A base sequencing verification method and system based on ion current and tunneling current characteristics

Abstract

The application relates to a base sequencing verification method based on ion current and tunneling current characteristics, which comprises the following steps: measuring ion current and tunneling current; comparing the drop of the ion current with a standard ion current drop to obtain the classification probability of the base corresponding to the ion current belonging to the base corresponding to the standard ion current drop; comparing the tunneling current with a standard tunneling current to obtain the classification probability of the base corresponding to the tunneling current belonging to the base corresponding to the standard tunneling current; distributing a time weight to the classification probability of the ion current according to the time of measuring the ion current relative to the time of measuring the tunneling current; inputting the classification probability of the ion current, the time weight of the classification probability of the ion current and the classification probability of the tunneling current into a neural network model to obtain a probability; and taking the base type corresponding to the maximum probability as the type of a base to be identified. The application can improve the accuracy and reliability of base identification and reduce the error rate of base identification.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a base sequencing verification method and system based on the characteristics of ion current and tunneling current. Background Technology

[0002] Nanopore sequencing is a representative of next-generation sequencing technologies. Its core principle is to identify bases by measuring the changes in ionic current caused by a single DNA or RNA molecule passing through a nanopore. However, traditional nanopore sequencing technology faces a key challenge: due to the extremely high measurement speed (the bases spend very little time inside the nanopore) and the presence of signal noise, the accuracy and reliability of base identification relying solely on ionic current are limited, especially when distinguishing structurally similar bases (such as cytosine C and thymine T), where the error rate is relatively high.

[0003] Therefore, how to improve the accuracy and reliability of base identification and reduce the error rate of base identification is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] This application provides a base sequencing verification method and system based on ion current and tunneling current characteristics to improve the accuracy and reliability of base identification and reduce the error rate of base identification.

[0005] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0006] A base sequencing verification method based on ion current and tunneling current characteristics includes the following steps: Step T10: During the time period when the base to be identified passes through the nanopore, the ion current is measured multiple times, and the tunneling current is measured at the moment when the base to be identified passes through the gap between the nanoelectrode pairs inside the nanopore; Step T20: The decrease in each ion current is calculated, and the decrease in each ion current is compared with the decrease in a standard ion current to obtain the classification probability of the base corresponding to each ion current belonging to the base corresponding to the decrease in the standard ion current; Step T30: The tunneling current is compared with the standard tunneling current to obtain the tunneling current... Step T40: Assign a time weight to the classification probability of each ion current corresponding to the base corresponding to the standard tunneling current, based on the time of measuring each ion current relative to the time of measuring the tunneling current; Step T50: Input the classification probability of each ion current, the time weight of the classification probability of each ion current, and the classification probability of the tunneling current into the neural network model to obtain the probability of the base to be identified belonging to each class of bases; Step T60: Take the type of the base corresponding to the highest probability as the type of the base to be identified, thereby realizing the identification of the base type.

[0007] The base sequencing verification method based on ion current and tunneling current characteristics described above preferably includes the following sub-steps for calculating the classification probability corresponding to the ion current: comparing the decrease in ion current with the corresponding standard ion current decrease in the base standard feature library; if the difference exceeds the measurement range of the standard ion current decrease, the classification probability of the base corresponding to the ion current belonging to the base corresponding to the standard ion current decrease is 0; if the difference does not exceed the measurement range of the standard ion current decrease, the classification probability of the base corresponding to the ion current belonging to the base corresponding to the standard ion current decrease is obtained according to the proportion of the difference relative to the measurement range of the standard ion current decrease.

[0008] The base sequencing verification method based on ion current and tunneling current characteristics described above preferably includes the following sub-steps for calculating the classification probability corresponding to the tunneling current: comparing the tunneling current with the corresponding standard tunneling current in the base standard feature library; if the difference exceeds the measurement range of the standard tunneling current, the base corresponding to the tunneling current is considered to have a classification probability of 0; if the difference does not exceed the measurement range of the standard tunneling current, the classification probability of the base corresponding to the tunneling current belonging to the base corresponding to the standard tunneling current is obtained according to the proportion of the difference relative to the measurement range of the standard tunneling current.

