WO2024178682A1 - 确定测序中反应时间的方法和测序方法及系统 - Google Patents

确定测序中反应时间的方法和测序方法及系统 Download PDF

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Publication number
WO2024178682A1
WO2024178682A1 PCT/CN2023/079100 CN2023079100W WO2024178682A1 WO 2024178682 A1 WO2024178682 A1 WO 2024178682A1 CN 2023079100 W CN2023079100 W CN 2023079100W WO 2024178682 A1 WO2024178682 A1 WO 2024178682A1
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Prior art keywords
cycle
reaction
sequencing
reaction time
time
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English (en)
French (fr)
Inventor
韦小芳
龚梅花
周爽
赵微
缪海涛
王静静
罗银铃
樊帆
赵胜明
徐崇钧
李计广
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202380068162.6A priority Critical patent/CN120019165A/zh
Priority to PCT/CN2023/079100 priority patent/WO2024178682A1/zh
Publication of WO2024178682A1 publication Critical patent/WO2024178682A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • 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.
  • the second generation sequencing technology is currently the most widely used sequencing technology due to its advantages such as low cost, fast sequencing speed and high throughput.
  • the second generation sequencing introduces reversible blocking groups and carries fluorescent marker molecules on the bases to read the DNA sequence by enhancing the intensity of the fluorescent signal.
  • this technology has certain limitations. As the read length increases, the fluorescent signal will decrease, resulting in a decrease in the quality of base sequencing, which limits the read length of the second generation sequencing. Therefore, the long read length sequencing of the second generation sequencing is a big challenge. It is very difficult to obtain an accurate long read length sequencing.
  • the present disclosure aims to solve the technical problems existing in the prior art at least to some extent.
  • Both polymerization reaction and regeneration reaction require a certain reaction time.
  • the prior art 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.
  • the reaction reagents affect the DNA template chain structure, photodamage, and enzyme activity, resulting in reduced polymerization and regeneration efficiency.
  • 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 sequencing back-end reaction time may result in The time for sequencing short reads is wasted, and at the end of sequencing (such as starting from the 150th cycle), the reaction efficiency decreases. If the reaction time of the short cycle is still used, it will lead to insufficient reaction and poor sequencing quality. In the prior art, when the total time is the same, the unreasonable allocation of reaction time leads to insufficient sequencing reaction and poor sequencing quality results.
  • 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.
  • the longer the DNA template chain reacts at high temperature the greater the damage to the nucleic acid sequence; the cumulative reaction time or temperature increases, the more serious the damage to the nucleic acid sequence.
  • the reaction time or reaction temperature of one chain will affect the results of one chain, the results of the second chain, and the overall result, and the reaction time or reaction temperature of the second chain will affect the results of the second chain and the overall result; for single-end sequencing, its reaction time or reaction temperature will directly affect the quality of the sequencing result. Poor distribution of reaction time or reaction temperature will lead to a waste of front-end cycle reaction time, insufficient back-end cycle reaction, long reaction time, low accuracy, and increased sequencing time.
  • the applicant adopts different reaction times or reaction temperatures for different cycles, constructs the relationship between the number of cycles-reaction time and the number of cycles-reaction temperature, and reasonably 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 sufficient, while reducing the damage to the nucleic acid sequence.
  • 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 read lengths, and protects the nucleic acid sequence from high temperature damage while ensuring a short reaction time in the early stage of sequencing, and the sequencing quality will not decrease much; it also reserves sufficient room for the later stage of sequencing, guarantees the overall sequencing level, and solves the influence of the early stage of sequencing on the later stage of sequencing and the overall sequencing.
  • the present disclosure proposes a sequencing method.
  • the sequencing method includes: (1) reacting a target nucleic acid fixed on a chip surface 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 be subjected to a next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles, and finally obtaining sequencing data; wherein the reaction time in at least one of the steps (1) and (3) is calculated based on a predetermined cycle number-reaction time relationship.
  • 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 complete, while reducing the damage to nucleic acids caused by long-term high temperature during the reaction, improving the sequencing quality, and facilitating long-read sequencing.
  • the above sequencing method may also have the following additional technical features:
  • 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.
  • the reaction time of each of the cycle segments increases as the number of cycle segments increases.
  • the reaction time between the cycle segments increases in an exponential function or a linear function distribution manner.
  • the linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.
  • 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.
  • 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 with 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.
  • the reaction in each cycle, includes immersing the chip in a reaction container storing 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.
  • the temperature of the reaction in at least one of the steps (1) and (3) is calculated by 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 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 linear function distribution mode; the linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.
  • a DNA polymerase with 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 time of each strand are The reaction time and/or reaction temperature are different from the corresponding reaction time and/or reaction temperature of each reaction of the two chains.
  • the present disclosure proposes a sequencing system.
  • the sequencing system includes: a chip; a sequencing device, the sequencing device is used to sequence the target nucleic acid fixed on the surface of the chip; one or more processors, the one or more processors are configured to perform: (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 be subjected to the next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles, and finally obtaining sequencing data; wherein the reaction time in at least one of the steps (1) and (3) is calculated by a predetermined cycle number-reaction time relationship.
  • 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.
  • 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 storing 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.
  • 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 being the same, The reaction temperatures between the various cycle segments are 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 temperatures between the various cycle segments increase in an exponential function or a linear function distribution mode; the linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.
  • the present disclosure provides an electronic device.
  • 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 includes computer instructions, and the one or more processors call the computer instructions to enable the electronic device to perform the sequencing method as described above.
  • the present disclosure provides a computer-readable storage medium, including 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.
  • FIG1 shows a schematic flow chart of a sequencing method according to an embodiment of the present disclosure
  • FIG2 shows a graph of cycle number-linear incubation time according to one embodiment of the present disclosure
  • FIG3 shows a graph of cycle number-exponential incubation time according to an 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 normalization value ⁇ coefficient B;
  • FIG4 shows a schematic diagram of a sequencing system according to an embodiment of the present disclosure
  • FIG5 shows a schematic diagram of the structure of an electronic device according to an embodiment of the present disclosure
  • FIG6 shows a constant incubation and gradient incubation curve according to Example 1 of the present disclosure, where the horizontal axis is the cycle number, and the vertical axis is the Q30% value of each cycle, indicating that the Q30% decreases with the increase of the cycle number, which can represent the quality of the sequencing result to a certain extent;
  • FIG7 shows an incubation time curve diagram according to Example 2 of the present disclosure
  • FIG8 shows a Q30% value analysis diagram according to Example 2 of the present disclosure, where the horizontal axis represents different incubation time modes.
  • the vertical axis is the Q30% index;
  • FIG9 shows an analysis diagram of the Q30% value of each cycle according to Example 2 of the present disclosure, wherein the horizontal axis is the cycle number, and the vertical axis is the Q30% value of each cycle, indicating that the Q30% decreases as the cycle number increases;
  • FIG10 shows an analysis diagram of the Q30% value of each cycle according to Example 3 of the present disclosure, where the horizontal axis is the cycle number, and the vertical axis is the Q30% value of each cycle, indicating that the Q30% decreases with the increase in the cycle number, which can represent the quality of the sequencing result to a certain extent;
  • 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, where 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.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present disclosure, unless otherwise specified, the meaning of "plurality” is two or more.
  • any values of the ranges disclosed in this article 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.
  • the endpoint values of each range, the endpoint values of each range and the 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 regarded as specifically disclosed in this article.
  • the present disclosure proposes 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.
  • the present disclosure provides a sequencing method.
  • the sequencing method includes: S100 reacting a target nucleic acid with a polymerization reagent, S200 detecting a fluorescent signal, S300 reacting a reaction product with a regeneration reagent, and S400 repeating steps S100 to S300, which will be described in detail below.
  • the target nucleic acid fixed on the chip surface reacts with a polymerization reagent (also referred to as In the process of "polymerization reaction", nucleotides or nucleotide analogs are added to obtain reaction products.
  • a polymerization reagent also referred to as In the process of "polymerization reaction”
  • nucleotides or nucleotide analogs are added to obtain reaction products.
  • the polymerization reagent continuously adds dNTP to the 3'-OH end of the primer/previously added nucleotide or nucleotide analog, extending the primer and synthesizing a new complementary DNA chain.
  • polymerization reagent refers to the reagent required to participate in the polymerization reaction. All reagents for the polymerization reaction disclosed in the art are included in the present disclosure, such as dNTP, DNA polymerase, and buffer.
  • the term “polymerization” can include both chain synthesis, i.e., chain extension, and the leveling process, i.e., to make up for the dNTP with a reversible blocking group and a fluorescent group that is not bound to the 3'-OH end during the chain synthesis process. In the leveling process, it can further bind to synchronize all reads signals.
  • step 100 may further include: cleaning the synthesis product with a cleaning reagent.
  • the sequencing chip connected to the synthesis product is placed in a reaction container containing a cleaning reagent to clean the free reaction reagent on the chip.
  • reaction container refers to a reaction site where polymerization and removal of reversible blocking groups and fluorescent groups can be carried out during the sequencing process.
  • reaction container can be a reagent tank on a sequencing platform.
  • the sequencing read information is obtained based on the signal color and intensity.
  • the reaction product is reacted with a regeneration agent (also referred to as "regeneration reaction” in the present disclosure) to obtain a product that can undergo the next round of polymerization reaction.
  • a regeneration agent also referred to as "regeneration reaction” in the present disclosure
  • the reaction product is reacted with the regeneration agent, the reversible blocking group and the fluorescent group carried on the reaction product can be removed. This helps the next round of polymerization reaction to occur.
  • blocking group binds to the 3' hydroxyl group or other sites of deoxyribose, causing deoxyribose to be unable to form a phosphodiester bond with subsequent dNTPs or to form a steric hindrance that prevents the polymerase from performing a polymerization reaction, thereby terminating chain extension.
