Method for determining reaction time in sequencing and sequencing method and system

Abstract

The invention relates to a sequencing method and a sequencing system, and the sequencing method comprises the following steps: (1) reacting target nucleic acid fixed on the surface of a chip with a polymerization reagent, and doping nucleotide or nucleotide analogues to obtain a reaction product; (2) detecting a fluorescence signal; (3) reacting the reaction product with a regeneration reagent to obtain a product which can be subjected to the next polymerization reaction; (4) repeating the step (1) to the step (3), and so on, and carrying out multiple cycles to finally obtain sequencing data; wherein the reaction time in at least one of the step (1) and the step (3) is calculated according to a predetermined cycle number-reaction time relationship.

Description

Method for determining reaction time in sequencing, sequencing method and system Technical Field

[0001] The present disclosure relates to the biological field. Specifically, the present disclosure relates to a method for determining reaction time in sequencing, and a sequencing method and system. Background Art

[0002] Second-generation sequencing (NGS) is currently the most widely used sequencing technology due to its low cost, fast sequencing speed, and high throughput. NGS introduces reversible blocking groups and carries fluorescent markers on bases, enhancing the intensity of the fluorescent signal to read the DNA sequence. However, this technology has certain limitations. As the read length increases, the fluorescent signal decreases, resulting in a decrease in base sequencing quality and limiting the read length of NGS. Therefore, long-read NGS sequencing is a significant challenge, and obtaining accurate long-read sequences is extremely difficult.

[0003] During the sequencing process, under the action of polymerase, complementary paired nucleotides will bind to the DNA chain. Because the nucleotides carry reversible blocking groups, only one nucleotide will bind in each cycle. The DNA sequence is read out after scanning the chip. After this cycle is completed, chemical reagents are added to cut off the reversible blocking group and the fluorescent group for regeneration and the next cycle is carried out.

[0004] The binding of nucleotides to the DNA template chain under the action of polymerase requires a certain reaction temperature and reaction time. At the same time, the reaction process of reversibly blocking groups and fluorescent groups cutting off also requires a certain reaction temperature and reaction time. Different reaction times may affect the effects of polymerization and regeneration (excision). As the sequencing read length increases, the efficiency of the reaction gradually decreases due to the influence of chemical reagents and the structure of the DNA template chain.

[0005] Therefore, how to determine the reaction time during sequencing remains to be studied.

[0006] Summary of the Invention

[0007] The present disclosure aims to solve the technical problems existing in the prior art at least to a certain extent.

[0008] It should be noted that the present disclosure is completed based on the following findings of the applicant:

[0009] Both polymerization and regeneration reactions require a certain reaction time. The existing technology uses the same polymerization reaction time and the same regeneration reaction time from the start cycle to the end cycle, and for double-end sequencing, the same reaction time is also used for the first and second chains.

[0010] As the sequencing read length increases, the reaction reagents affect the structure of the DNA template chain, photodamage, and reduction of enzyme activity, resulting in reduced efficiency of polymerization and regeneration. Using the same reaction time will lead to a waste of sequencing front-end time, because when sequencing short read lengths, the reaction can be completed in a shorter reaction time, but using the reaction time of the sequencing back-end may result in a waste of time when sequencing short read lengths, and at the sequencing back-end (such as starting from the 150th cycle), the reaction efficiency decreases, and still using the short cycle reaction time will lead to insufficient reaction and reduced sequencing quality. In the existing technology, when the total time is the same, the unreasonable allocation of reaction time leads to insufficient sequencing reaction and poor sequencing quality results.

[0011] If the sequencing reaction time is blindly increased in the later stages of sequencing in order to obtain better results, although better quality data can sometimes be obtained with short read lengths, in the long run, sequencing with long read lengths will bring certain disadvantages. Due to the excessive increase in reaction time, the nucleic acid sequence is kept at high temperature for a long time, which will cause irreversible damage to the nucleic acid sequence, making it unable to maintain its unique structural characteristics to complete sequencing, resulting in poor sequencing quality.

[0012] Similarly, if the same reaction temperature is used during the sequencing process, it is easy for the reaction temperature to be low and cause insufficient reaction, or for the reaction temperature to be high and cause damage to the nucleic acid sequence, thus affecting the sequencing quality.

[0013] Theoretically, the longer the DNA template strand reacts at high temperature, the greater the damage to the nucleic acid sequence; increasing the cumulative reaction time or temperature increases the damage to the nucleic acid sequence. For paired-end sequencing, the reaction time or temperature of one strand will affect the results of one strand, the results of the second strand, and the overall result; the reaction time or temperature of the second strand will affect the results of the second strand and the overall result. For single-end sequencing, the reaction time or temperature will directly affect the quality of the sequencing result. Poorly allocated reaction time or temperature will lead to wasted front-end cycle reaction time, insufficient back-end cycle reaction, long reaction time, low accuracy, and increased sequencing time.

[0014] In view of this, the applicant adopts different reaction times or reaction temperatures for different cycles, constructs a cycle number-reaction time relationship and a cycle number-reaction temperature relationship, and rationally allocates the reaction time and reaction temperature of different cycles based on the pre-constructed relationship, so that the reaction of each cycle is more complete, while reducing the damage to the nucleic acid sequence. Thus, the present disclosure solves the problems caused by too long or too short reaction time, too high or too low reaction temperature, can better balance the reaction time, is suitable for sequencing with long reads, and protects the nucleic acid sequence from high temperature damage while ensuring a short reaction time in the early stage of sequencing. At the same time, the sequencing quality will not be greatly reduced; it also leaves sufficient room for the later stage of sequencing, guarantees the overall sequencing level, and solves the impact of the early stage of sequencing on the later stage of sequencing and the overall sequencing.

[0015] To this end, in one aspect of the present disclosure, a sequencing method is provided. According to an embodiment of the present disclosure, the sequencing method comprises: (1) reacting a target nucleic acid immobilized on a chip surface with a polymerization reagent to incorporate nucleotides or nucleotide analogs to obtain a reaction product; (2) detecting a fluorescent signal; (3) reacting the reaction product with a regeneration reagent to obtain a product that can undergo the next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles to ultimately obtain sequencing data; wherein the reaction time in at least one of steps (1) and (3) is calculated based on a predetermined cycle number-reaction time relationship.

[0016] According to the method of the embodiment of the present disclosure, different reaction times are used for different cycles, and a cycle number-reaction time relationship is constructed. Based on the pre-constructed relationship, the reaction times of different cycles are reasonably allocated, so that the reaction of each cycle is more sufficient, while reducing the damage to nucleic acids caused by long-term high temperature during the reaction, improving sequencing quality, and facilitating long-read sequencing.

[0017] According to one embodiment of the present disclosure, the above sequencing method may also have the following additional technical features:

[0018] According to an embodiment of the present disclosure, the total number of cycles is divided into N cycle segments, the reaction time of each cycle in each cycle segment is the same, and the reaction time between each cycle segment is different; N is an integer greater than 1.