[0009] The base sequencing verification method based on ion current and tunneling current characteristics described above preferably assigns time weights to the classification probabilities corresponding to ion currents, including the following sub-steps: taking the time of measuring the tunneling current as the reference time for the corresponding base, calculating the time interval between the time of measuring multiple ion currents corresponding to the base and the reference time; if the time interval is not zero, using the reciprocal of the time interval as the time weight of the classification probability corresponding to the corresponding ion current; if the time interval is zero, using multiples of the sum of the reciprocals of adjacent time intervals as the time weight of the classification probability corresponding to the corresponding ion current.

[0010] In the base sequencing verification method based on ion current and tunneling current characteristics described above, preferably, the expression of the neural network model is as follows:

[0011] ;

[0012] in, For the first The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. ; For the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X; For the first ion current Corresponding classification probability Time weighting; The quantity of ion current; tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X.

[0013] A base sequencing verification system based on ion current and tunneling current characteristics includes: a nanopore gene sequencer and a sequencing computing center; the nanopore gene sequencer measures the ion current multiple times during the time period when the base to be identified passes through the nanopore, and measures the tunneling current at the moment when the base to be identified passes through the gap between the nanoelectrode pairs inside the nanopore; the sequencing computing center calculates the decrease in each ion current, compares the decrease in each ion current with the decrease in a standard ion current, and obtains the classification probability that the base corresponding to each ion current belongs to the base corresponding to the decrease in the standard ion current; the tunneling current is compared with the standard tunneling current to obtain the classification probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current.

[0014] The sequencing computing center assigns a time weight to the classification probability corresponding to each ion current based on the time of measurement of each ion current relative to the time of measurement of the tunneling current. The classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current are input into the neural network model to obtain the probability that the base to be identified belongs to each type of base. The type of the base with the highest probability is taken as the type of the base to be identified, thereby realizing the identification of the base type.

[0015] In the base sequencing verification system based on ion current and tunneling current characteristics described above, preferably, the sequencing computing center compares the decrease in ion current with the corresponding standard ion current decrease in the base standard feature library. If the difference exceeds the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is 0. If the difference does not exceed the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is obtained based on the proportion of the difference relative to the measurement range of the standard ion current decrease.

[0016] In the base sequencing verification system based on ion current and tunneling current characteristics described above, preferably, the sequencing computing center compares the tunneling current with the corresponding standard tunneling current in the base standard feature library. If the difference exceeds the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the classification of the base corresponding to the standard tunneling current is considered to be 0. If the difference does not exceed the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the classification of the base corresponding to the standard tunneling current is obtained according to the proportion of the difference relative to the measurement range of the standard tunneling current.

[0017] In the base sequencing verification system based on ion current and tunneling current characteristics described above, preferably, the sequencing computing center uses the time when the tunneling current is measured as the reference time for the corresponding base, calculates the time interval between the time when multiple ion currents corresponding to the base are measured and the reference time, and if the time interval is not zero, the reciprocal of the time interval is used as the time weight of the classification probability corresponding to the corresponding ion current; if the time interval is zero, the sum of the reciprocals of the adjacent time intervals is used as the time weight of the classification probability corresponding to the corresponding ion current.

[0018] In the base sequencing verification system based on ion current and tunneling current characteristics described above, preferably, the expression of the neural network model is as follows:

[0019] ;

[0020] in, For the first The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. ; For the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X; For the first ion current Corresponding classification probability Time weighting; The quantity of ion current; tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X.