  • the 3' terminal hydroxyl structure can be restored, and a phosphodiester bond can be formed with subsequent dNTPs to complete chain extension.
  • it can be reversibly removed to restore the spatial structure of DNA, and a polymerization reaction can occur with subsequent dNTPs and polymerases to complete chain extension.
  • the blocking group can be any group used in the art to block dNTPs, typically but not limitingly including azido methylene, and some fluorescent groups can also be used as blocking groups.
  • Other available blocking groups also include, for example, those disclosed in the 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 dNTPs can be used as a blocking group in the present disclosure. These blocking groups can all be reversibly detached from the dNTPs.
  • 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.
  • steps S100 to S300 are repeated, and so on, for multiple cycles to finally obtain sequencing data.
  • the reaction time in at least one of steps S100 and S300 is calculated by a predetermined cycle number-reaction time relationship.
  • 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 time of different cycles is reasonably allocated, so that the reaction of each cycle is more sufficient, while reducing the damage to the nucleic acid caused by the long-term high temperature in the reaction, improving the sequencing quality, and facilitating the sequencing of long reads.
  • 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.
  • 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.
  • the reaction time of each cycle segment increases as the number of cycle segments increases.
  • the reaction time between each cycle segment increases in an arithmetic progression or a geometric progression distribution.
  • the reaction time of the polymerization and regeneration reaction can be carried out in a gradient increasing manner within a certain range.
  • the gradient increase can be carried out in an arithmetic progression or a geometric progression or in other ways, as follows:
  • the total number of cycles can be divided into different segments, the number of cycles in a cycle segment uses the same reaction time, and the reaction time between different cycle segments is inconsistent; the segments can be divided equally or by custom segmentation;
  • Specific examples include: one segmentation method is to divide the 300 cycles into 2 segments, each segment is 150 cycles; another segmentation method is to divide the 300 cycles into 5 segments, each segment is 60 cycles; another segmentation method is to divide the 300 cycles into 6 segments, each segment is 50 cycles; and so on.
  • 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 in a segment can be adjusted as needed, and the number of cycles in each segment does not need to be consistent.
  • the loops in the same loop segment use the same reaction time, and different reaction times are used between different loop segments.
  • the reaction time can be increased according to a certain arithmetic or geometric progression, or the reaction time of each loop segment can be customized.
  • the increment method of arithmetic progression can be:
  • 300 cycles are divided into 5 cycle segments, each cycle segment is 60 cycles, and the reaction time is increased in an arithmetic progression in the range of 20s-100s, then the reaction times of the 5 cycle segments are 20s, 40s, 60s, 80s and 100s respectively.
  • the above is just an example, and the segmentation method and reaction time can be other similar settings.
  • each cycle segment is 50 cycles
  • the reaction time increases in a geometric progression in the range of 15s-120s
  • the reaction times of the 4 cycle segments are 15s, 30s, 60s and 120s respectively.
  • the above is just an example, and the segmentation method and reaction time can be other similar settings.
  • the custom increment method can be:
  • 300 cycles are divided into 5 cycle segments, each cycle segment is 60 cycles, and the reaction time is in a gradient increase mode within the range of 20s-100s, then the reaction times of the 5 cycle segments are 20s, 30s, 60s, 80s and 100s respectively.
  • the segmentation mode and reaction time can be other similar settings.
  • the segmentation method and reaction time settings of polymerization and regeneration reactions can be different and can be edited and set separately.
  • the segmentation method and reaction time settings of the first chain and the second chain can be different and can be edited and set separately.
  • 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 sequencing cycle number; wherein the reaction time is the rounded result of the formula.
  • the polymerization and regeneration reactions are carried out in a linear manner within a certain range:
  • the exposure time for each current cycle is determined according to the following formula:
  • Reaction time (s) A + (B-A) / D ⁇ (C-1)
  • 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
  • D represents the total sequencing cycle number
  • the reaction time is the rounded result of the formula.
  • the linear reaction time settings for polymerization and regeneration reactions can be different and can be edited and set separately.
  • the linear reaction time settings for the first and second chains can be different and can be edited separately.
  • FIG. 2 shows a reaction time setting of a linear reaction, but it does not mean that the present disclosure has only this linear reaction mode.
  • a method for determining the relationship between the number of cycles and the 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 with the obtained normalized value and the number of cycles, wherein the coefficient P is the reaction time expected to be increased in 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 relationship between the number of cycles and the reaction time.
  • the reaction time of the polymerization and regeneration reactions is carried out in an exponential reaction manner within a certain range.
  • the formula of the exponential reaction can be set according to the following method, but is not limited to this setting method:
  • the sequencing is performed under the condition of fixed reaction time. According to the error rate curve of sequencing, the error rate of the last cycle is normalized. The normalization coefficient is Q. Then the error rates corresponding to all cycles are divided by the coefficient Q to obtain a normalized curve.
  • the coefficient P is the planned increase in incubation time of the last cycle; an exponential curve multiplied by the P coefficient is obtained;
  • the exponential incubation formula is:
  • FIG. 3 shows a time setting of an exponential reaction, but it does not mean that the present disclosure has only this exponential reaction mode.
  • 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.
  • the exponential reaction time settings of the first chain and the second chain can be different and can be edited and set separately.
  • the sequencing method disclosed herein can be a single-end sequencing or a double-end sequencing method, including sequencing by primer extension using labeled or unlabeled nucleotides, such as sequencing-by-ligation or pyrophosphate sequencing, for example, using any of the Sanger dideoxy sequencing, nanopore or "NexGen” sequencing methods in the art (e.g., using the sequencing platform of MGI, ROCHE
  • the method can be implemented by using the 454 sequencing platform, ILLUMINATM SOLEXATM sequencing platform, LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLIDTM sequencing platform, PACIFIC BIOSCIENCES' SMRTTM sequencing platform, POLLONATOR Polony sequencing platform, COMPLETE GENOMICS sequencing platform, INTELLIGENT BIOSYSTEMS' sequencing platform, HELICOS sequencing platform or any other sequencer and system in the art).
  • the present disclosure does not strictly limit the occurrence manner of the polymerization reaction and the regeneration reaction of steps S100 and S300.
  • Some sequencing platforms inject reaction reagents or regeneration reagents onto the sequencing chip through a syringe pump, such as MGI's sequencing platforms: MGISEQ-200RS, MGISEQ-2000RS, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-E25 and other platforms; some sequencing platforms adopt an immersion method, that is, an immersion biochemical platform, which is to immerse the chip in a reaction container containing reaction reagents or regeneration reagents. After a certain reaction is completed, the chip is transferred to another reaction container to complete the next step, such as the DNBSEQ-T10 ⁇ 4RS sequencing platform.
  • the reaction in each cycle, includes immersing the chip in a reaction container storing 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.
  • the new reaction container such as a reagent tank
  • the reagent active ingredients in some key reagent tanks will also change.
  • the concentration of the reagent to be reacted in the reaction container is too low or the active ingredient deteriorates to be unfavorable for the reaction to occur, it is necessary to replace a new reagent, and the soaking time can be appropriately shortened at this time.
  • the biochemical parameters in the sequencing platform for example: immersion biochemical platform
  • different temperatures can be set in different sequencing stages to achieve more suitable sequencing results, and in the same round of reagents, different soaking times are set to achieve the best experimental effect.
  • the initial biochemical time of biochemical tank 1 is 50s and the final biochemical time is 60s.
  • the number of soaking times is set to 78;
  • reaction time (s) 50 + 10 (X-1) / (78-1).
  • the reaction time is 50 seconds.
  • the reaction time is 60 seconds.
  • the reaction time increases linearly with the increase of the number of immersions.
  • reaction temperature can also be adjusted according to the number of soaking times.
  • the reaction temperature in at least one of step S100 and step S300 is calculated based on a predetermined cycle number-reaction temperature relationship.
  • 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; with the increase in the number of cycle segments, 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.
  • a DNA polymerase with strand displacement activity is used to perform multiple displacement amplification reactions on the target nucleic acid fixed 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 of each strand is different from the corresponding reaction time and/or reaction temperature of each strand.
  • the reaction time/reaction temperature of the nth cycle of the first strand is different from the reaction time/reaction temperature of the nth cycle of the second strand.
  • the present disclosure provides a sequencing system.
  • 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 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, and obtaining a product that can be subjected to the next round of polymerization reaction; (4) repeating steps (1) to (3), and so on, for multiple cycles, and finally obtaining sequencing data; wherein the reaction time in at least one of steps (1) and (3) is calculated by a predetermined cycle number-reaction time relationship.
  • one or more processors 300 include:
  • the first module 210 is used to react the target nucleic acid fixed on the chip surface with a polymerization reagent, incorporate nucleotides or nucleotide analogs, and obtain a reaction product;
  • a second module 220, the second module 220 is used to detect a fluorescent signal
  • 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;
  • the fourth module 240 is used to make the first module 210, the second module 220 and the third module 230 The executed operations are repeated in sequence, and so on, for multiple rounds of cycles, and finally sequencing data is obtained;
  • 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.
  • 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.
  • the reaction time between each cycle segment increases in an exponential function or a linear function distribution manner.
  • one or more processors 300 are configured to perform: 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 number of cycles, where 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.
  • the temperature of the reaction in at least one of step (1) and step (3) is calculated by 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 mode; the linear function distribution mode is selected from an arithmetic progression or a geometric progression distribution mode.
  • the one or more processors 300 may be general-purpose processors or special-purpose processors. For example, they may be baseband processors or central processing units.
  • the baseband processor may be used to process communication protocols and communication data
  • the central processing unit may be used to process communication protocols and communication data. 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 data of computer programs.
  • the processor 300 can be implemented in 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.