[0019] According to an embodiment of the present disclosure, the reaction time of each cycle segment increases as the number of cycle segments increases.

[0020] According to an embodiment of the present disclosure, as the number of cycle segments increases, the reaction time between the cycle segments increases in an exponential function or linear function distribution manner.

[0021] According to an embodiment of the present disclosure, the linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.

[0022] According to one embodiment of the present disclosure, the cycle number-reaction time relationship is selected from the following formula: reaction time (s) = A + (B-A) / N×(C-1); wherein A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and N represents the total sequencing cycle number; wherein the reaction time is the rounded result of the formula.

[0023] According to one embodiment of the present disclosure, a method for determining the cycle number-reaction time relationship includes: performing steps (1) to (4) under the condition that the reaction time of each cycle is the same, and obtaining a normalized curve based on the obtained sequencing error rate; multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve using the obtained normalized value and the cycle number, wherein the coefficient P is the expected increase in reaction time for the last cycle; and adding the reaction time required for the first cycle to the exponential formula corresponding to the exponential curve to obtain the cycle number-reaction time relationship.

[0024] According to one embodiment of the present disclosure, in each cycle, the reaction includes immersing the chip in a reaction container containing a polymerization reagent or a regeneration reagent. After the reaction is completed, the sequencing chip is transferred to another reaction container; as the number of cycles increases, the number of immersions in each reaction container increases, and the time of each immersion is extended.

[0025] According to one embodiment of the present disclosure, the time for each immersion is determined according to the following formula: immersion time (s) = 50 + 10 (X-1) / (Y-1), where X is the number of immersions of the same reaction container, and Y is the total number of immersions of the same reaction container.

[0026] According to an embodiment of the present disclosure, the temperature of the reaction in at least one of the steps (1) and (3) is calculated based on a predetermined cycle number-reaction temperature relationship. The total number of cycles of the cycle is divided into N cycle segments, the reaction temperature of each cycle within each cycle segment is the same, and the reaction temperature between each cycle segment is different; N is an integer greater than 1; the reaction temperature of each cycle segment increases with the increase in the number of cycle segments; as the number of cycle segments increases, the reaction temperature between each cycle segment increases in an exponential function or linear function distribution manner; the linear function distribution manner is selected from an arithmetic progression or a geometric progression distribution manner.

[0027] According to one embodiment of the present disclosure, after multiple cycles, a DNA polymerase having strand displacement activity is used to perform multiple displacement amplification reactions on the target nucleic acid fixed on the chip, which can also be referred to as one strand, to obtain a complementary strand, which can also be referred to as two strands; the two strands are subjected to steps (1) to (4); wherein the reaction time and / or reaction temperature for each reaction of the one strand is different from the corresponding reaction time and / or reaction temperature for each reaction of the two strands.

[0028] In another aspect of the present disclosure, a sequencing system is provided. According to one embodiment of the present disclosure, the sequencing system includes: a chip; a sequencing device, the sequencing device being used to sequence a target nucleic acid fixed on the surface of the chip; one or more processors, the one or more processors being configured to execute: (1) reacting the target nucleic acid fixed on the surface of the chip with a polymerization reagent, incorporating nucleotides or nucleotide analogs, and obtaining a reaction product; (2) detecting a fluorescent signal; (3) reacting the reaction product with a regeneration reagent to obtain a product that can undergo a next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles, and ultimately obtaining sequencing data; wherein the reaction time in at least one of steps (1) and (3) is calculated based on a predetermined cycle number-reaction time relationship.

[0029] According to one embodiment of the present disclosure, the one or more processors are configured to execute: dividing the total number of cycles of the loop into N cycle segments, the reaction time of each cycle in each cycle segment is the same, and the reaction time between each cycle segment is different; N is an integer greater than 1; as the number of cycle segments increases, the reaction time between each cycle segment increases in an exponential function or linear function distribution manner.

[0030] According to one embodiment of the present disclosure, the one or more processors are configured to determine the reaction time according to the following formula: reaction time (s) = A + (B-A) / D × (C-1); wherein A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total number of sequencing cycles; wherein the reaction time is the rounded result of the formula; or the one or more processors are configured to execute: obtaining a normalized curve based on the sequencing error rate obtained by sequencing under the condition that the reaction time of each cycle is the same; multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve with the obtained normalized value and the cycle number, wherein the coefficient P is the expected increase in reaction time for the last cycle; adding the reaction time required for the first cycle to the exponential formula corresponding to the exponential curve to obtain the cycle number-reaction time relationship.

[0031] According to one embodiment of the present disclosure, the one or more processors are configured to perform the reaction in each round of cyclic reaction, including: immersing the chip in a reaction container containing a polymerization reagent or a regeneration reagent, and after the reaction is completed, transferring the chip to another reaction container; as the number of cycles increases, the number of immersions in each reaction container increases, and the time of each immersion is extended.

[0032] According to one embodiment of the present disclosure, the one or more processors are configured to determine the time of each immersion according to the following formula: immersion time (s) = 50 + 10 (X-1) / (Y-1), where X is the number of times the same reaction container is immersed, and Y is the total number of times the same reaction container is immersed.

[0033] According to an embodiment of the present disclosure, the temperature of the reaction in at least one of the steps (1) and (3) is calculated based on a predetermined cycle number-reaction temperature relationship; the one or more processors are configured to execute: dividing the total number of cycles of the cycle into N cycle segments, the reaction temperature of each cycle in each cycle segment is the same, and the reaction temperature between each cycle segment is different; N is an integer greater than 1; the reaction temperature of each cycle segment increases with the increase of the number of cycle segments; with the increase of the number of cycle segments, the reaction temperature between each cycle segment increases in an exponential function or a linear function distribution manner; the linear function distribution manner is selected from an arithmetic progression or a geometric progression distribution manner.

[0034] In yet another aspect, the present disclosure provides an electronic device. According to one embodiment of the present disclosure, the electronic device includes: a memory and one or more processors; the memory is coupled to the one or more processors, the memory is configured to store computer program code, the computer program code including computer instructions, and the one or more processors invoke the computer instructions to cause the electronic device to perform the sequencing method described above.

[0035] In another aspect of the present disclosure, a computer-readable storage medium is provided, comprising computer instructions. According to an embodiment of the present disclosure, when the computer instructions are executed on an electronic device, the electronic device executes the sequencing method as described above.

[0036] In another aspect of the present disclosure, a computer program product is provided. According to an embodiment of the present disclosure, when the computer program product is run on a computer, the computer is enabled to perform the sequencing method as described above.