[0021] Compared to the aforementioned background technology, the base sequencing verification method and system based on ion current and tunneling current characteristics provided in this application identify base types by combining ion current and tunneling current. Since tunneling current is extremely sensitive to the electronic structure of DNA / RNA molecules, this application can provide high base resolution and accurately distinguish between structurally similar bases. Furthermore, since this application uses multiple measurements of ion current when identifying base types, it can improve the accuracy and reliability of base type identification and avoid identification errors caused by using a single ion current for base type identification. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 This is a flowchart of a base sequencing verification method based on ion current and tunneling current characteristics;

[0024] Figure 2 This is a schematic diagram of a base sequencing verification system based on the characteristics of ion current and tunneling current. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] Example 1

[0027] like Figure 1 As shown, this application provides a base sequencing verification method based on ion current and tunneling current characteristics, comprising the following steps:

[0028] Step T10: During the time period when the base to be identified passes through the nanopore, the ion current is measured multiple times, and the tunneling current is measured at the moment when the base to be identified passes through the gap between the nanoelectrode pairs in the nanopore.

[0029] A nanopore with a diameter of approximately 1 nm to 3 nm is fabricated on a silicon nitride thin film. Electrodes (e.g., Ag / AgCl electrodes) are placed on both sides of the nanopore to apply a voltage (e.g., 200 mV) across the nanopore. Charged ions (K+) in an electrolyte solution (e.g., KCl solution) are then introduced. + Cl- The current will pass through the nanopore due to the electric field generated by the electrodes on both sides of the nanopore, forming an ion current. For ease of distinction, this ion current is referred to as the original ion current in this application. At the inner wall of the middle part of the nanopore, a pair of extremely close (nanoscale spacing) nanoelectrodes (e.g., tip spacing of about 1 nm) are integrated by microfabrication techniques (e.g., EBL and FIB), and this pair of nanoelectrodes is connected to a high-gain, high-bandwidth transimpedance amplifier to apply an independent voltage (e.g., 0.1-0.5V).

[0030] During sequencing, DNA / RNA molecules are captured under an electric field, and their bases (A / T / C / G) sequentially enter the nanopore at a predetermined velocity. The entry of these bases partially blocks the ion channels provided by the nanopore for charged ions, causing a decrease in the permeability of charged ions through the nanopore. This results in a characteristic "drop peak" in the ion current. Because different types of bases block the ion channels to varying degrees, the resulting ion currents also differ. Therefore, in the DNA / RNA molecule... The time it takes for a base to pass through a nanopore was measured. ion current ,in For the first The first ion current corresponding to each base. For the first The second ion current corresponding to each base. For the first The first base corresponds to the first ion current The number of ion currents, by the first Measured by each base ion current This can be used to identify the first The type of the base. Optionally, the first base is measured at the same time interval. Each base corresponds to Ion current.

[0031] When the bases of a DMA / RNA molecule pass sequentially through the gaps between nanoelectrode pairs on the inner wall of a nanopore, the electrons of the bases themselves will form a weak tunneling current between the two nanoelectrodes due to the "quantum tunneling effect" (electrons jumping over an energy barrier that would otherwise be insurmountable). Because different types of bases have different molecular structures, the intensity and characteristics of the tunneling current will also differ. Therefore, in the DNA / RNA molecule... When a base passes through the gap between the nanoelectrode pairs, the tunneling current generated by that base between the nanoelectrode pairs is measured. Through the first Tunneling current measured by each base It can also be used to identify the first The type of base.

[0032] The speed at which bases pass through the nanopore can be controlled by enzymes or by optimizing the voltage across the nanopore. This speed can be controlled at the millisecond / base level. By controlling the speed at which bases pass through the nanopore, it can be ensured that each base can remain in the ion detection region and tunneling detection region within the nanopore for a longer period of time, thus ensuring that the ion current of each base can be detected more times.

[0033] Step T20: Calculate the decrease of each ion current and compare the decrease of each ion current with the decrease of the standard ion current to obtain the classification probability of the base corresponding to each ion current belonging to the base corresponding to the decrease of the standard ion current.

[0034] In the measurement After each ion current is measured, the decrease in each ion current is compared with the original ion current (i.e., the ion current when no bases pass through the nanopore) to obtain the decrease in each ion current.