  • IC integrated circuit
  • ASIC application specific integrated circuit
  • PCB printed circuit board
  • the processor and the transceiver can also be manufactured using various IC process technologies, such as complementary metal oxide semiconductor (CMOS), N-type metal oxide semiconductor (nMetal-oxide-semiconductor, NMOS), P-type metal oxide semiconductor (positive channel metal oxide semiconductor, PMOS), bipolar junction transistor (bipolar junction transistor, BJT), bipolar CMOS (BiCMOS), silicon germanium (SiGe), gallium arsenide (GaAs), etc.
  • CMOS complementary metal oxide semiconductor
  • N-type metal oxide semiconductor nMetal-oxide-semiconductor
  • PMOS bipolar junction transistor
  • BJT bipolar CMOS
  • SiGe silicon germanium
  • GaAs gallium arsenide
  • the present disclosure proposes an electronic device.
  • the electronic device includes: a memory, 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 perform the sequencing method as described above.
  • the electronic device can be any intelligent terminal including a sequencer, a tablet computer, a computing cluster, etc.
  • memory refers to any computer program product, device, and/or apparatus (e.g., disk, optical disk, memory, 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).
  • ROM read-only memory
  • RAM random access memory
  • the memory can store an operating system and other application programs.
  • 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 in communication within the device through the bus 450.
  • the processor 410 can be implemented by 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.
  • a general-purpose CPU Central Processing Unit
  • ASIC application-specific integrated circuit
  • the memory 420 may be implemented in the form of ROM (Read Only Memory), RAM (Random Access Memory), static storage device, dynamic storage device, etc.
  • the memory 420 may store an operating system and other application programs.
  • the relevant program codes are stored in the memory 420 and are called and executed by the processor 410.
  • the input/output interface 430 is used to connect the input/output module to realize information input and output.
  • the input/output module can be configured in the device as a component (not shown in the figure), or it can be externally connected to the device to provide corresponding functions.
  • the input device may include a keyboard, a mouse, a touch screen, a microphone, various sensors, etc.
  • the output device may include a display, a speaker, a vibrator, an indicator light, etc.
  • the communication interface 440 is used to connect a communication module (not shown) to realize communication interaction between the device and other devices.
  • the communication module can realize communication through a wired mode (such as USB, network cable, etc.) or a wireless mode (such as mobile network, WIFI, Bluetooth, etc.).
  • the bus 450 comprises a pathway for transmitting information between the various components of the device (eg, the processor 410, the memory 420, the input/output interface 430, and the communication interface 440).
  • 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 the specific implementation process, the device may also include other components necessary for normal operation.
  • the above device may also only include the components necessary for implementing the embodiments of the present specification, and does not necessarily include all the components shown in the figure.
  • the computer program product includes one or more computer programs.
  • 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.
  • the computer program can be transmitted from a website site, computer, server or data center by wired (e.g., coaxial cable, optical fiber, digital subscriber line (digital subscriber line, DSL)) or wireless (e.g., infrared, wireless, microwave, etc.) mode to another website site, computer, server or data center.
  • wired e.g., coaxial cable, optical fiber, digital subscriber line (digital subscriber line, DSL)
  • wireless e.g., infrared, wireless, microwave, etc.
  • the present disclosure proposes a computer-readable storage medium, including computer instructions.
  • the electronic device executes 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 data center that includes one or more available media integrated.
  • the available medium can be a magnetic medium (e.g., a floppy disk, a hard disk, a magnetic tape), an optical medium (e.g., a high-density digital video disc (digital video disc, DVD)), or a semiconductor medium (e.g., a solid state drive (solid state disk, SSD)), etc.
  • the present disclosure provides a computer program product.
  • the computer program product when the computer program product is run on a computer, the computer is enabled to perform the sequencing method as described above.
  • the relevant hardware is completed by a computer program, which can be stored in a computer-readable storage medium.
  • the program When executed, it can include the processes of the above-mentioned method embodiments.
  • the aforementioned storage medium includes: ROM or random access memory RAM, magnetic disk or optical disk and other media that can store program codes.
  • MGISEQ-2000RS sequencer FTAT sequencer
  • PCR instrument PCR eight-tube, 3.0 fluorescence quantifier, a set of pipettes, a high-speed centrifuge, 200 ⁇ L wide-mouth pipette tips, and an ice box.
  • This implementation case is based on the MGISEQ-2000RS platform, and the reagents used are all from the library construction kit and single-end sequencing kit (hereinafter referred to as SE400 kit) used with the sequencer.
  • SE400 kit single-end sequencing kit
  • the sample comes from Escherichia coli; during the verification process, refer to the "MGISEQ-2000RS High-throughput (Rapid) Sequencing Reagent Set User Manual" to prepare and load the DNB required by SE400 and prepare the SE400 reagent tank;
  • SE400 sequencing takes SE400 sequencing as an example, sets the incubation time by gradient incubation, and changes the SE400 biochemical script required for sequencing;
  • This example designs two groups of incubation times for comparison:
  • the first group the overall synthesis time was maintained at 60 s, that is, the incubation time was maintained constant.
  • the second group 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 setting is shown in Table 3.
  • 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 decreases more slowly than the Q30% of the constant incubation time, proving that the gradient incubation time method produces better results than constant incubation.
  • Table 4 Comparison of the effects of constant incubation time and gradient incubation time on overall sequencing quality in this example.
  • ESR (%) Filter reads according to Q value, the proportion of reads greater than Q30.
  • This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI, and the verification sample used is derived from Escherichia coli.
  • the preparation, loading and reagent tank configuration of DNBs are common to other MGI platforms and can be adapted.
  • This implementation case takes SE150 sequencing as an example, sets the incubation time in a linear incubation manner, and changes the SE150 biochemical script required for sequencing.
  • This example designs two groups of incubation times for comparison:
  • the first group maintain the overall synthesis time at 20s, the filling time at 30s, and the regeneration time at 30s, that is, maintain a constant incubation time.
  • the second group the incubation time was carried out in a linear incubation manner, and the incubation time was calculated according to the formula, as shown in the following formula: Down:
  • 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.
  • the incubation time for synthesis is 13+(43-13)/150 ⁇ (C-1)
  • the incubation time for filling is 22+(52-22)/150 ⁇ (C-1)
  • the incubation time for regeneration is 27+(57-27)/150 ⁇ (C-1)
  • This implementation case is based on the sequencer MGISEQ-2000RS platform manufactured by MGI, and the reagents used are all derived from the library construction kit and the double-end sequencing kit (hereinafter referred to as the PE300 kit) used in conjunction with the sequencer; the verification sample used is derived from Escherichia coli; during the verification process, the "MGISEQ-2000RS High-throughput (Rapid) Sequencing Reagent Kit User Manual" 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, sets the incubation time by exponential incubation, and changes the biochemical script of PE300 required for sequencing.
  • This example designs two groups of incubation times for comparison:
  • the first group the overall synthesis (chain synthesis) time was maintained at 60 s, the filling time was maintained at 120 s, and the regeneration time was maintained at 60 s, that is, the incubation time was maintained constant.
  • Group 2 Referring to Figure 3, the incubation time was carried out in an exponential incubation manner, and the incubation time was calculated according to the formula, which is as follows:
  • K is the current cycle
  • Y is the incubation time of the cycle
  • T is the incubation time of the first cycle.
  • 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 decreases more slowly than the Q30% of the constant incubation time, proving that the exponential incubation time method produces better results than constant incubation.
  • Table 5 shows the comparison of the effects of constant incubation time and exponential incubation time on sequencing quality in this example.
  • RecoverValue This indicator is only for the PE sequencing part and reflects the recovery of the second-strand signal.
  • This implementation case is based on the MGISEQ-2000RS platform, a sequencer manufactured by MGI.
  • the reagents used are all from the library construction kit and double-end sequencing kit used with the sequencer.
  • the verification sample used is from Escherichia coli.
  • the DNB required for PE250 was prepared, loaded, and the PE250 reagent tank was prepared with reference to the "MGISEQ-2000RS High-throughput (Fast) Sequencing Reagent Set Instructions".
  • This implementation case takes PE250 sequencing as an example to set the gradient reaction temperature and change the biochemical script of PE250 required for sequencing.
  • This example designs two groups of incubation temperatures for comparison:
  • Group 1 Maintain the overall leveling temperature at 60°C, that is, maintain a constant reaction temperature.
  • the second group 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.
  • 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, which proves that the gradient temperature method produces better results than the constant temperature.
  • Table 7 Comparison of the effects of constant incubation temperature and gradient incubation temperature on overall sequencing quality in this example.