[0037] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0039] FIG1 shows a schematic flow chart of a sequencing method according to one embodiment of the present disclosure;

[0040] FIG2 shows a graph of cycle number-linear incubation time according to one embodiment of the present disclosure;

[0041] FIG3 shows a graph of cycle number-exponential incubation time according to one embodiment of the present disclosure, wherein a represents the error rate curve of one strand after sequencing; b represents the curve obtained after normalization of the error rate curve; and c represents the exponential curve obtained by multiplying the normalization value by the coefficient B;

[0042] FIG4 shows a schematic diagram of a sequencing system according to an embodiment of the present disclosure;

[0043] FIG5 shows a schematic structural diagram of an electronic device according to an embodiment of the present disclosure;

[0044] FIG6 shows a graph of constant incubation and gradient incubation according to Example 1 of the present disclosure, where the horizontal axis represents the cycle number and the vertical axis represents the Q30% value of each cycle, indicating that the Q30% decreases with increasing cycle number, which can represent the quality of the sequencing results to a certain extent;

[0045] FIG7 shows an incubation time curve according to Example 2 of the present disclosure;

[0046] FIG8 shows a Q30% value analysis diagram according to Example 2 of the present disclosure, wherein the horizontal axis represents different incubation time modes, and the vertical axis represents the Q30% index;

[0047] FIG9 shows an analysis chart of the Q30% value of each cycle according to Example 2 of the present disclosure, where the horizontal axis represents the cycle number and the vertical axis represents the Q30% value of each cycle, indicating that the Q30% decreases with increasing cycle number;

[0048] FIG10 shows an analysis chart of the Q30% value of each cycle according to Example 3 of the present disclosure, where the horizontal axis represents the cycle number and the vertical axis represents the Q30% value of each cycle, indicating that the Q30% decreases with increasing cycle number, which can represent the quality of the sequencing results to a certain extent;

[0049] Figure 11 shows a comparative analysis of the effects of constant reaction temperature and gradient reaction temperature on the degradation of sequencing quality according to Example 4 of the present disclosure. The horizontal axis is the number of cycles, and the vertical axis is the Q30% value of each cycle, indicating that Q30% decreases with increasing cycle number, which can represent the quality of the sequencing results to a certain extent. DETAILED DESCRIPTION

[0050] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0051] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0052] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0053] In this document, the term "including" is an open expression, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0054] The present disclosure provides a sequencing method, a method for determining reaction time in sequencing, a sequencing system, an electronic device, and a computer-readable storage medium, which will be described in detail below.

[0055] Sequencing methods

[0056] In one aspect, the present disclosure provides a sequencing method. According to an embodiment of the present disclosure, referring to FIG1 , the sequencing method includes: reacting a target nucleic acid with a polymerization reagent at step S100; detecting a fluorescent signal at step S200; reacting the reaction product with a regeneration reagent at step S300; and repeating steps S100 to S300 at step S400. Each of these steps will be described in detail below.

[0057] S100 reacts the target nucleic acid with the polymerization reagent

[0058] In this step, the target nucleic acid immobilized on the chip surface reacts with a polymerization reagent (also referred to as a "polymerization reaction" in this invention), incorporating nucleotides or nucleotide analogs to produce a reaction product. Under the guidance of the template, the polymerization reagent continuously adds dNTPs to the 3'-OH terminus of the primer / previously incorporated nucleotide or nucleotide analog, extending the primer and synthesizing a new complementary DNA strand.

[0059] In this disclosure, the term "polymerization reagent" refers to reagents required for polymerization reactions. This includes all reagents known in the art for polymerization reactions, such as dNTPs, DNA polymerase, and buffers. The term "polymerization" encompasses both chain synthesis, i.e., chain extension, and the leveling process, i.e., compensating for unbound dNTPs with reversible blocking groups and fluorescent groups at the 3'-OH end during chain synthesis. This leveling process allows for further binding, synchronizing all read signals.

[0060] According to an embodiment of the present disclosure, step 100 may further include: cleaning the synthesis product using a cleaning reagent. Specifically, the sequencing chip connected to the synthesis product is placed in a reaction container containing a cleaning reagent to clean the reaction reagent free on the chip.

[0061] In the present disclosure, the term "reaction vessel" refers to a reaction site where polymerization and removal of reversible blocking groups and fluorescent groups can occur during the sequencing process. There is no strict limitation on the type of specific reaction vessel. For example, it can be a reagent tank on a sequencing platform.

[0062] S200 detection of fluorescence signals

[0063] By detecting the fluorescent signal, the sequencing read information is obtained based on the signal color and intensity.

[0064] S300 reacts the reaction product with a regeneration reagent

[0065] In this step, the reaction product is reacted with a regeneration agent (also referred to herein as a "regeneration reaction") to obtain a product ready for the next round of polymerization. By reacting the reaction product with the regeneration agent, the reversible blocking group and fluorescent group carried by the reaction product are removed, thereby facilitating the next round of polymerization.

[0066] In the present disclosure, the term "blocking group" binds to the 3' hydroxyl group or other sites of deoxyribose, preventing the deoxyribose from forming a phosphodiester bond with subsequent dNTPs or forming a steric hindrance that prevents the polymerase from performing a polymerization reaction, thereby terminating chain extension. When it is reversibly removed, the 3' terminal hydroxyl structure can be restored, allowing it to form a phosphodiester bond with subsequent dNTPs, thereby completing chain extension. Or, it can be reversibly removed to restore the spatial structure of DNA, allowing it to undergo a polymerization reaction with subsequent dNTPs and polymerase, thereby completing chain extension. The blocking group can be any group used in the art to block dNTPs, typically but not limited to azido methylene, and some fluorescent groups can also serve as blocking groups. Other available blocking groups include, for example, those disclosed in international application WO2014139596A1. The present disclosure does not particularly limit the type of blocking group, and any group used in the art to block the 3' hydroxyl group of a dNTP can be used as a blocking group in the present disclosure. These blocking groups can all be reversibly detached from the dNTP.

[0067] The present disclosure does not strictly limit the type of "regeneration reagent", and all substances disclosed in the art that can remove reversible blocking groups and fluorescent groups are included in the present disclosure.

[0068] S400 repeats steps S100 to S300

[0069] In this step, steps S100 to S300 are repeated, and so on, for multiple cycles to finally obtain sequencing data.

[0070] According to one embodiment of the present disclosure, the reaction time in at least one of steps S100 and S300 is calculated based on a predetermined cycle number-reaction time relationship. In the method according to an embodiment of the present disclosure, different reaction times are used for different cycles, and a cycle number-reaction time relationship is constructed. Based on this pre-constructed relationship, the reaction times of different cycles are rationally allocated, so that the reaction of each cycle is more complete, while reducing damage to nucleic acids caused by prolonged high temperatures during the reaction, improving sequencing quality, and facilitating long-read sequencing.

[0071] According to an embodiment of the present disclosure, the total number of cycles is divided into N cycle segments, the reaction time of each cycle in each cycle segment is the same, and the reaction time between each cycle segment is different; N is an integer greater than 1.

[0072] Different cycles may use different reaction times, and the cycle may be divided into different cycle segments according to certain rules. The number of cycles within a cycle segment uses the same reaction time, and the reaction times between different cycle segments are inconsistent; or the reaction time of each cycle may be inconsistent, and the reaction time and the number of cycles are calculated according to a certain formula, that is, the total number of cycles is the same as the number of cycle segments.