[0035] Furthermore, the first The first base corresponds to the first ion current The decline The expression is as follows:

[0036] ;

[0037] in, This represents the original ion current.

[0038] This application has pre-constructed a standard feature library of bases (A / T / C / G), which specifies the standard ion current reduction and standard tunneling current corresponding to each type of base, for example: , , , ,in This represents the decrease in standard ion current corresponding to base A. This represents the standard tunneling current corresponding to base A. This represents the decrease in standard ion current corresponding to base C. This represents the standard tunneling current corresponding to the base C.

[0039] The decrease in ion current is compared with the corresponding decrease in standard ion current in the base (A / T / C / G) standard feature library. If the difference (the difference between the decrease in ion current and the corresponding decrease in standard ion current) exceeds the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is considered to be 0. If the difference does not exceed the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is obtained based on the proportion of the difference relative to the measurement range of the standard ion current decrease.

[0040] Furthermore, the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X The expression is as follows:

[0041] ;

[0042] in, This represents the decrease in standard ion current corresponding to base X. for Scope of measurement .

[0043] Step T30: Compare the tunneling current with the standard tunneling current to obtain the classification probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current.

[0044] The tunneling current is compared with the corresponding standard tunneling current in the base (A / T / C / G) standard feature library. If the difference (the difference between the tunneling current and the corresponding standard tunneling current) exceeds the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current is considered to be 0. If the difference does not exceed the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current is obtained according to the proportion of the difference relative to the measurement range of the standard tunneling current.

[0045] Furthermore, tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X The expression is as follows:

[0046] ;

[0047] in, This represents the standard tunneling current corresponding to base X. for The range of measurement.

[0048] Step T40: Assign time weights to the classification probabilities corresponding to each ion current based on the time when each ion current is measured relative to the time when the tunneling current is measured.

[0049] To measure the first The tunneling current corresponding to each base The time is the The reference time of each base Calculate and measure the first Each base corresponds to ion current The moment Compared with the reference time The time interval, where To measure the first The first ion current corresponding to each base At that moment, To measure the first The second ionic current corresponding to each base At that moment, To measure the first The first base corresponds to the first ion current At that moment.

[0050] Furthermore, the measurement of the first The first base corresponds to the first ion current The moment Compared with the reference time time interval The expression is as follows:

[0051] ;

[0052] If the time interval is not zero, the reciprocal of the time interval is used as the time weight of the classification probability corresponding to the corresponding ion current. If the time interval is zero, the sum of the reciprocals of the adjacent time intervals is used as the time weight of the classification probability corresponding to the corresponding ion current.

[0053] Furthermore, ion current Corresponding classification probability Time weight The expression is as follows:

[0054] ;

[0055] in, To measure the first The first base corresponds to the first ion current The moment Compared with the reference time The time interval, To measure the first The first base corresponds to the first ion current The moment Compared with the reference time The time interval, To adjust the parameters.

[0056] Step T50: Input the classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current into the neural network model to obtain the probability that the base to be identified belongs to each class of bases.

[0057] In this application, a neural network model is constructed and trained. The classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current are taken as inputs into the neural network model to obtain the output of the neural network model. The output of the neural network model is used as the probability that the base passing through the nanopore belongs to each class of bases.

[0058] Furthermore, the expression for the constructed neural network model is as follows:

[0059] ;

[0060] in, The output of the neural network represents the first... The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. , and These are constants obtained through training.

[0061] Step T60: The type of the base with the highest probability is taken as the type of the base to be identified, thereby realizing the identification of the base type;

[0062] The first of the DNA / RNA molecules Arrange the bases that belong to the base group (A / T / C / G) from highest to lowest probability, and select the base type corresponding to the highest probability as the first base of the DNA / RNA molecule. The bases belong to a certain type of base. By identifying the type of all bases in the DNA / RNA molecule in the manner described above, the base sequencing results are obtained.

[0063] Example 2

[0064] like Figure 2 As shown, this application provides a base sequencing verification system 200 based on the characteristics of ion current and tunneling current, including: a nanopore gene sequencer 210 and a sequencing computing center 220, wherein the sequencing computing center 220 and the nanopore gene sequencer 210 can be an integrated device or a separate device.

