ideal signal value for a single strand is 10, and the measured signal for a certain cycle is 8, then in the next cycle, the concentration of dNTPs and DNA polymerase should be increased by 0.2 times (i.e., 120% concentration) to enhance the signal.
[0089] If the ideal signal value for strand 1 is 10, and the measured signal for a certain cycle is 12, then in the next cycle, reduce the concentration of dNTPs and DNA polymerase by 0.2 times (i.e., 80% concentration) to avoid signal overexposure.
[0090] Results analysis:
[0091] The changes in Hot dNTP concentration in the test cases are shown in Figure 1. The signal values and Q30 comparison results for each test case are shown in Figures 2-3.
[0092] As shown in Figure 2, for the SE or PE chain, since Test Example 2 / 3 used a gradually increasing concentration scheme, the initial concentration was low, resulting in a low signal in the early stage; however, since Test Example 2 / 3 used a linear / exponential concentration increasing scheme, the signal value decreased more slowly than that of Test Example 1.
[0093] As shown in Figure 3, since the PE / SE initial template (DNB or DNA clusters, etc.) is relatively fresh, reducing the Hot dNTP concentration has little effect on the initial Q30. Even though test examples 2 / 3 used a variable concentration scheme with a lower initial concentration, the effect on the initial Q30 was relatively small, and the initial Q30 was not significantly different from that of test example 1.
[0094] As shown in Figure 3, Test Examples 2 / 3 used an independent linear / exponential PE / SE chain synthesis scheme, employing a lower concentration of Hot dNTPs in the early stages of SE / PE synthesis, which reduced the accumulation of fluorescent scars. As the SE / PE cycle number increased, the Hot dNTP concentration gradually increased, mitigating the signal decline that led to the Q30 decrease. Combined with the reduction in scars and the gradual enhancement of the signal, the Q30 decline rate in Test Examples 2 / 3 was lower than that in Test Example 1.
[0095] As shown in Figures 1-3, the variable concentration sequencing schemes in Test Examples 2 / 3 outperformed the fixed concentration scheme in Test Example 1 in some metrics and stages. Compared with sequencing schemes with fixed reagent concentrations, sequencing with dynamically adjusted reagent concentrations provides a more flexible sequencing method. It allows for adaptive adjustments to different concentration methods for different libraries and applications, unlocking greater potential for optimizing sequencing quality and cost.
[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.
[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A sequencing method, characterized in that, include: Step 1: The nucleic acid to be tested is incubated and subjected to base extension reaction in the presence of nucleotides carrying modifying groups and DNA polymerase; wherein the modifying group is selected from at least one of the following: fluorescent group, affinity group or reversible blocking group; Step 2: Perform signal generation processing on the extended reaction products; Step 3: Detect the signal and its intensity generated after processing, determine the type of nucleotide added to the extension reaction product, thereby determining the type of nucleotide at the extension reaction position of the nucleic acid to be tested, and determine the concentration of nucleotides carrying the modification group and / or DNA polymerase required for the next round of extension reaction based on the signal intensity and / or sequencing cycle number.
2. The method according to claim 1, characterized in that, Further includes: Step 4: Remove the modifying groups carried by the added nucleotides in the extended reaction product; Optionally, step 5: repeat steps 1 to 4 to determine the nucleic acid sequence to be tested.
3. The method according to claim 1, characterized in that, The concentration of nucleotides and / or DNA polymerases carrying the modifying groups required for the next round of extension reaction may be the same as or different from the concentration of nucleotides and / or DNA polymerases carrying the modifying groups in the current extension reaction.
4. The method according to claim 3, characterized in that, Further includes: Based on the concentration of the nucleotide and / or DNA polymerase carrying the modifying group, at least one of the following conditions may be further adjusted: 1) Incubation treatment time; 2) Incubation temperature.
5. The method according to claim 3 or 4, characterized in that, Based on the increase in sequencing cycle number, the concentration of the nucleotides and / or DNA polymerases carrying the modification groups in the incubation treatment system is increased.
6. The method according to claim 3 or 4, characterized in that, Based on the difference between the signal intensity and the predetermined signal threshold, the concentration of the nucleotides and / or DNA polymerases carrying the modified groups in the incubation treatment system is changed.
7. The method according to claim 1, characterized in that, In the incubation process, the four nucleotides carrying the modifying groups are added simultaneously or not simultaneously.