[0073] According to one embodiment of the present disclosure, the reaction time of each cycle segment increases as the number of cycle segments increases. According to another embodiment of the present disclosure, as the number of cycle segments increases, the reaction time between each cycle segment increases in an arithmetic progression or a geometric progression distribution.

[0074] During the sequencing reaction, from the start cycle to the end cycle, the reaction time of the polymerization and regeneration reactions can be increased in a gradient within a certain range. The gradient increase can be performed in an arithmetic progression or a geometric progression or in other ways, as follows:

[0075] 1. First set the number of segments. You can divide the total number of cycles into different segments. The number of cycles within a segment uses the same reaction time, and the reaction time between different segments is inconsistent. The segments can be divided equally or by custom.

[0076] 1) The rules for splitting the segmentation are:

[0077] For example, if the total number of cycles is 300, the 300 cycles can be divided into X segments, and the number of cycles in each segment is Y, where Y = 300 / X. Specific examples include: one segmentation method is to divide the 300 cycles into 2 segments, each with 150 cycles; another segmentation method is to divide the 300 cycles into 5 segments, each with 60 cycles; another segmentation method is to divide the 300 cycles into 6 segments, each with 50 cycles; and so on.

[0078] 2) Custom segmentation rules are:

[0079] For example, 300 cycles can be divided into the first cycle segment of 1-150 cycles, the second cycle segment of 151-200 cycles, the third cycle segment of 201-250 cycles, the fourth cycle segment of 251-300 cycles; and so on. The number of segments and the number of cycles within a segment can be adjusted as needed, and the number of cycles in each segment does not need to be the same.

[0080] 2. After setting the segments, the loops within the same loop segment use the same reaction time, and different loop segments use different reaction times. The reaction time can be increased according to a certain arithmetic progression or geometric progression, or the reaction time of each loop segment can be customized.

[0081] 1) The increasing method of an arithmetic progression can be:

[0082] For example, if 300 cycles are divided into five segments, each consisting of 60 cycles, and the reaction time increases in an arithmetic progression from 20s to 100s, the reaction times of the five segments are 20s, 40s, 60s, 80s, and 100s, respectively. The above is just an example; the segmentation method and reaction time can be set in other similar ways.

[0083] 2) The increasing method of geometric progression can be:

[0084] For example, if 200 cycles are divided into four segments, each consisting of 50 cycles, and the reaction time increases in a geometric progression from 15s to 120s, then the reaction times for the four segments are 15s, 30s, 60s, and 120s, respectively. The above is just an example; the segmentation method and reaction time can be set in other similar ways.

[0085] 3) The custom increment method can be:

[0086] For example, if 300 cycles are divided into five segments, each consisting of 60 cycles, and the reaction time is increased in a gradient manner within the range of 20s-100s, then the reaction times of the five segments are 20s, 30s, 60s, 80s, and 100s, respectively. The above is just an example, and the segmentation method and reaction time can be other similar settings.

[0087] The segmentation mode and reaction time settings of polymerization and regeneration reactions can be different and can be edited and set separately.

[0088] The segmentation method and reaction time settings of the first and second chains can be different and can be edited and set separately.

[0089] For example, see Table 1 below:

[0090] Table 1: One way to set up a gradient reaction

[0091] According to one embodiment of the present disclosure, the relationship between the number of cycles and the reaction time is selected from the following formula: reaction time (s) = A + (B-A) / D × (C-1); wherein A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and N represents the total number of sequencing cycles; wherein the reaction time is the rounded result of the formula.

[0092] During the sequencing reaction, from the start cycle to the end cycle, the polymerization and regeneration reactions proceed in a linear manner within a certain range:

[0093] The exposure time for each current cycle is determined according to the following formula:

[0094] Reaction time (s) = A + (BA) / D × (C-1)

[0095] Where A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total sequencing cycle number. The reaction time is the rounded result of the formula.

[0096] The linear reaction time settings for polymerization and regeneration reactions can be different and can be edited and set separately.

[0097] The linear reaction time settings for the first and second chains can be different and can be edited separately.

[0098] FIG. 2 shows a reaction time setting for a linear reaction, but it does not mean that the present disclosure only has this linear reaction mode.

[0099] According to one embodiment of the present disclosure, a method for determining the relationship between cycle number and reaction time includes: performing steps (1) to (4) under the condition that the reaction time of each cycle is the same, and obtaining a normalized curve based on the obtained sequencing error rate; multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve using the obtained normalized value and the cycle number, where the coefficient P is the expected increase in reaction time for the last cycle; and adding the reaction time required for the first cycle to the exponential formula corresponding to the exponential curve to obtain the cycle number-reaction time relationship.

[0100] During the sequencing reaction, from the start cycle to the end cycle, the polymerization and regeneration reactions proceed in an exponential manner within a certain range. The exponential reaction formula can be set as follows, but is not limited to this setting method:

[0101] Sequencing is performed under fixed reaction time conditions. According to the sequencing error rate curve, the error rate of the last cycle is normalized. The normalization coefficient is Q. The corresponding error rates of all cycles are divided by the coefficient Q to obtain a normalized curve.

[0102] Furthermore, for each cycle's normalized value *P, the coefficient P is the planned increase in incubation time for the last cycle; an exponential curve is obtained by multiplying the coefficient P;

[0103] Furthermore, the formula of the exponential curve is found using Excel, MATLAB or other tools to obtain the exponential formula;

[0104] Furthermore, by adding the reaction time required for the first cycle to the exponential formula, an exponential reaction time curve is obtained.

[0105] Use Excel, MATLAB or other tools to find the formula for the exponential curve. The formula is:

[0106] S=a*K^3+b*K^2+c;

[0107] Adding the incubation time T of the first cycle to this formula, the exponential incubation formula is:

[0108] S = a*K^3+b*K^2+c. In this example, T = 50, that is, the formula for exponential incubation is: S = a*K^3+b*K^2+c+50, where K is the current cycle, S is the incubation time of the cycle, and a and b are constants.

[0109] FIG3 shows a time setting of an exponential reaction, but it does not mean that the present disclosure only has this exponential reaction mode.

[0110] The exponential reactions of the polymerization and regeneration reactions in the present disclosure may be set in different ways and may be edited and set separately.

[0111] Furthermore, the exponential reaction time settings for the first and second chains can be different and can be edited and set separately.

[0112] The sequencing methods disclosed herein can be single-end sequencing or double-end sequencing methods, including sequencing by primer extension using labeled or unlabeled nucleotides, such as sequencing-by-ligation or pyrosequencing, and can be achieved, for example, using any of the Sanger dideoxy sequencing, nanopore, or "NexGen" sequencing methods known in the art (e.g., using MGI's sequencing platform, the ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™ sequencing platform, the SOLID™ sequencing platform of LIFE TECHNOLOGIES / APPLIED BIOSYSTEMS, the SMRT™ sequencing platform of PACIFIC BIOSCIENCES, the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICS sequencing platform, the INTELLIGENT BIOSYSTEMS sequencing platform, the HELICOS sequencing platform, or any other sequencer and system known in the art). The present disclosure does not strictly limit the manner in which the polymerization reaction and regeneration reaction of steps S100 and S300 occur. Some sequencing platforms use a syringe pump to inject reaction reagents or regeneration reagents onto the sequencing chip, such as MGI's sequencing platforms: MGISEQ-200RS, MGISEQ-2000RS, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-E25, etc.; some sequencing platforms use an immersion method, that is, an immersion biochemical platform, in which the chip is immersed in a reaction vessel containing reaction reagents or regeneration reagents. After a certain reaction is completed, the chip is transferred to another reaction vessel to complete the next operation, such as the DNBSEQ-T10×4RS sequencing platform.