[0065] The Nanopore Gene Sequencing 210 measures the ion current multiple times during the time period when the base to be identified passes through the nanopore, and measures the tunneling current at the moment when the base to be identified passes through the gap between the nanoelectrode pairs inside the nanopore.

[0066] The nanopore gene sequencer 210 has a silicon nitride film 211 on which a nanopore with a diameter of approximately 1 nm-3 nm is fabricated. Electrodes (e.g., Ag / AgCl electrodes) 212 are disposed on both sides of the nanopore for applying a voltage (e.g., 200 mV) across the nanopore. An electrolyte solution (e.g., KCl solution) 213 is contained within the nanopore. The electrolyte solution 213 contains charged ions (KCl). + Cl - The current will pass through the nanopore due to the electric field generated by the electrodes 212 on both sides of the nanopore, forming an ion current. For ease of distinction, this ion current is referred to as the original ion current in this application. At the inner wall of the middle part of the nanopore, a pair of very close (nanoscale spacing) nanoelectrodes (e.g., tip spacing of about 1 nm) 214 are integrated by microfabrication techniques (e.g., EBL and FIB), and this pair of nanoelectrodes 214 are connected to a high-gain, high-bandwidth transimpedance amplifier 215 to apply an independent voltage (e.g., 0.1-0.5V).

[0067] During sequencing, DNA / RNA molecules are captured under an electric field, and their bases (A / T / C / G) sequentially enter the nanopore at a predetermined velocity. The entry of these bases partially blocks the ion channels provided by the nanopore for charged ions, causing a decrease in the permeability of charged ions through the nanopore. This results in a characteristic "drop peak" in the ion current. Because different types of bases block the ion channels to varying degrees, the resulting ion currents also differ. Therefore, in the DNA / RNA molecule... Nanopore genome sequencer 210 measured the time it takes for a single base to pass through a nanopore. ion current ,in For the first The first ion current corresponding to each base. For the first The second ion current corresponding to each base. For the first The first base corresponds to the first ion current The number of ion currents, by the first Measured by each base ion current This can be used to identify the first The type of the base. Optionally, the first base is measured at the same time interval. Each base corresponds to Ion current.

[0068] When the bases of a DMA / RNA molecule pass sequentially through the gaps between the nanoelectrode pairs on the inner wall of a nanopore, the electrons of the bases themselves will form a weak tunneling current between the two nanoelectrodes due to the "quantum tunneling effect" (electrons jumping over an energy barrier that would otherwise be insurmountable). Because different types of bases have different molecular structures, the intensity and characteristics of the tunneling current will also differ. Therefore, in the DNA / RNA molecule... When a base passes through the gap between the nanoelectrode pairs, the nanopore gene sequencer 210 measures the tunneling current generated between the nanoelectrode pairs by that base. Through the first Tunneling current measured by each base It can also be used to identify the first The type of base.

[0069] The speed at which bases pass through the nanopore can be controlled by enzymes or by optimizing the voltage across the nanopore. This speed can be controlled at the millisecond / base level. By controlling the speed at which bases pass through the nanopore, it can be ensured that each base can remain in the ion detection region and tunneling detection region within the nanopore for a longer period of time, thus ensuring that the ion current of each base can be detected more times.

[0070] The sequencing computing center 220 calculates the decrease in each ion current and compares the decrease in each ion current with the decrease in the standard ion current to obtain the classification probability that the base corresponding to each ion current belongs to the base corresponding to the decrease in the standard ion current.

[0071] In the measurement After each ion current, the sequencing computing center 220 compares each ion current with the original ion current (i.e., the ion current when no bases pass through the nanopore) to obtain the decrease in each ion current.

[0072] Furthermore, the first The first base corresponds to the first ion current The decline The expression is as follows:

[0073] ;

[0074] in, This represents the original ion current.