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Abstract

一种测序方法和测序方法及系统，该测序方法包括：（1）将固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；（2）检测荧光信号；（3）将反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；（4）重复步骤（1）到步骤（3），以此类推，进行多轮循环，最终获得测序数据；其中，步骤（1）和步骤（3）至少之一中反应的时间是通过预先确定的循环数-反应时间关系计算获得。

Description

确定测序中反应时间的方法和测序方法及系统 技术领域
本公开涉及生物领域。具体地，本公开涉及确定测序中反应时间的方法和测序方法及系统。
背景技术
二代测序技术因其费用低、测序速度快及高通量等优点，是目前应用最为广泛的测序技术。二代测序引入了可逆阻断基团，同时在碱基上携带荧光标记分子，通过增强荧光信号强度从而读出DNA序列，但这样的技术存在一定的限制，随着读长增长，荧光信号会随之下降，导致碱基测序质量下降，限制了二代测序的读长，因此二代测序的长读长测序是一个很大的挑战，想要获得一个准确的长读长测序是非常困难的。
测序过程中在聚合酶的作用下，互补配对的核苷酸会结合到DNA链上，因为核苷酸带有可逆阻断基团，每个循环只会结合一种核苷酸，对芯片进行扫描后读出DNA的序列，在这个循环完成后，加入化学试剂将可逆阻断基团和荧光基团切断进行再生，进行下一个循环。
核苷酸在聚合酶的作用下结合到DNA模板链上，需要一定的反应温度和反应时间，同时可逆阻断基团和荧光基团切断的反应过程也需要一定的反应温度和反应时间；其中不同的反应时间可能影响到聚合和再生(切除)的效果，随着测序读长增长，由于化学试剂和DNA模板链结构的影响，反应的效率逐渐降低。
因此，如何确定测序过程中反应时间仍有待研究。
发明内容
本公开旨在至少在一定程度上解决现有技术中存在的技术问题。
需要说明的是，本公开是基于申请人的下列发现而完成的：
聚合反应和再生反应都需要一定的反应时间，现有技术是从起始循环到终止循环，使用相同的聚合反应时间和相同的再生反应时间，并且对于双端测序，一链和二链也使用相同的反应时间。
随着测序读长增长，反应试剂影响DNA模板链结构、光损伤以及酶活性的降低，导致聚合和再生的效率降低。使用相同的反应时间，会导致测序前端时间的浪费，因为在测序短读长时，在较短的反应时间内即可完成反应，但是使用测序后端的反应时间可能会导致 测序短读长时时间的浪费，而到了测序后端(如从第150个循环开始)，反应效率下降，仍然使用短循环的反应时间会导致反应不充分，测序质量下降。现有技术在使用总时间相同的情况下，反应时间分配不合理导致测序反应不充分，测序质量结果不佳。
如果为了得到较好的结果而盲目的在测序后期增加测序的反应时间，虽然有时在短读长上可以得到质量较好的数据，但是从长远来看，对于长读长的测序会带来一定的弊端，由于过度的增加反应时间，使核酸序列在高温下处于较长的时间，会对核酸序列造成不可逆的伤害，使其不能够维持特有的结构特征完成测序，造成测序质量变差。
同样地，在测序过程中若使用相同的反应温度，也容易出现因反应温度低而造成反应不充分或者因反应温度高而造成核酸序列损伤，影响测序质量。
理论上，DNA模板链在高温下反应的时间越长，对于核酸序列的伤害更大；累计反应时间或温度增加，对核酸序列的损伤更严重。针对双端测序，一链的反应时间或反应温度会影响一链的结果、二链的结果以及整体的结果，二链的反应时间或反应温度会影响二链的结果和整体的结果；针对单端测序，其反应时间或反应温度将直接影响该测序结果的好坏。反应时间或反应温度分配不好导致前端循环反应时间的浪费，后端循环反应不充分，反应时间长、准确度低，测序时长增加。
有鉴于此，申请人针对不同循环采用不同的反应时间或反应温度，构建循环数-反应时间关系和循环数-反应温度关系，基于该预先构建的关系合理分配不同循环的反应时间和反应温度，使得每个循环的反应更加充分，同时减少核酸序列的损伤。由此，本公开解决了反应时间过长或过短、反应温度过高或过低引起的问题，可以较好的平衡反应时间，适用于长读长的测序，在测序前期保证反应时间较短的情况下，保护核酸序列不受高温损伤，同时测序质量也不会下降很多；也为测序后期保留充分的余地，保障整体测序水平，解决测序前期对测序后期以及整体测序的影响。
为此，在本公开的一个方面，本公开提出了一种测序方法。根据本公开的实施例，所述测序方法包括：(1)将固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；(2)检测荧光信号；(3)将所述反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；(4)重复步骤(1)到步骤(3)，以此类推，进行多轮循环，最终获得测序数据；其中，所述步骤(1)和步骤(3)至少之一中所述反应的时间是通过预先确定的循环数-反应时间关系计算获得。
根据本公开实施例的方法中，针对不同循环采用不同的反应时间，构建循环数-反应时间关系，基于该预先构建的关系合理分配不同循环的反应时间，使得每个循环的反应更加充分，同时减少反应中长时间的高温对核酸的损伤，提高测序质量，有利于长读长的测序。
根据本公开的一种实施例，上述测序方法还可以具有下列附加技术特征：
根据本公开的一种实施例，将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应时间相同，各个所述循环段之间的反应时间不同；N为大于1的整数。
根据本公开的一种实施例，每个所述循环段的反应时间随着循环段数的增加而增加。
根据本公开的一种实施例，随着循环段数的增加，各个所述循环段之间的反应时间呈指数函数或者线性函数分布方式增加。
根据本公开的一种实施例，所述线性函数分布方式选自等差数列或者等比数列分布方式。
根据本公开的一种实施例，所述循环数-反应时间关系选自如下公式：反应时间(s)＝A+(B-A)/N×(C-1)；其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，N表示测序总循环数；其中反应时间为公式结果取整。
根据本公开的一种实施例，确定所述循环数-反应时间关系的方法包括：在各个循环的反应时间相同的条件下，进行步骤(1)～(4)，根据获得的测序错误率，得到归一化曲线；将所述归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，所述系数P为最后一个循环预计增加的反应时间；将所述指数曲线对应的指数公式中加上第一个循环所需反应时间，得到所述循环数-反应时间关系。
根据本公开的一种实施例，每轮循环中，所述反应包括将所述芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，所述反应完成后，将所述测序芯片转移至另外的反应容器中；随着循环次数增加，每个反应容器内发生浸泡次数增加，每次浸泡的时间延长。
根据本公开的一种实施例，每次浸泡的时间按照下列公式确定：浸泡时间(s)＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，Y为同一反应容器总浸泡次数。
根据本公开的一种实施例，所述步骤(1)和步骤(3)至少之一中所述反应的温度是通过预先确定的循环数-反应温度关系计算获得。将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应温度相同，各个所述循环段之间的反应温度不同；N为大于1的整数；每个所述循环段的反应温度随着循环段数的增加而增加；随着循环段数的增加，各个所述循环段之间的反应温度呈指数函数或者线性函数分布方式增加；所述线性函数分布方式选自等差数列或者等比数列分布方式。
根据本公开的一种实施例，进行多轮循环后，采用具有链置换活性的DNA聚合酶对固定在芯片上的目标核酸也可以称之为一链，进行多重置换扩增反应，得到互补链也可以称之为二链；将所述二链进行步骤(1)～(4)；其中，所述一链每次进行的反应时间和/或反 应温度与所述二链每次进行相应的反应时间和/或反应温度不同。
在本公开的又一方面，本公开提出了一种测序系统。根据本公开的一种实施例，所述测序系统包括：芯片；测序设备，所述测序设备用于对固定于所述芯片表面的目标核酸进行测序；一个或多个处理器，所述一个或多个处理器被配置为用于执行：(1)使固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；(2)检测荧光信号；(3)使所述反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；(4)重复步骤(1)到步骤(3)，以此类推，进行多轮循环，最终获得测序数据；其中，所述步骤(1)和步骤(3)至少之一中所述反应的时间是通过预先确定的循环数-反应时间关系计算获得。
根据本公开的一种实施例，所述一个或多个处理器被配置为用于执行：将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应时间相同，各个所述循环段之间的反应时间不同；N为大于1的整数；随着循环段数的增加，各个所述循环段之间的反应时间呈指数函数或者线性函数分布方式增加。
根据本公开的一种实施例，所述一个或多个处理器被配置为用于执行按照如下公式确定反应时间：反应时间(s)＝A+(B-A)/D×(C-1)；其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整；或者所述一个或多个处理器被配置为用于执行：根据在各个循环的反应时间相同的条件下进行测序获得的测序错误率，得到归一化曲线；将所述归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，所述系数P为最后一个循环预计增加的反应时间；将所述指数曲线对应的指数公式中加上第一个循环所需反应时间，得到所述循环数-反应时间关系。
根据本公开的一种实施例，所述一个或多个处理器被配置为用于执行的每轮循环反应中的所述反应包括：使所述芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，所述反应完成后，使所述芯片转移至另外的反应容器中；随着循环次数增加，每个反应容器内发生浸泡次数的增加，每次浸泡的时间延长。
根据本公开的一种实施例，所述一个或多个处理器被配置为用于执行按照下列公式确定每次浸泡的时间：浸泡时间(s)＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，Y为同一反应容器总浸泡次数。