[0113] According to one embodiment of the present disclosure, in each cycle, the reaction includes immersing the chip in a reaction container containing a polymerization reagent or a regeneration reagent. After the reaction is completed, the sequencing chip is transferred to another reaction container; as the number of cycles increases, the number of immersions in each reaction container increases, and the time of each immersion is extended.

[0114] According to one embodiment of the present disclosure, the time for each immersion is determined according to the following formula: immersion time (s) = 50 + 10 (X-1) / (Y-1), where X is the number of immersions of the same reaction container, and Y is the total number of immersions of the same reaction container.

[0115] When operating in a reagent soaking mode on a sequencing platform, a small amount of residual liquid will be carried into a new reaction vessel (such as a reagent tank) during the chip transfer process, resulting in a decrease in the concentration of polymerization reagent or regeneration reagent in the new reaction vessel. Moreover, during the heating time, the active ingredients of the reagents in some key reagent tanks will also change. In order to allow the reaction to fully occur, it is necessary to extend the time of each soaking. When the concentration of the reagent to be reacted in the reaction vessel is too low or the active ingredient deteriorates to the point that it is not conducive to the reaction, it is necessary to replace it with a new reagent, and the soaking time can be appropriately shortened at this time. Specifically, by setting the biochemical parameters in the sequencing platform (for example: immersion biochemical platform) software, different temperatures can be set in different sequencing stages to achieve more appropriate sequencing results, and in the same round of reagents, different reaction times are set for different soaking times to achieve the best experimental results.

[0116] Specific plan:

[0117] Set the initial biochemical time and final biochemical time of the biochemical tank. For example, the initial biochemical time of biochemical tank 1 is 50s and the final biochemical time is 60s.

[0118] For example, when 6 slides are sequenced simultaneously, the number of soaking times is set to 78;

[0119] When the reagents in the biochemical tank are soaked for the Xth time, the reaction time (s) = 50 + 10 (X-1) / (78-1).

[0120] When the slide is immersed in biochemical tank 1 for the first time, the reaction time is 50 seconds. When the slide is immersed for 78 times, the reaction time is 60 seconds. The reaction time increases linearly with the increase in the number of immersions.

[0121] Similarly, the reaction temperature can also be adjusted according to the number of soaking times.

[0122] According to an embodiment of the present disclosure, the reaction temperature in at least one of step S100 and step S300 is calculated based on a predetermined cycle number-reaction temperature relationship.

[0123] According to one embodiment of the present disclosure, the total number of cycles is divided into N cycle segments, the reaction temperature of each cycle in each cycle segment is the same, and the reaction temperature between each cycle segment is different; N is an integer greater than 1; the reaction temperature of each cycle segment increases with the increase in the number of cycle segments; as the number of cycle segments increases, the reaction temperature between each cycle segment increases in an exponential function or linear function distribution manner; the linear function distribution manner is selected from an arithmetic progression or a geometric progression distribution manner.

[0124] According to one embodiment of the present disclosure, after multiple cycles, a DNA polymerase with strand displacement activity is used to perform a multiple displacement amplification reaction on the target nucleic acid immobilized on the chip to obtain complementary strands; the two strands are subjected to steps S100 to S400; wherein the reaction time and / or reaction temperature for each reaction of the first strand are different from the corresponding reaction time and / or reaction temperature for each reaction of the second strand. Specifically, the reaction time / reaction temperature for the nth cycle of the first strand is different from the reaction time / reaction temperature for the nth cycle of the second strand.

[0125] Sequencing system, electronic device, computer readable storage medium, and computer program product

[0126] In another aspect of the present disclosure, a sequencing system is provided. According to an embodiment of the present disclosure, referring to FIG4 , the sequencing system 1000 includes: a chip 100, a sequencing device 200, and one or more processors 300, wherein the one or more processors 300 are configured to execute: (1) reacting a target nucleic acid immobilized on a chip surface with a polymerization reagent to incorporate nucleotides or nucleotide analogs to obtain a reaction product; (2) detecting a fluorescent signal; (3) reacting the reaction product with a regeneration reagent to obtain a product that can undergo a next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles to ultimately obtain sequencing data; wherein the reaction time in at least one of steps (1) and (3) is calculated based on a predetermined cycle number-reaction time relationship.

[0127] Different reaction times are used for different cycles, and a cycle number-reaction time relationship is constructed. Based on this pre-constructed relationship, the reaction time of different cycles is reasonably allocated to make the reaction of each cycle more complete. At the same time, the damage to nucleic acids caused by long-term high temperature during the reaction is reduced, the sequencing quality is improved, and it is conducive to long-read sequencing.

[0128] According to an embodiment of the present disclosure, referring to FIG4 , one or more processors 300 include:

[0129] The first module 210 is used to react the target nucleic acid fixed on the chip surface with a polymerization reagent to incorporate nucleotides or nucleotide analogs to obtain a reaction product;

[0130] A second module 220, the second module 220 is used to detect fluorescence signals;

[0131] The third module 230 is used to react the reaction product with a regeneration agent to obtain a product that can be used for the next round of polymerization reaction;

[0132] The fourth module 240 is used to repeat the operations performed in the first module 210, the second module 220 and the third module 230 in sequence, and so on, for multiple cycles to finally obtain sequencing data;

[0133] The reaction time in at least one of the first module 210 and the third module 230 is calculated based on a predetermined relationship between the number of cycles and the reaction time.

[0134] According to one embodiment of the present disclosure, one or more processors 300 are configured to execute: dividing the total number of loops into N loop segments, the reaction time of each loop in each loop segment is the same, and the reaction time between each loop segment is different; N is an integer greater than 1.

[0135] According to an embodiment of the present disclosure, as the number of cycle segments increases, the reaction time between each cycle segment increases in an exponential function or linear function distribution manner.

[0136] According to one embodiment of the present disclosure, one or more processors 300 are configured to execute the determination of the reaction time according to the following formula: reaction time (s) = A + (B-A) / D × (C-1); wherein A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total sequencing cycle number; wherein the reaction time is the rounded result of the formula.

[0137] According to one embodiment of the present disclosure, one or more processors 300 are configured to perform the following steps: obtaining a normalized curve based on the sequencing error rate obtained by sequencing under conditions in which the reaction time of each cycle is the same; multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve using the obtained normalized value and the number of cycles, where the coefficient P is the expected increase in reaction time for the last cycle; and adding the reaction time required for the first cycle to the exponential formula corresponding to the exponential curve to obtain a cycle number-reaction time relationship.