8. A high-throughput sequencing system, characterized in that, include: Sequencing chip, reagent fluid system, signal detection system, and control system; in, The reagent fluid system includes multiple reagent storage containers for storing different types of sequencing reagents and buffer solutions; The control system controls the supply of various sequencing reagents and buffers during the sequencing process. The control system supplies sequencing reagents of different concentrations to the sequencing chip based on the sequencing reagent concentration information provided by the detection system, or the control system supplies sequencing reagents of different concentrations to the sequencing chip based on a preset concentration calculation formula. The sequencing reagent is selected from at least one of nucleotides carrying modifying groups, polymerases, or affinity reagents; The provision of sequencing reagents of different concentrations is achieved by the control system supplying the sequencing chip with different volumes of sequencing reagent stock solution and different volumes of buffer solution, resulting in sequencing reagents of different concentrations.
9. The system according to claim 8, characterized in that, The sequencing chip provides an incubation reaction carrier for the nucleic acid to be tested, the sequencing reagents, and the buffer solution; Optionally, the four nucleotides carrying the modifying groups are added simultaneously or not simultaneously to the incubation reaction carrier; Optionally, the modifying group is selected from at least one of the following: a fluorescent group, an affinity group, or a reversible blocking group.
10. The system according to claim 9, characterized in that, The nucleic acid to be tested is immobilized on the surface of the sequencing chip; Optionally, the sequencing chip is a closed flow cell chip or an open chip.
11. The system according to claim 8, characterized in that, The signal detection system is used for sequencing signal detection; Optionally, the signal detection system is used to determine the concentration information of the sequencing reagents for the next round.
12. The system according to claim 11, characterized in that, The concentration of the nucleotides and / or DNA polymerase carrying the modifying groups required for the next round of extension reaction can be the same as or different from the concentration in the previous round.
13. The system according to claim 12, characterized in that, Based on the difference between the signal intensity and a predetermined signal threshold, the concentrations of the nucleotides and / or DNA polymerases carrying the modified groups in the incubation system are varied; and / or Based on the increase in sequencing cycle number, the concentration of the nucleotides and / or DNA polymerases carrying the modification groups in the incubation treatment system is increased.
14. The system according to claim 8, characterized in that, The control system includes: based on the concentration of the nucleotide and / or DNA polymerase carrying the modifying group, further adjusting at least one of the following conditions: 1) Incubation treatment time; 2) Incubation temperature.
15. A method for dynamically adjusting the concentration of sequencing reagents, characterized in that, include: a) The nucleic acid to be tested is incubated and subjected to base extension reaction in the presence of nucleotides carrying modifying groups and DNA polymerase; wherein the modifying group is selected from at least one of the following: fluorescent group, affinity group or reversible blocking group; b) Detect the signal and intensity of the extended reaction products; c) Determine the concentration of nucleotides and / or DNA polymerases carrying the modification groups required for the next round of extension reaction based on signal intensity and / or sequencing cycle number.
16. The method according to claim 15, characterized in that, Based on signal intensity, determine the required concentrations of nucleotides carrying modified groups and / or DNA polymerase for the next round of extension reactions, including: Based on the difference between the signal intensity and a predetermined signal threshold, the concentration of nucleotides and / or DNA polymerases carrying modifying groups or the concentration of other sequencing reagents required for the next round of extension reaction are determined.
17. The method according to claim 15 or 16, characterized in that, If the signal intensity is higher than a predetermined signal threshold, reduce the concentration of nucleotides and / or DNA polymerase carrying the modified groups required for the next round of extension reaction; If the signal intensity is lower than a predetermined signal threshold, the concentration of nucleotides and / or DNA polymerase carrying the modifying group required for the next round of extension reaction is increased.
18. The method according to claim 17, characterized in that, If the signal intensity is X, the predetermined signal threshold is Y, and X is greater than Y, then the concentration of the nucleotide and / or DNA polymerase carrying the modified group required for the next round of extension reaction is reduced to Y / X times the original concentration.
19. The method according to claim 17, characterized in that, If the signal intensity is X, the predetermined signal threshold is Y, and X is less than Y, then the concentration of the nucleotide and / or DNA polymerase carrying the modified group required for the next round of extension reaction is increased to Y / X times the original concentration.
20. The method according to claim 15, characterized in that, Based on the sequencing cycle number, determine the concentration of nucleotides and / or DNA polymerase carrying the modified groups required for the next round of extension reaction, including: adjusting the concentration of nucleotides and / or DNA polymerase carrying the modified groups required for the next round of extension reaction proportionally based on a predetermined linear or exponential concentration change formula.