根据本公开的一种实施例，所述步骤(1)和步骤(3)至少之一中所述反应的温度是通过预先确定的循环数-反应温度关系计算获得；所述一个或多个处理器被配置为用于执行：将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应温度相同， 各个所述循环段之间的反应温度不同；N为大于1的整数；每个所述循环段的反应温度随着循环段数的增加而增加；随着循环段数的增加，各个所述循环段之间的反应温度呈指数函数或者线性函数分布方式增加；所述线性函数分布方式选自等差数列或者等比数列分布方式。
在本公开的又一方面，本公开提出了一种电子设备。根据本公开的一种实施例，所述电子设备包括：存储器、一个或多个处理器；所述存储器与所述一个或多个处理器耦合，所述存储器用于存储计算机程序代码，所述计算机程序代码包括计算机指令，所述一个或多个处理器调用所述计算机指令以使得所述电子设备执行如前面所述测序方法。
在本公开的又一方面，本公开提出了一种计算机可读存储介质，包括计算机指令。根据本公开的一种实施例，当所述计算机指令在电子设备上运行时，使得所述电子设备执行如前面所述测序方法。
在本公开的又一方面，本公开提出了一种计算机程序产品。根据本公开的实施例，当所述计算机程序产品在计算机上运行时，使得计算机执行如前面所述测序方法。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本公开一个实施例的测序方法流程示意图；
图2显示了根据本公开一个实施例的循环数-线性孵育时间曲线图；
图3显示了根据本公开一个实施例的循环数-指数孵育时间曲线图，a表示测序后一链的错误率曲线；b表示错误率曲线归一化后获得的曲线；c表示通过归一化值×系数B，获得的指数曲线；
图4显示了根据本公开一个实施例的测序系统示意图；
图5显示了根据本公开一个实施例的电子设备结构示意图；
图6显示了根据本公开实施例1的恒定孵育和梯度孵育曲线图，横坐标为循环数，纵坐标为每个循环的Q30％的值，表示Q30％随着循环数增加而下降的情况，在一定程度上可以代表测序结果的好坏程度；
图7显示了根据本公开实施例2的孵育时间曲线图；
图8显示了根据本公开实施例2的Q30％值分析图，横坐标为不同的孵育时间方式， 纵坐标为Q30％指标；
图9显示了根据本公开实施例2的每个循环的Q30％值分析图，横坐标为循环数，纵坐标为每个循环的Q30％的值，表示Q30％随着循环数增加而下降的情况；
图10显示了根据本公开实施例3的每个循环的Q30％值分析图，横坐标为循环数，纵坐标为每个循环的Q30％的值，表示Q30％随着循环数增加而下降的情况，在一定程度上可以代表测序结果的好坏程度；
图11显示了根据本公开实施例4的中恒定反应温度和梯度反应温度对测序质量下降的比对分析图，横坐标为循环数，纵坐标为每个循环的Q30％的值，表示Q30％随着循环数增加而下降的情况，在一定程度上可以代表测序结果的好坏程度。
具体实施方式
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本公开的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在本文中，术语“包括”为开放式表达，即包括本公开所指明的内容，但并不排除其他方面的内容。
本公开提出了测序方法、确定测序中反应时间的方法、测序系统、电子设备和计算机可读存储介质，下面将分别对其进行详细描述。
测序方法
在本公开的一个方面，本公开提出了一种测序方法。根据本公开的实施例，参见图1，该测序方法包括：S100将目标核酸与聚合试剂进行反应、S200检测荧光信号、S300将反应产物与再生试剂进行反应、S400重复步骤S100到S300，下面将分别进行详细描述。
S100将目标核酸与聚合试剂进行反应
在该步骤中，将固定于芯片表面的目标核酸与聚合试剂进行反应(在本发明中，亦成 为“聚合反应”)，掺入核苷酸或核苷酸类似物，得到反应产物。在模板指导下，聚合试剂不断将dNTP加到引物/前一个掺入的核苷酸或核苷酸类似物的3’-OH末端，使引物延长，合成出新的互补的DNA链。
在本公开中，术语“聚合试剂”是指参与聚合反应所需试剂，凡是本领域所公开的聚合反应发生的试剂均包含在本公开中，例如dNTP、DNA聚合酶、缓冲液。术语“聚合”既可以包括链合成，即发生链延伸，也可以包括补平过程，即弥补链合成过程中3’-OH端未结合上带有可逆阻断基团和荧光基团的dNTP，在补平过程中可以进一步结合，让所有reads信号同步。
根据本公开的一种实施例，步骤100还可以包括：利用清洗试剂清洗合成产物，具体地，将连接有合成产物的测序芯片于含有清洗试剂的反应容器中，清洗芯片上游离的反应试剂。
在本公开中，术语“反应容器”是指测序过程中，可以进行聚合以及去除可逆阻断基团和荧光基团的反应场所，对于具体的反应容器的种类不作严格限定，例如，可以为测序平台上的试剂槽。
S200检测荧光信号
通过检测荧光信号，基于信号颜色和强度等，获知测序读段信息。
S300将反应产物与再生试剂进行反应
在该步骤中，将反应产物与再生试剂进行反应(在本公开中，亦称为“再生反应”)，获得可以进行下一轮聚合反应的产物。通过将反应产物与再生试剂反应，可以去除反应产物上携带的可逆阻断基团和荧光基团。由此，有助于发生下一轮的聚合反应。
在本公开中，术语“阻断基团”通过与脱氧核糖的3’羟基或其它位点结合，导致脱氧核糖无法同后续的dNTP形成磷酸二酯键或形成空间位阻使聚合酶无法进行聚合反应，进而终止链延伸。当将其可逆地去除，可以恢复3’端羟基结构，可以与后续dNTP形成磷酸二酯键，进而完成链延伸。或将其可逆的去除，恢复DNA的空间结构，可以与后续dNTP和聚合酶发生聚合反应，进而完成链延伸。阻断基团可以是任何本领域中用于阻断dNTP的的基团，典型但非限定性的包括叠氮亚甲基，有一些荧光基团也可以作为阻断基团。其它的可用阻断基团还包括例如国际申请WO2014139596A1中公开的那些阻断基团。本公开对阻断基团的种类没有特别限定，任何本领域中用于阻断dNTP的3’羟基的基团都可以作为本公开中的阻断基团。这些阻断基团都能够可逆地从dNTP上脱落下来。
本公开对于“再生试剂”的类型不作严格限定，凡是本领域所公开的可以去除可逆阻断基团和荧光基团的物质均包含在本公开中。
S400重复步骤S100到S300
在该步骤中，重复步骤S100到步骤S300，以此类推，进行多轮循环，最终获得测序数据。
根据本公开的一种实施例，步骤S100和S300至少之一中反应的时间是通过预先确定的循环数-反应时间关系计算获得的。根据本公开实施例的方法中，针对不同循环采用不同的反应时间，构建循环数-反应时间关系，基于该预先构建的关系合理分配不同循环的反应时间，使得每个循环的反应更加充分，同时减少反应中长时间的高温对核酸的损伤，提高测序质量，有利于长读长的测序。
根据本公开的一种实施例，将循环的总循环数划分为N个循环段，每个循环段内各循环的反应时间相同，各个循环段之间的反应时间不同；N为大于1的整数。
不同循环可采用不同的反应时间，循环可以按照一定规则分成不同循环段，循环段内的循环数采用相同的反应时间，不同循环段之间的反应时间不一致；也可以是每个循环其反应时间都不一致，反应时间与循环数按照一定的公式进行，即总循环数与循环段数相同。
根据本公开的一种实施例，每个循环段的反应时间随着循环段数的增加而增加。根据本公开的另一种实施例，随着循环段数的增加，各个循环段之间的反应时间呈等差数列或者等比数列分布方式增加。
在测序反应过程中，从起始循环到终止循环，聚合及再生反应的反应时间可以按照在一定范围内梯度增加的方式进行，梯度增加可以是按照等差数列或等比数列的梯度或其他方式进行，具体如下：
1.先设置分段的数目，可以将总的循环数分成不同段，循环段内的循环数采用相同的反应时间，不同循环段之间的反应时间不一致；可以有平分的方式进行分段，也可以自定义分段；
1)平分的分段规则有：
如总循环数是300，可将300个循环平分成X段，则每个循环段的循环数是Y，Y＝300/X，具体的例子有，一种分段方式是将300个循环分为2个循环段，每个循环段为150个循环；另一种分段方式是将300循环分为5个循环段，每个循环段为60个循环；再一种方式是将300循环分为6个循环段，每个循环段为50个循环；等等类似这样的分段方式。
2)自定义的分段规则有：
如300个循环数，可以分为第一个循环段是1-150个循环，第二个循环段是151-200个循环，第三个循环段是201-250个循环，第四个循环段是251-300个循环；等等类似的分段方式；段数和段内的循环数可根据需要进行调整，不需要每段的循环数一致。
2.设置好分段后，同一循环段内的循环采用相同的反应时间，不同循环段的之间采用不同的反应时间，反应时间可以按照一定的等差数列或等比数列的来增加反应的时间，也可以是自定义每个循环段的反应时间。
1)等差数列的递增方式可以是：
如300循环分为5个循环段，每个循环段为60个循环，反应时间在20s-100s的范围内按照等差数列的梯度增加方式，则5个循环段的反应时间分别是20s、40s、60s、80s和100s。以上只是举例说明，分段方式和反应时间可以是其他类似设置。
2)等比数列的递增方式可以是：
如200循环分为4个循环段，每个循环段为50个循环，反应时间在15s-120s的范围内按照等比数列的梯度增加方式，则4个循环段的反应时间分别是15s、30s、60s和120s。以上只是举例说明，分段方式和反应时间可以是其他类似设置。
3)自定义的递增方式可以是：
如300循环分为5个循环段，每个循环段为60个循环，反应时间在20s-100s的范围内梯度增加方式，则5个循环段的反应时间分别是20s、30s、60s、80s和100s。以上只是举例说明，分段方式和反应时间可以是其他类似设置。
聚合及再生反应的分段方式和反应时间设置可以不同，可分别进行编辑设置。
一链和二链的分段方式和反应时间设置可以不同，可分别进行编辑设置。
举例说明，如下表1所示：
表1：梯度反应的一种设置方式
根据本公开的一种实施例，循环数-反应时间关系选自如下公式：反应时间(s)＝A+(B-A)/D×(C-1)；其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，N表示测序总循环数；其中反应时间为公式结果取整。
在测序反应过程中，从起始循环到终止循环，聚合及再生反应的反应时间按照在一定范围内的线性反应的方式进行：
按照如下公式确定每一当前循环的曝光时间：
反应时间(s)＝A+(B-A)/D×(C-1)
其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整。
聚合及再生反应的线性反应时间设置可以不同，可分别进行编辑设置。
一链和二链的线性反应时间设置可以不同，可分别进行编辑设置。
图2所示的是一种线性反应的反应时间设置情况，但不代表本公开只有这一种线性反应的方式。
根据本公开的一种实施例，确定循环数-反应时间关系的方法包括：在各个循环的反应时间相同的条件下，进行步骤(1)～(4)，根据获得的测序错误率，得到归一化曲线；将归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，系数P为最后一个循环预计增加的反应时间；将指数曲线对应的指数公式中加上第一个循环所需反应时间，得到循环数-反应时间关系。