[0138] According to one embodiment of the present disclosure, one or more processors 300 are configured to execute the reaction in each round of cyclic reaction, including: immersing the chip in a reaction container containing a polymerization reagent or a regeneration reagent, and after the reaction is completed, removing the chip from the reaction container; as the number of cycles increases, the number of immersions in each reaction container increases, and the time of each immersion is extended; the one or more processors are configured to execute the following formula to determine the time of each immersion: immersion time = 50 + 10 (X-1) / (Y-1), X is the number of immersions of the same reaction container, the immersion time is in seconds, and Y is the total number of immersions of the same reaction container.

[0139] According to one embodiment of the present disclosure, the temperature of the reaction in at least one of step (1) and step (3) is calculated based on a predetermined cycle number-reaction temperature relationship; one or more processors are configured to execute: dividing the total number of cycles into N cycle segments, the reaction temperature of each cycle in each cycle segment is the same, and the reaction temperature between each cycle segment is different; N is an integer greater than 1; the reaction temperature of each cycle segment increases with the increase of the number of cycle segments; with the increase of the number of cycle segments, the reaction temperature between each cycle segment increases in an exponential function or a linear function distribution manner; the linear function distribution manner is selected from an arithmetic progression or a geometric progression distribution manner.

[0140] One or more processors 300 may be general-purpose processors or dedicated processors, etc. For example, they may be baseband processors or central processing units. The baseband processor may be used to process communication protocols and communication data, and the central processing unit may be used to control communication equipment (such as base stations, baseband chips, terminal equipment, terminal equipment chips, DU or CU, etc.), execute computer programs, and process computer program data. The processor 300 may be implemented on an integrated circuit (IC), an analog IC, a radio frequency integrated circuit RFIC, a mixed signal IC, an application specific integrated circuit (ASIC), a printed circuit board (PCB), an electronic device, etc. The processor and transceiver may also be manufactured using various IC process technologies, such as complementary metal oxide semiconductor (CMOS), N-type metal oxide semiconductor (NMOS), positive channel metal oxide semiconductor (PMOS), bipolar junction transistor (BJT), bipolar CMOS (BiCMOS), silicon germanium (SiGe), gallium arsenide (GaAs), etc.

[0141] In another aspect of the present disclosure, an electronic device is provided. According to an embodiment of the present disclosure, the electronic device includes: a memory and one or more processors; the memory is coupled to the one or more processors, the memory is used to store computer program code, the computer program code including computer instructions, and the one or more processors invoke the computer instructions to cause the electronic device to perform the sequencing method described above. Specifically, the electronic device can be any intelligent terminal, including a sequencer, a tablet computer, a computing cluster, and the like.

[0142] The term "memory" as used in this disclosure refers to any computer program product, device, and / or apparatus (e.g., a disk, an optical disk, a memory, a programmable logic device (PLD)) for providing machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as machine-readable signals. The memory can be implemented in the form of a read-only memory (ROM), a static storage device, a dynamic storage device, or a random access memory (RAM). The memory can store an operating system and other application programs. When the technical solutions provided in the embodiments of this specification are implemented by software or firmware, the relevant program code is stored in the memory and is called by the processor to execute the sequencing method of the embodiments of the present application.

[0143] 5 , the electronic device 400 may include a processor 410, a memory 420, an input / output interface 430, a communication interface 440, and a bus 450. The processor 410, the memory 420, the input / output interface 430, and the communication interface 440 are connected to each other within the device via the bus 450.

[0144] The processor 410 can be implemented using a general-purpose CPU (Central Processing Unit), a microprocessor, an application-specific integrated circuit (ASIC), or one or more integrated circuits, and is used to execute relevant programs to implement the technical solutions provided in the embodiments of this specification.

[0145] The memory 420 can be implemented in the form of ROM (Read Only Memory), RAM (Random Access Memory), static storage devices, dynamic storage devices, etc. The memory 420 can store an operating system and other application programs. When the technical solutions provided in the embodiments of this specification are implemented through software or firmware, the relevant program code is stored in the memory 420 and is called and executed by the processor 410.

[0146] The input / output interface 430 is used to connect an input / output module to implement information input and output. The input / output module can be configured as a component in the device (not shown in the figure) or can be externally connected to the device to provide corresponding functions. Input devices may include a keyboard, mouse, touch screen, microphone, various sensors, etc., and output devices may include a display, speaker, vibrator, indicator light, etc.

[0147] The communication interface 440 is used to connect to a communication module (not shown) to enable communication between the device and other devices. The communication module can communicate via a wired method (such as USB, network cable, etc.) or a wireless method (such as mobile network, WiFi, Bluetooth, etc.).

[0148] The bus 450 comprises a pathway for transmitting information between the various components of the device, such as the processor 410 , the memory 420 , the input / output interface 430 , and the communication interface 440 .

[0149] It should be noted that although the above device only shows the processor 410, the memory 420, the input / output interface 430, the communication interface 440, and the bus 450, in a specific implementation, the device may also include other components necessary for normal operation. In addition, it will be understood by those skilled in the art that the above device may only include the components necessary to implement the embodiments of this specification, and does not necessarily include all the components shown in the figure.

[0150] In the above embodiments, it can be implemented in whole or in part by software, hardware, firmware or any combination thereof. When implemented using software, it can be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer programs. When the computer program is loaded and executed on a computer, the process or function according to the embodiment of the present application is generated in whole or in part. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable device. The computer program can be stored in a computer-readable storage medium or transmitted from one computer-readable storage medium to another computer-readable storage medium. For example, the computer program can be transmitted from one website, computer, server or data center to another website, computer, server or data center via wired (e.g., coaxial cable, optical fiber, digital subscriber line (DSL)) or wireless (e.g., infrared, wireless, microwave, etc.) means.

[0151] To this end, in another aspect of the present disclosure, the present disclosure proposes a computer-readable storage medium, including computer instructions. According to an embodiment of the present disclosure, when the computer instructions are run on an electronic device, the electronic device is caused to perform the sequencing method as described above. The computer-readable storage medium can be any available medium that can be accessed by a computer or a data storage device such as a server or a data center that includes one or more available media integrations. Available media can be magnetic media (e.g., floppy disks, hard disks, tapes), optical media (e.g., high-density digital video discs (DVDs)), or semiconductor media (e.g., solid-state drives (SSDs)), etc.

[0152] In another aspect of the present disclosure, a computer program product is provided. According to an embodiment of the present disclosure, when the computer program product is run on a computer, the computer is enabled to perform the sequencing method as described above.

[0153] Those skilled in the art will appreciate that all or part of the process steps in the above-described method embodiments can be implemented by a computer program instructing the relevant hardware. The program can be stored in a computer-readable storage medium, and when executed, the program can include the process steps in the above-described method embodiments. The aforementioned storage medium includes various media capable of storing program code, such as ROM or random access memory (RAM), magnetic disks, or optical disks.