[0075] In this application, a standard feature library of bases (A / T / C / G) has been pre-constructed in the sequencing computing center 220. This library specifies the standard ion current reduction and standard tunneling current corresponding to each type of base. For example: , , , ,in This represents the decrease in standard ion current corresponding to base A. This represents the standard tunneling current corresponding to base A. This represents the decrease in standard ion current corresponding to base C. This represents the standard tunneling current corresponding to the base C.

[0076] The sequencing computing center 220 compares the decrease in ion current with the corresponding standard ion current decrease in the base (A / T / C / G) standard feature library. If the difference (the difference between the decrease in ion current and the corresponding standard ion current decrease) exceeds the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is considered to be 0. If the difference does not exceed the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is obtained based on the proportion of the difference relative to the measurement range of the standard ion current decrease.

[0077] Furthermore, the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X The expression is as follows:

[0078] ;

[0079] in, This represents the decrease in standard ion current corresponding to base X. for Scope of measurement .

[0080] The sequencing computing center 220 compares the tunneling current with the standard tunneling current to obtain the classification probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current.

[0081] The sequencing computing center 220 compares the tunneling current with the corresponding standard tunneling current in the standard feature library of bases (A / T / C / G). If the difference (the difference between the tunneling current and the corresponding standard tunneling current) exceeds the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the classification of the base corresponding to the standard tunneling current is considered to be 0. If the difference does not exceed the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the classification of the base corresponding to the standard tunneling current is obtained according to the proportion of the difference relative to the measurement range of the standard tunneling current.

[0082] Furthermore, tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X The expression is as follows:

[0083] ;

[0084] in, This represents the standard tunneling current corresponding to base X. for The range of measurement.

[0085] The sequencing computing center 220 assigns time weights to the classification probability corresponding to each ion current based on the time when each ion current is measured relative to the time when the tunneling current is measured.

[0086] The sequencing computing center 220 has measured the first... The tunneling current corresponding to each base The time is the The reference time of each base Calculate and measure the first Each base corresponds to ion current The moment Compared with the reference time The time interval, where To measure the first The first ion current corresponding to each base At that moment, To measure the first The second ionic current corresponding to each base At that moment, To measure the first The first base corresponds to the first ion current At that moment.

[0087] Furthermore, the measurement of the first The first base corresponds to the first ion current The moment Compared with the reference time time interval The expression is as follows:

[0088] ;

[0089] If the time interval is not zero, the sequencing computing center 220 uses the reciprocal of the time interval as the time weight of the classification probability corresponding to the corresponding ion current. If the time interval is zero, the sequencing computing center 220 uses multiples of the sum of the reciprocals of the adjacent time intervals as the time weight of the classification probability corresponding to the corresponding ion current.

[0090] Furthermore, ion current Corresponding classification probability Time weight The expression is as follows:

[0091] ;

[0092] in, To measure the first The first base corresponds to the first ion current The moment Compared with the reference time The time interval, To measure the first The first base corresponds to the first ion current The moment Compared with the reference time The time interval, To adjust the parameters.

[0093] The sequencing computing center 220 inputs the classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current into the neural network model to obtain the probability that the base to be identified belongs to each class of bases.

[0094] In this application, a neural network model is pre-built and trained in the sequencing computing center 220. The sequencing computing center 220 takes the classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current as inputs into the neural network model, thereby obtaining the output of the neural network model. The output of the neural network model is used as the probability that the base passing through the nanopore belongs to each class of bases.

[0095] Furthermore, the expression for the constructed neural network model is as follows:

[0096] ;

[0097] in, The output of the neural network represents the first... The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. , and These are constants obtained through training.

[0098] The sequencing computing center 220 uses the type of the base with the highest probability as the type of the base to be identified, thereby realizing the identification of the base type.

[0099] The sequencing computing center 220 will sequence the DNA / RNA molecules' first... Arrange the bases that belong to the base group (A / T / C / G) from highest to lowest probability, and select the base type corresponding to the highest probability as the first base of the DNA / RNA molecule. The bases belong to a certain type of base. By identifying the type of all bases in the DNA / RNA molecule in the manner described above, the base sequencing results are obtained.