在测序反应过程中，从起始循环到终止循环，聚合及再生反应的反应时间按照在一定范围内的指数反应的方式进行，指数反应的公式可以按照以下方法设置，但不局限于只有这种设置方式：
在固定反应时间的条件下进行测序，根据测序的错误率曲线，对最后一个循环的错误率归一化，归一化的系数为Q，则所有循环相应的错误率除以系数Q，获得归一化曲线；
进一步地，对每个循环的归一化数值*P，系数P为最后一个循环计划增加的孵育时间；获得乘以P系数的指数曲线；
进一步地，使用Excel、MATLAB或其他工具求指数曲线的公式，获得指数公式；
进一步地，在指数公式的基础上加上第一个循环需要反应的时间，则获得一个指数反应时间的曲线。
使用Excel、MATLAB或其他工具求指数曲线的公式，求公式得：
S＝a*K^3+b*K^2+c；
在该公式基础上加上第一个循环的孵育时间T，则指数孵育得公式为：
S＝a*K^3+b*K^2+c，在该例子中T＝50，即指数孵育的公式为：S＝a*K^3+b*K^2+c+50，其中K为当前循环，S为该循环的孵育时间，a、b为常数。
图3所示的是一种指数反应的时间设置情况，但并不代表本公开只有这一种指数反应的方式。
本公开中的聚合及再生反应的指数反应的设置方式可以不同，可分别进行编辑设置。
进一步地，一链和二链的指数反应时间设置可以不同，可分别进行编辑设置。
本公开的测序方法可以是单端测序或双端测序方法，包括使用经标记或未经标记的核苷酸通过引物延伸进行测序，如边测序边连接或者焦磷酸测序等，例如使用Sanger双脱氧法、纳米孔或本领域的“NexGen”测序方法中的任一种(例如，使用MGI的测序平台、ROCHE 454测序平台、ILLUMINATM SOLEXATM测序平台、LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS的SOLIDTM测序平台、PACIFIC BIOSCIENCES的SMRTTM测序平台、POLLONATOR Polony测序平台、COMPLETE GENOMICS测序平台、INTELLIGENT BIOSYSTEMS的测序平台、HELICOS测序平台或本领域的任何其它测序仪和系统)实现。本公开对于步骤S100和S300的聚合反应和再生反应的发生方式不做严格限定，一些测序平台是通过注射泵将反应试剂或者再生试剂打在测序芯片上，例如MGI的测序平台：MGISEQ-200RS、MGISEQ-2000RS、DNBSEQ-T7、DNBSEQ-G99、DNBSEQ-E25等平台；也有一些测序平台是采用浸泡方式，即浸没式生化平台，是将芯片浸泡在含有反应试剂或者再生试剂的反应容器中，待某一反应完成后，将芯片转移至另一反应容器中完成下一步操作，例如DNBSEQ-T10×4RS测序平台。
根据本公开的一种实施例，每轮循环中，反应包括将芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，反应完成后，将测序芯片转移至另外的反应容器中；随着循环次数增加，每个反应容器内发生浸泡次数增加，每次浸泡的时间延长。
根据本公开的一种实施例，每次浸泡的时间按照下列公式确定：浸泡时间(s)＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，Y为同一反应容器总浸泡次数。
当在测序平台上使用试剂浸泡的方式操作时，在芯片转移过程中，会携带少量残留液进入新的反应容器(例如试剂槽)中，导致新的反应容器中聚合试剂或者再生试剂浓度有所降低。而且加热时间的过程中，一些关键试剂槽中的试剂有效成分也会发生变化。为了使得反应充分发生，需要延长每次浸泡的时间。当反应容器内待反应的试剂浓度过低或者有效成分变质至不利于反应发生，则需要重新更换新的试剂，此时可以适当缩短浸泡时间。具体地，通过测序平台(例如：浸没式生化平台)软件中的生化参数设置，可以在不同的测序阶段设置不同的温度，来达到比较合适的测序结果，并在同一轮试剂中，不同的浸泡次数设置不同的反应时间，来达到最佳的实验效果。
具体的方案：
设置生化槽初始生化时间和末阶段生化时间，比如生化槽1的初始生化时间是50s，末阶段生化时间是60s；
比如有6张载片同时进行测序时，浸泡次数设定为78次；
生化槽中的试剂进行第X次浸泡时，反应时间(s)＝50+10(X-1)/(78-1)。
则载片在生化槽1进行第一次浸泡时，反应时间是50s，当进行78次浸泡时，反应时间是60s，反应时间随着浸泡次数的增加线性增加。
同理反应温度也可以按照浸泡次数进行调整。
根据本公开的一种实施例，步骤S100和步骤S300至少之一中反应的温度是通过预先确定的循环数-反应温度关系计算获得。
根据本公开的一种实施例，将循环的总循环数划分为N个循环段，每个循环段内各循环的反应温度相同，各个循环段之间的反应温度不同；N为大于1的整数；每个循环段的反应温度随着循环段数的增加而增加；随着循环段数的增加，各个循环段之间的反应温度呈指数函数或者线性函数分布方式增加；线性函数分布方式选自等差数列或者等比数列分布方式。
根据本公开的一种实施例，进行多轮循环后，采用具有链置换活性的DNA聚合酶对固定在芯片上的目标核酸进行多重置换扩增反应，得到互补链；将二链进行步骤S100～S400；其中，一链每次进行的反应时间和/或反应温度与二链每次进行相应的反应时间和/或反应温度不同。具体地，一链进行第n轮循环的反应时间/反应温度与二链进行第n轮循环的反应时间/反应温度不同。
测序系统、电子设备、计算机可读存储介质和计算机程序产品
在本公开的又一方面，本公开提出了一种测序系统。根据本公开的实施例，参见图4，该测序系统1000包括：芯片100、测序设备200和一个或多个处理器300，一个或多个处理器300被配置为用于执行：(1)使固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；(2)检测荧光信号；(3)使反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；(4)重复步骤(1)到步骤(3)，以此类推，进行多轮循环，最终获得测序数据；其中，步骤(1)和步骤(3)至少之一中反应的时间是通过预先确定的循环数-反应时间关系计算获得。
针对不同循环采用不同的反应时间，构建循环数-反应时间关系，基于该预先构建的关系合理分配不同循环的反应时间，使得每个循环的反应更加充分，同时减少反应中长时间的高温对核酸的损伤，提高测序质量，有利于长读长的测序。
根据本公开的一种实施例，参见图4，一个或多个处理器300包括：
第一模块210，该第一模块210用于使固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；
第二模块220，该第二模块220用于检测荧光信号；
第三模块230，该第三模块230用于使反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；
第四模块240，该第四模块240用于使第一模块210、第二模块220和第三模块230中 执行的操作依次重复，以此类推，进行多轮循环，最终获得测序数据；
其中，第一模块210和第三模块230至少之一中反应的时间是通过预先确定的循环数-反应时间关系计算获得。
根据本公开的一种实施例，一个或多个处理器300被配置为用于执行：将循环的总循环数划分为N个循环段，每个循环段内各循环的反应时间相同，各个循环段之间的反应时间不同；N为大于1的整数。
根据本公开的一种实施例，随着循环段数的增加，各个循环段之间的反应时间呈指数函数或者线性函数分布方式增加。
根据本公开的一种实施例，一个或多个处理器300被配置为用于执行按照如下公式确定反应时间：反应时间(s)＝A+(B-A)/D×(C-1)；其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整。
根据本公开的一种实施例，一个或多个处理器300被配置为用于执行：根据在各个循环的反应时间相同的条件下进行测序获得的测序错误率，得到归一化曲线；将归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，系数P为最后一个循环预计增加的反应时间；将指数曲线对应的指数公式中加上第一个循环所需反应时间，得到循环数-反应时间关系。
根据本公开的一种实施例，一个或多个处理器300被配置为用于执行的每轮循环反应中的反应包括：使芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，反应完成后，使芯片从反应容器中取出；随着循环次数增加，每个反应容器内发生浸泡次数的增加，每次浸泡的时间延长；一个或多个处理器被配置为用于执行按照下列公式确定每次浸泡的时间：浸泡时间＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，浸泡时间单位为秒，Y为同一反应容器总浸泡次数。
根据本公开的一种实施例，步骤(1)和步骤(3)至少之一中反应的温度是通过预先确定的循环数-反应温度关系计算获得；一个或多个处理器被配置为用于执行：将循环的总循环数划分为N个循环段，每个循环段内各循环的反应温度相同，各个循环段之间的反应温度不同；N为大于1的整数；每个循环段的反应温度随着循环段数的增加而增加；随着循环段数的增加，各个循环段之间的反应温度呈指数函数或者线性函数分布方式增加；线性函数分布方式选自等差数列或者等比数列分布方式。
一个或多个处理器300可以是通用处理器或者专用处理器等。例如可以是基带处理器或中央处理器。基带处理器可以用于对通信协议以及通信数据进行处理，中央处理器可以 用于对通信设备(如，基站、基带芯片，终端设备、终端设备芯片，DU或CU等)进行控制，执行计算机程序，处理计算机程序的数据。处理器300可实现在集成电路(integrated circuit，IC)、模拟IC、射频集成电路RFIC、混合信号IC、专用集成电路(application specific integrated circuit，ASIC)、印刷电路板(printed circuit board，PCB)、电子设备等上。该处理器和收发器也可以用各种IC工艺技术来制造，例如互补金属氧化物半导体(complementary metal oxide semiconductor，CMOS)、N型金属氧化物半导体(nMetal-oxide-semiconductor，NMOS)、P型金属氧化物半导体(positive channel metal oxide semiconductor，PMOS)、双极结型晶体管(bipolar junction transistor，BJT)、双极CMOS(BiCMOS)、硅锗(SiGe)、砷化镓(GaAs)等。
在本公开的又一方面，本公开提出了一种电子设备。根据本公开的实施例，电子设备包括：存储器、一个或多个处理器；存储器与一个或多个处理器耦合，存储器用于存储计算机程序代码，计算机程序代码包括计算机指令，一个或多个处理器调用计算机指令以使得电子设备执行如前面所述测序方法。具体地，电子设备可以为包括测序仪、平板电脑、计算集群等任意智能终端。