[0154] It should be noted that the features and advantages described above for the sequencing method and the method for determining the reaction time in sequencing are also applicable to the sequencing system, electronic device, computer-readable storage medium and computer program product, and will not be repeated here.

[0155] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0156] In the following examples, the key equipment and key reagents used are as follows:

[0157] 1. Key equipment:

[0158] MGISEQ-2000RS sequencer, FTAT sequencer, PCR instrument, PCR eight-tube, 3.0 fluorescence quantitative instrument, a set of pipettes, a high-speed centrifuge, 200μL wide-mouth pipette tips, and an ice box.

[0159] 2. Key reagents are shown in Table 2 below:

[0160] Table 2 Required Reagents

[0161] Example 1

[0162] This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI. The reagents used are all derived from the library preparation kit and single-end sequencing kit (hereinafter referred to as the SE400 kit) used with the sequencer. The verification sample used was derived from Escherichia coli. During the verification process, the DNB preparation and loading required by the SE400 and the SE400 reagent tank were performed with reference to the "MGISEQ-2000RS High-Throughput (Rapid) Sequencing Reagent Set User Manual". This implementation case uses SE400 sequencing as an example, setting the incubation time using a gradient incubation method and changing the SE400 biochemical script required for sequencing.

[0163] This example designs two groups of incubation times for comparison:

[0164] Group 1: Maintain the overall synthesis time at 60 s, that is, maintain a constant incubation time.

[0165] Group 2: The incubation time was carried out in a gradient incubation manner, with 100 cycles as one gradient. The incubation time of each gradient was different. The specific incubation time settings are shown in Table 3.

[0166] The results are shown in Table 4 and Figure 6. The gradient incubation time set according to the method of the embodiment of the present disclosure decreased Q30% more slowly than the constant incubation time, proving that the gradient incubation time method produces better results than constant incubation.

[0167] Table 3: SE400 gradient incubation time settings

[0168] Table 4: Comparison of the effects of constant incubation time and gradient incubation time on overall sequencing quality in this example.

[0169] Note: ESR (%): Filter reads according to Q value, the proportion of reads greater than Q30.

[0170] Example 2

[0171] This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI, and uses a validation sample derived from E. coli. The DNB preparation, loading, and reagent tank configuration are common and adaptable to other MGI platforms. This implementation case uses SE150 sequencing as an example, setting the incubation time using a linear incubation method and modifying the SE150 biochemical script required for sequencing.

[0172] This example designs two groups of incubation times for comparison:

[0173] Group 1: Maintain the overall synthesis time at 20 s, the leveling time at 30 s, and the regeneration time at 30 s, that is, maintain a constant incubation time.

[0174] Group 2: The incubation time was carried out in a linear incubation manner. The incubation time was calculated according to the formula:

[0175] Reaction time (s) = A + (B-A) / D * (C-1), where A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total sequencing cycle number; the reaction time is the rounded result of the formula.

[0176] The incubation time for synthesis is 13+(43-13) / 150×(C-1)

[0177] The incubation time for filling is 22+(52-22) / 150×(C-1)

[0178] The incubation time for regeneration is 27+(57-27) / 150×(C-1)

[0179] The specific incubation time is shown in Figure 7.

[0180] The results are shown in Figures 8 and 9. The linear incubation time set according to the method of the embodiment of the present disclosure decreases more slowly than the Q30% of the constant incubation time, proving that the linear incubation time method produces better results than constant incubation.

[0181] Example 3

[0182] This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI. The reagents used are all derived from the library construction kit and paired-end sequencing kit (hereinafter referred to as the PE300 kit) used with the sequencer. The verification sample used was derived from Escherichia coli. During the verification process, the MGISEQ-2000RS High-Throughput (Rapid) Sequencing Reagent Set Instructions was referred to for the preparation and loading of DNBs required for PE300 and the preparation of the PE300 reagent tank. This implementation case takes PE300 sequencing as an example, setting the incubation time using an exponential incubation method and changing the PE300 biochemical script required for sequencing.

[0183] This example designs two groups of incubation times for comparison:

[0184] Group 1: Maintain the overall synthesis (chain synthesis) time at 60 s, the filling time at 120 s, and the regeneration time at 60 s, that is, maintain a constant incubation time.

[0185] Group 2: Referring to Figure 3, the incubation time was carried out in an exponential incubation manner. The incubation time was calculated according to the formula:

[0186] S=3*10^-6*K^3-0.0001*K^2-0.0089*K+1.3741+T

[0187] Where K is the current cycle, Y is the incubation time of the cycle, and T is the incubation time of the first cycle.

[0188] The synthetic incubation time formula is: y = 3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+19

[0189] The incubation time formula for filling level is: y=3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+60

[0190] The incubation time formula for regeneration is: y = 3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+40

[0191] The results are shown in Table 5 and Figure 10. The exponential incubation time set according to the method of the embodiment of the present disclosure decreased more slowly than the Q30% of the constant incubation time, proving that the exponential incubation time method produces better results than constant incubation.

[0192] Table 5 shows the comparison of the effects of constant incubation time and exponential incubation time on sequencing quality in this example.

[0193] Note: SplitRate (%): Split rate, the proportion of sequences with successfully removed labels in the data to the total data.

[0194] RecoverValue (AVG): This indicator is only for the PE sequencing part and reflects the recovery of the second-strand signal.

[0195] Example 4

[0196] This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI. The reagents used are all from the library preparation kit and paired-end sequencing kit that come with the sequencer. The validation sample used is from Escherichia coli. During the validation process, the DNB preparation and loading required for PE250 and the preparation of the PE250 reagent tank were performed with reference to the "MGISEQ-2000RS High-Throughput (Rapid) Sequencing Reagent Set Instructions." This implementation case uses PE250 sequencing as an example to set the gradient reaction temperature and modify the PE250 biochemical script required for sequencing.

[0197] This example designs two groups of incubation temperatures for comparison:

[0198] Group 1: Maintain the overall leveling temperature at 60°C, that is, maintain a constant reaction temperature.

[0199] Group 2: The reaction temperature was set in a gradient temperature manner. The incubation temperature of each gradient was different. The specific incubation time settings are shown in Table 6.

[0200] The results are shown in Table 7 and Figure 11. The gradient reaction temperature set according to the method of the embodiment of the present disclosure has a better recovery of the second-strand signal, a slower decrease in Q30%, and a higher overall data volume than the constant incubation temperature. This proves that the gradient temperature method produces better results than the constant temperature method.

[0201] Table 6: PE250 first chain gradient temperature settings

[0202] Table 7: Comparison of the effects of constant incubation temperature and gradient incubation temperature on overall sequencing quality in this example.

[0203] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

[0204] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in this disclosure can be achieved. This is not limited herein.