本公开所使用的术语“存储器”指的是用于将机器指令和/或数据提供给可编程处理器的任何计算机程序产品、设备、和/或装置(例如，磁盘、光盘、存储器、可编程逻辑装置(PLD))，包括，接收作为机器可读信号的机器指令的机器可读介质。存储器可以采用只读存储器(Read Only Memory，ROM)、静态存储设备、动态存储设备或者随机存取存储器(Random Access Memory，RAM)等形式实现。存储器可以存储操作系统和其他应用程序，在通过软件或者固件来实现本说明书实施例所提供的技术方案时，相关的程序代码保存在存储器中，并由处理器来调用执行本申请实施例的测序方法。
在一些实施例中，参见图5，该电子设备400可以包括：处理器410、存储器420、输入/输出接口430、通信接口440和总线450。其中处理器410、存储器420、输入/输出接口430和通信接口440通过总线450实现彼此之间在设备内部的通信连接。
处理器410可以采用通用的CPU(Central Processing Unit，中央处理器)、微处理器、应用专用集成电路(Application Specific Integrated Circuit，ASIC)、或者一个或多个集成电路等方式实现，用于执行相关程序，以实现本说明书实施例所提供的技术方案。
存储器420可以采用ROM(Read Only Memory，只读存储器)、RAM(Random Access Memory，随机存取存储器)、静态存储设备，动态存储设备等形式实现。存储器420可以存储操作系统和其他应用程序，在通过软件或者固件来实现本说明书实施例所提供的技术方案时，相关的程序代码保存在存储器420中，并由处理器410来调用执行。
输入/输出接口430用于连接输入/输出模块，以实现信息输入及输出。输入/输出模块可以作为组件配置在设备中(图中未示出)，也可以外接于设备以提供相应功能。其中输入设备可以包括键盘、鼠标、触摸屏、麦克风、各类传感器等，输出设备可以包括显示器、扬声器、振动器、指示灯等。
通信接口440用于连接通信模块(图中未示出)，以实现本设备与其他设备的通信交互。其中通信模块可以通过有线方式(例如USB、网线等)实现通信，也可以通过无线方式(例如移动网络、WIFI、蓝牙等)实现通信。
总线450包括一通路，在设备的各个组件(例如处理器410、存储器420、输入/输出接口430和通信接口440)之间传输信息。
需要说明的是，尽管上述设备仅示出了处理器410、存储器420、输入/输出接口430、通信接口440以及总线450，但是在具体实施过程中，该设备还可以包括实现正常运行所必需的其他组件。此外，本领域的技术人员可以理解的是，上述设备中也可以仅包含实现本说明书实施例方案所必需的组件，而不必包含图中所示的全部组件。
在上述实施例中，可以全部或部分地通过软件、硬件、固件或者其任意组合来实现。当使用软件实现时，可以全部或部分地以计算机程序产品的形式实现。计算机程序产品包括一个或多个计算机程序。在计算机上加载和执行计算机程序时，全部或部分地产生按照本申请实施例的流程或功能。计算机可以是通用计算机、专用计算机、计算机网络、或者其他可编程装置。计算机程序可以存储在计算机可读存储介质中，或者从一个计算机可读存储介质向另一个计算机可读存储介质传输，例如，计算机程序可以从一个网站站点、计算机、服务器或数据中心通过有线(例如同轴电缆、光纤、数字用户线(digital subscriber line，DSL))或无线(例如红外、无线、微波等)方式向另一个网站站点、计算机、服务器或数据中心进行传输。
为此，在本公开的又一方面，本公开提出了一种计算机可读存储介质，包括计算机指令。根据本公开的实施例，当计算机指令在电子设备上运行时，使得电子设备执行如前面所述测序方法。计算机可读存储介质可以是计算机能够存取的任何可用介质或者是包含一个或多个可用介质集成的服务器、数据中心等数据存储设备。可用介质可以是磁性介质(例如，软盘、硬盘、磁带)、光介质(例如，高密度数字视频光盘(digital video disc，DVD))、或者半导体介质(例如，固态硬盘(solid state disk，SSD))等。
在本公开的又一方面，本公开提出了一种计算机程序产品。根据本公开的实施例，当计算机程序产品在计算机上运行时，使得计算机执行如前面所述测序方法。
本领域普通技术人员可以理解实现上述实施例方法中的全部或部分流程，该流程可以 由计算机程序来指令相关的硬件完成，该程序可存储于计算机可读取存储介质中，该程序在执行时，可包括如上述各方法实施例的流程。而前述的存储介质包括：ROM或随机存储记忆体RAM、磁碟或者光盘等各种可存储程序代码的介质。
需要说明的是，前面针对测序方法和确定测序中反应时间的方法中所描述的特征和优点，同样适用于该测序系统、电子设备、计算机可读存储介质和计算机程序产品，在此不再赘述。
下面将结合实施例对本公开的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
下述实施例中，使用的关键器材和关键试剂如下：
1、关键器材：
MGISEQ-2000RS测序仪，FTAT测序仪、PCR仪，PCR八连管，3.0荧光定量仪，移液器一套，高速离心机，200μL阔口吸头，冰盒。
2、关键试剂如下表2所示：
表2所需的试剂
实施例1
本实施案例是基于华大智造的测序仪MGISEQ-2000RS平台，使用的试剂均来源于该测序仪配套使用的建库试剂盒以及单端测序试剂盒(以下简称SE400试剂盒)；使用的验证 样本来源于大肠杆菌；在验证过程中参考《MGISEQ-2000RS高通量(快速)测序试剂套装使用说明书》，进行SE400所需的DNB的制备、加载及SE400试剂槽的配制；本实施案例以SE400测序为例，进行梯度孵育的方式进行孵育时间的设置，更改测序需要的SE400的生化脚本；
本实施例设计两组孵育时间进行对比：
第一组：保持整体合成时间维持在60s，即维持恒定孵育时间。
第二组：将孵育时间按照梯度孵育的方式进行，每100个循环为一个梯度，每个梯度的孵育时间不同，具体的孵育时间设置见表3。
结果见表4和图6，根据本公开实施例的方法设置的梯度孵育时间，比恒定孵育时间的Q30％下降缓慢，证明梯度孵育时间的方法比恒定孵育会产生更好的效果。
表3：SE400梯度孵育的时间设置情况
表4：本实施例中恒定孵育时间和梯度孵育时间对整体测序质量的比对情况。
注：ESR(％)：按照Q值对reads进行过滤，大于Q30的reads所占的比例。
实施例2
本实施案例是基于华大智造的测序仪MGISEQ-2000RS平台，使用的验证样本来源于大肠杆菌；DNB制备，加载及试剂槽的配制与MGI其他平台通用，可以适配；本实施案例以SE150测序为例，进行线性孵育的方式进行孵育时间的设置，更改测序需要的SE150的生化脚本。
本实施例设计两组孵育时间进行对比：
第一组：保持整体合成时间维持在20s，补平时间维持在30s，再生时间维持在30s，即维持恒定孵育时间。
第二组：将孵育时间按照线性孵育的方式进行，孵育时间按照公式进行计算，公式如 下：
反应时间(s)＝A+(B-A)/D*(C-1)，其中，A表示第一个循环的反应时间常数，B表示最后循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整。
合成的孵育时间为13+(43-13)/150×(C-1)
补平的孵育时间为22+(52-22)/150×(C-1)
再生的孵育时间为27+(57-27)/150×(C-1)
具体的孵育时间见图7。
结果见图8和图9，根据本公开实施例的方法设置的线性孵育时间，比恒定孵育时间的Q30％下降缓慢，证明线性孵育时间的方法比恒定孵育会产生更好的效果。
实施例3
本实施案例是基于华大智造的测序仪MGISEQ-2000RS平台，使用的试剂均来源于该测序仪配套使用的建库试剂盒以及双端测序试剂盒(以下简称PE300试剂盒)；使用的验证样本来源于大肠杆菌；在验证过程中参考《MGISEQ-2000RS高通量(快速)测序试剂套装使用说明书》，进行PE300所需的DNB的制备、加载及PE300试剂槽的配制；本实施案例以PE300测序为例，进行指数孵育的方式进行孵育时间的设置，更改测序需要的PE300的生化脚本。
本实施例设计两组孵育时间进行对比：
第一组：保持整体合成(链合成)时间维持在60s，补平时间维持在120s，再生时间维持在60s，即维持恒定孵育时间。
第二组：参考图3，将孵育时间按照指数孵育的方式进行，孵育时间按照公式进行计算，公式如下：
S＝3*10^-6*K^3-0.0001*K^2-0.0089*K+1.3741+T
其中K为当前循环，Y为该循环的孵育时间，T为第一个循环的孵育时间，则
合成的孵育时间公式为：y＝3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+19
补平的孵育时间公式为：y＝3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+60
再生的孵育时间公式为：y＝3*10^-6*A^3-0.0001*A^2-0.0089*A+1.3741+40
结果如表5和图10，根据本公开实施例的方法设置的指数孵育时间，比恒定孵育时间的Q30％下降缓慢，证明指数孵育时间的方法比恒定孵育会产生更好的效果。
表5示出了本实施例中恒定孵育时间和指数孵育时间对测序质量的比对情况

注：SplitRate(％)：拆分率，数据中成功拆除标签的序列占总数据的比例。
RecoverValue(AVG)：该指标仅针对PE测序部分，反映二链信号回升情况。
实施例4
本实施案例是基于华大智造的测序仪MGISEQ-2000RS平台，使用的试剂均来源于该测序仪配套使用的建库试剂盒以及双端测序试剂盒；使用的验证样本来源于大肠杆菌；在验证过程中参考《MGISEQ-2000RS高通量(快速)测序试剂套装使用说明书》，进行PE250所需的DNB的制备、加载及PE250试剂槽的配制；本实施案例以PE250测序为例，进行梯度反应温度的设置，更改测序需要的PE250的生化脚本；
本实施例设计两组孵育温度进行对比：
第一组：保持整体补平温度维持在60℃，即维持恒定反应温度。
第二组：将反应温度按照梯度温度的方式进行，每个梯度的孵育温度不同，具体的孵育时间设置见表6。
结果见表7和图11，根据本公开实施例的方法设置的梯度反应温度，比起恒定孵育温度，梯度反应温度的二链信号回升更好，Q30％下降缓慢，整体数据量更高，证明梯度温度的方法比恒定温度会产生更好的效果。
表6：PE250一链梯度温度设置情况
表7：本实施例中恒定孵育温度和梯度孵育温度对整体测序质量的比对情况。

尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。
应该理解，可以使用上面所示的各种形式的流程，重新排序、增加或删除步骤。例如，本公开中记载的各步骤可以并行地执行也可以顺序地执行也可以不同的次序执行，只要能够实现本公开公开的技术方案所期望的结果，本文在此不进行限制。
需要说明的是，在本文中，诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上仅是本公开的具体实施方式，使本领域技术人员能够理解或实现本公开。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本公开的精神或范围的情况下，在其它实施例中实现。因此，本公开将不会被限制于本文的这些实施例，而是要符合与本文所申请的原理和新颖特点相一致的最宽的范围。

Claims (18)

  1. 一种测序方法，其特征在于，包括：
    (1)将固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；
    (2)检测荧光信号；
    (3)将所述反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；
    (4)重复步骤(1)到步骤(3)，以此类推，进行多轮循环，最终获得测序数据；
    其中，所述步骤(1)和步骤(3)至少之一中反应的时间是通过预先确定的循环数-反应时间关系计算获得。
  2. 根据权利要求1所述的方法，其特征在于，将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应时间相同，各个所述循环段之间的反应时间不同；
    N为大于1的整数。
  3. 根据权利要求2所述的方法，其特征在于，每个所述循环段的反应时间随着循环段数的增加而增加；
    随着循环段数的增加，各个所述循环段之间的反应时间呈指数函数或者线性函数分布方式增加；
    所述线性函数分布方式选自等差数列或者等比数列分布方式。
  4. 根据权利要求1所述的方法，其特征在于，所述循环数-反应时间关系选自如下公式：
    反应时间(s)＝A+(B-A)/D×(C-1)；
    其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整。
  5. 根据权利要求1所述的方法，其特征在于，确定所述循环数-反应时间关系的方法包括：
    在各个循环的反应时间相同的条件下，进行步骤(1)～(4)，根据获得的测序错误率，得到归一化曲线；
    将所述归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，所述系数P为最后一个循环预计增加的反应时间；
    将所述指数曲线对应的指数公式中加上第一个循环所需反应时间，得到所述循环数-反应时间关系。
  6. 根据权利要求1所述的方法，其特征在于，每轮循环中，所述反应包括将所述芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，所述反应完成后，将所述测序芯片转移至另外的反应容器中；
    随着循环次数增加，每个反应容器内发生浸泡次数增加，每次浸泡的时间延长。
  7. 根据权利要求6所述的方法，其特征在于，每次浸泡的时间按照下列公式确定：
    浸泡时间(s)＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，Y为同一反应容器总浸泡次数。
  8. 根据权利要求1所述的方法，其特征在于，所述步骤(1)和步骤(3)至少之一中所述反应的温度是通过预先确定的循环数-反应温度关系计算获得。
  9. 根据权利要求1所述的方法，其特征在于，将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应温度相同，各个所述循环段之间的反应温度不同；
    N为大于1的整数；
    每个所述循环段的反应温度随着循环段数的增加而增加；
    随着循环段数的增加，各个所述循环段之间的反应温度呈指数函数或者线性函数分布方式增加；
    所述线性函数分布方式选自等差数列或者等比数列分布方式。
  10. 根据权利要求1-9任一项所述的方法，其特征在于，进行多轮循环后，采用具有链置换活性的DNA聚合酶对目标核酸进行多重置换扩增反应，得到互补二链；将所述二链进行步骤(1)～(4)；
    其中，所述一链每次进行的反应时间和/或反应温度与所述二链每次进行相应的反应时间和/或反应温度不同。
  11. 一种测序系统，其特征在于，包括：
    芯片；
    测序设备，所述测序设备用于对固定于所述芯片表面的目标核酸进行测序；
    一个或多个处理器，所述一个或多个处理器被配置为用于执行：
    (1)使固定于芯片表面的目标核酸与聚合试剂进行反应，掺入核苷酸或核苷酸类似物，得到反应产物；
    (2)检测荧光信号；
    (3)使所述反应产物与再生试剂进行反应，获得可以进行下一轮聚合反应的产物；
    (4)重复步骤(1)到步骤(3)，以此类推，进行多轮循环，最终获得测序数据；
    其中，所述步骤(1)和步骤(3)至少之一中所述反应的时间是通过预先确定的循环 数-反应时间关系计算获得。
  12. 根据权利要求11所述的测序系统，其特征在于，所述一个或多个处理器被配置为用于执行：
    将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应时间相同，各个所述循环段之间的反应时间不同；
    N为大于1的整数；
    随着循环段数的增加，各个所述循环段之间的反应时间呈指数函数或者线性函数分布方式增加。
  13. 根据权利要求11所述的测序系统，其特征在于，所述一个或多个处理器被配置为用于执行按照如下公式确定反应时间：
    反应时间(s)＝A+(B-A)/D×(C-1)；
    其中，A表示第一个循环的反应时间常数，B表示最后一个循环的反应时间常数，C表示当前测序循环数，D表示测序总循环数；其中反应时间为公式结果取整；或者
    所述一个或多个处理器被配置为用于执行：
    根据在各个循环的反应时间相同的条件下进行测序获得的测序错误率，得到归一化曲线；
    将所述归一化曲线中每个循环的归一化数值乘以系数P，并将所得归一化数值与循环数构建指数曲线，所述系数P为最后一个循环预计增加的反应时间；
    将所述指数曲线对应的指数公式中加上第一个循环所需反应时间，得到所述循环数-反应时间关系。
  14. 根据权利要求11所述的测序系统，其特征在于，所述一个或多个处理器被配置为用于执行的每轮循环反应中的所述反应包括：使所述芯片浸泡于存放有聚合试剂或再生试剂的反应容器中，所述反应完成后，使所述芯片从所述反应容器中取出；
    随着循环次数增加，每个反应容器内发生浸泡次数的增加，每次浸泡的时间延长；
    所述一个或多个处理器被配置为用于执行按照下列公式确定每次浸泡的时间：
    浸泡时间＝50+10(X-1)/(Y-1)，X为同一反应容器浸泡的次数，浸泡时间单位为秒，Y为同一反应容器总浸泡次数。
  15. 根据权利要求11所述的系统，其特征在于，所述步骤(1)和步骤(3)至少之一中所述反应的温度是通过预先确定的循环数-反应温度关系计算获得；
    所述一个或多个处理器被配置为用于执行：
    将所述循环的总循环数划分为N个循环段，每个所述循环段内各循环的反应温度相同， 各个所述循环段之间的反应温度不同；
    N为大于1的整数；
    每个所述循环段的反应温度随着循环段数的增加而增加；
    随着循环段数的增加，各个所述循环段之间的反应温度呈指数函数或者线性函数分布方式增加；
    所述线性函数分布方式选自等差数列或者等比数列分布方式。
  16. 一种电子设备，其特征在于，包括：存储器、一个或多个处理器；所述存储器与所述一个或多个处理器耦合，所述存储器用于存储计算机程序代码，所述计算机程序代码包括计算机指令，所述一个或多个处理器调用所述计算机指令以使得所述电子设备执行如权利要求1～10任一项所述测序方法。
  17. 一种计算机可读存储介质，包括计算机指令，其特征在于，当所述计算机指令在电子设备上运行时，使得所述电子设备执行如权利要求1～10任一项所述测序方法。
  18. 一种计算机程序产品，其特征在于，当所述计算机程序产品在计算机上运行时，使得计算机执行如权利要求1～10任一项所述测序方法。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103917654A (zh) * 2011-04-01 2014-07-09 桑特里莱恩科技控股公司 用于对长核酸进行测序的方法和系统
CN105001292A (zh) * 2015-07-14 2015-10-28 深圳市瀚海基因生物科技有限公司 一种光可断裂的荧光标记可逆终端化合物及其在dna或rna测序中的用途
WO2016038381A1 (en) * 2014-09-11 2016-03-17 Illumina Cambridge Limited Method of obtaining paired-end sequencing information
CN108603227A (zh) * 2015-11-18 2018-09-28 卡利姆·U·米尔 超分辨率测序
US20200056229A1 (en) * 2017-11-29 2020-02-20 Xgenomes Corp. Sequencing by emergence
WO2020243187A1 (en) * 2019-05-29 2020-12-03 Xgenomes Corp. Sequencing by emergence

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103917654A (zh) * 2011-04-01 2014-07-09 桑特里莱恩科技控股公司 用于对长核酸进行测序的方法和系统
WO2016038381A1 (en) * 2014-09-11 2016-03-17 Illumina Cambridge Limited Method of obtaining paired-end sequencing information
CN105001292A (zh) * 2015-07-14 2015-10-28 深圳市瀚海基因生物科技有限公司 一种光可断裂的荧光标记可逆终端化合物及其在dna或rna测序中的用途
CN108603227A (zh) * 2015-11-18 2018-09-28 卡利姆·U·米尔 超分辨率测序
US20200056229A1 (en) * 2017-11-29 2020-02-20 Xgenomes Corp. Sequencing by emergence
WO2020243187A1 (en) * 2019-05-29 2020-12-03 Xgenomes Corp. Sequencing by emergence

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