[0205] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article or device. In the absence of further limitations, an element defined by the sentence "comprises a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0206] The foregoing are merely specific embodiments of the present disclosure, intended to enable those skilled in the art to understand and implement the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is not to be limited to these embodiments, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A sequencing method, It is characterized in that include: (1) reacting the target nucleic acid fixed on the chip surface with a polymerization reagent, incorporating nucleotides or nucleotide analogs, and obtaining a reaction product; (2) detecting fluorescence signals; (3) reacting the reaction product with a regeneration agent to obtain a product that can be subjected to a next round of polymerization reaction; (4) Repeat steps (1) to (3), and so on, for multiple cycles to finally obtain sequencing data; The reaction time in at least one of step (1) and step (3) is calculated based on a predetermined cycle number-reaction time relationship.

2. The method according to claim 1, It is characterized in that Dividing the total number of cycles into N cycle segments, wherein the reaction time of each cycle in each cycle segment is the same, and the reaction time between each cycle segment is different; N is an integer greater than 1.

3. The method according to claim 2, It is characterized in that The reaction time of each of the cycle segments increases as the number of cycle segments increases; As the number of cycle segments increases, the reaction time between each cycle segment increases in an exponential function or linear function distribution manner; The linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.

4. The method according to claim 1, It is characterized in that The cycle number-reaction time relationship is selected from the following formula: Reaction time (s) = A + (BA) / D × (C-1); Wherein, A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total sequencing cycle number; the reaction time is the rounded result of the formula.

5. The method according to claim 1, It is characterized in that The method for determining the cycle number-reaction time relationship comprises: Under the condition that the reaction time of each cycle is the same, steps (1) to (4) are performed, and a normalized curve is obtained according to the obtained sequencing error rate; Multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve using the obtained normalized value and the cycle number, wherein the coefficient P is the reaction time expected to be increased in the last cycle; The reaction time required for the first cycle is added to the exponential formula corresponding to the exponential curve to obtain the cycle number-reaction time relationship.

6. The method according to claim 1, It is characterized in that In each cycle, the reaction includes immersing the chip in a reaction container storing a polymerization reagent or a regeneration reagent, and after the reaction is completed, transferring the sequencing chip to another reaction container; As the number of cycles increases, the number of immersions in each reaction vessel increases and the duration of each immersion increases.

7. The method according to claim 6, It is characterized in that The soaking time for each time is determined according to the following formula: Soaking time (s) = 50 + 10 (X-1) / (Y-1), X is the number of times the same reaction container is soaked, and Y is the total number of times the same reaction container is soaked.

8. The method according to claim 1, It is characterized in that The temperature of the reaction in at least one of the steps (1) and (3) is calculated based on a predetermined cycle number-reaction temperature relationship.

9. The method according to claim 1, It is characterized in that The total number of cycles of the cycle is divided into N cycle segments, the reaction temperature of each cycle in each cycle segment is the same, and the reaction temperature between each cycle segment is different; N is an integer greater than 1; The reaction temperature of each of the cycle segments increases as the number of cycle segments increases; As the number of cycle segments increases, the reaction temperature between the cycle segments increases in an exponential or linear distribution manner; The linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.

10. The method according to any one of claims 1 to 9, It is characterized in that After multiple cycles, a DNA polymerase with strand displacement activity is used to perform multiple displacement amplification reactions on the target nucleic acid to obtain complementary double strands; the double strands are subjected to steps (1) to (4); The reaction time and / or reaction temperature of each reaction of the first chain is different from the corresponding reaction time and / or reaction temperature of each reaction of the second chain.

11. A sequencing system, It is characterized in that include: chip; A sequencing device, which is used to sequence the target nucleic acid fixed on the surface of the chip; One or more processors configured to perform: (1) reacting the target nucleic acid fixed on the chip surface with a polymerization reagent, incorporating nucleotides or nucleotide analogs, and obtaining a reaction product; (2) detecting fluorescence signals; (3) reacting the reaction product with a regeneration agent to obtain a product that can be subjected to a next round of polymerization reaction; (4) Repeat steps (1) to (3), and so on, for multiple cycles to finally obtain sequencing data; The reaction time in at least one of the steps (1) and (3) is determined by a predetermined cycle. The number-reaction time relationship was calculated.

12. The sequencing system according to claim 11, It is characterized in that The one or more processors are configured to perform: Dividing the total number of cycles into N cycle segments, wherein the reaction time of each cycle in each cycle segment is the same, and the reaction time between each cycle segment is different; N is an integer greater than 1; As the number of cycle segments increases, the reaction time between the cycle segments increases in an exponential or linear distribution.

13. The sequencing system according to claim 11, It is characterized in that The one or more processors are configured to determine the reaction time according to the following formula: reaction time (s) = A + (BA) / D × (C-1); Where A represents the reaction time constant of the first cycle, B represents the reaction time constant of the last cycle, C represents the current sequencing cycle number, and D represents the total sequencing cycle number; the reaction time is the rounded result of the formula; or The one or more processors are configured to perform: A normalized curve is obtained based on the sequencing error rate obtained by sequencing under the same reaction time in each cycle; Multiplying the normalized value of each cycle in the normalized curve by a coefficient P, and constructing an exponential curve using the obtained normalized value and the cycle number, wherein the coefficient P is the reaction time expected to be increased in the last cycle; The reaction time required for the first cycle is added to the exponential formula corresponding to the exponential curve to obtain the cycle number-reaction time relationship.

14. The sequencing system according to claim 11, It is characterized in that The one or more processors are configured to perform the reaction in each round of cyclic reaction, comprising: immersing the chip in a reaction container storing a polymerization reagent or a regeneration reagent, and after the reaction is completed, taking the chip out of the reaction container; As the number of cycles increases, the number of immersions in each reaction vessel increases, and the duration of each immersion is prolonged; The one or more processors are configured to determine the duration of each soaking according to the following formula: Soaking time = 50 + 10 (X-1) / (Y-1), X is the number of times the same reaction container is soaked, the soaking time unit is seconds, and Y is the total number of times the same reaction container is soaked.

15. The system according to claim 11, It is characterized in that The temperature of the reaction in at least one of the steps (1) and (3) is calculated based on a predetermined cycle number-reaction temperature relationship; The one or more processors are configured to perform: The total number of cycles of the cycle is divided into N cycle segments, and the reaction temperature of each cycle in each cycle segment is the same. The reaction temperature between each of the cycle sections is different; N is an integer greater than 1; The reaction temperature of each of the cycle segments increases as the number of cycle segments increases; As the number of cycle segments increases, the reaction temperature between the cycle segments increases in an exponential or linear distribution manner; The linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.

16. An electronic device, It is characterized in that include: A memory and one or more processors; the memory is coupled to the one or more processors, the memory is used to store computer program code, the computer program code includes computer instructions, and the one or more processors call the computer instructions to enable the electronic device to execute the sequencing method according to any one of claims 1 to 10.

17. A computer-readable storage medium comprising computer instructions, It is characterized in that When the computer instructions are executed on an electronic device, the electronic device executes the sequencing method according to any one of claims 1 to 10.

18. A computer program product, It is characterized in that When the computer program product is run on a computer, the computer is enabled to execute the sequencing method according to any one of claims 1 to 10.

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