[0100] In this application, base type identification is performed by combining ion current and tunneling current. Since tunneling current is extremely sensitive to the electronic structure of DNA / RNA molecules, this application can provide high base resolution and accurately distinguish structurally similar bases (such as cytosine C and thymine T). Furthermore, since this application uses multiple measurements of ion current when identifying base types, the accuracy and reliability of base type identification can be improved, avoiding identification errors caused by using a single ion current.

[0101] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0102] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A base sequencing verification method based on ion current and tunneling current characteristics, characterized in that, Includes the following steps: Step T10: During the time period when the base to be identified passes through the nanopore, the ion current is measured multiple times, and the tunneling current is measured at the moment when the base to be identified passes through the gap between the nanoelectrode pairs in the nanopore. Step T20: Calculate the decrease of each ion current, compare the decrease of each ion current with the decrease of the standard ion current, and obtain the classification probability of the base corresponding to each ion current belonging to the base corresponding to the decrease of the standard ion current. Step T30: Compare the tunneling current with the standard tunneling current to obtain the classification probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current. Step T40: Assign time weights to the classification probabilities corresponding to each ion current based on the time when each ion current is measured relative to the time when the tunneling current is measured. Step T50: Input the classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current into the neural network model to obtain the probability that the base to be identified belongs to each class of bases. Step T60: The type of the base with the highest probability is taken as the type of the base to be identified, thereby realizing the identification of the base type.

2. The base sequencing verification method based on ion current and tunneling current characteristics according to claim 1, characterized in that, The calculation of the classification probability corresponding to the ion current includes the following sub-steps: The decrease in ion current is compared with the decrease in standard ion current in the base standard feature library; If the difference exceeds the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the classification of the base corresponding to the standard ion current decrease is 0. If the difference does not exceed the measurement range of the standard ion current decrease, then the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is obtained based on the proportion of the difference relative to the measurement range of the standard ion current decrease.

3. The base sequencing verification method based on ion current and tunneling current characteristics according to claim 1 or 2, characterized in that, The calculation of the classification probability corresponding to the tunneling current includes the following sub-steps: The tunneling current is compared with the corresponding standard tunneling current in the base standard feature library; If the difference exceeds the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the classification of the base corresponding to the standard tunneling current is considered to be 0. If the difference does not exceed the measurement range of the standard tunneling current, the probability of the base corresponding to the tunneling current belonging to the base corresponding to the standard tunneling current is obtained based on the proportion of the difference relative to the measurement range of the standard tunneling current.

4. The base sequencing verification method based on ion current and tunneling current characteristics according to claim 1 or 2, characterized in that, Assigning time weights to the classification probabilities corresponding to ion currents includes the following sub-steps: Using the time when the tunneling current is measured as the reference time for the corresponding base, the time interval between the time when the multiple ion currents corresponding to the base are measured and the reference time is calculated respectively. If the time interval is not zero, the reciprocal of the time interval is used as the time weight of the classification probability corresponding to the ion current. If the time interval is zero, then the sum of the reciprocals of the adjacent time intervals is used as the time weight of the classification probability corresponding to the ion current.

5. The base sequencing verification method based on ion current and tunneling current characteristics according to claim 1 or 2, characterized in that, The expression for the neural network model is as follows: ； in, For the first The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. ; For the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X; For the first ion current Corresponding classification probability Time weighting; The quantity of ion current; tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X.

6. A base sequencing verification system based on ion current and tunneling current characteristics, characterized in that, include: Nanopore gene sequencer and sequencing computing center; The nanopore gene sequencer measures the ion current multiple times during the time it takes for the base to be identified to pass through the nanopore, and measures the tunneling current at the moment when the base to be identified passes through the gap between the nanoelectrode pairs inside the nanopore. The sequencing computing center calculates the decrease in current for each ion and compares it with the decrease in current for a standard ion to obtain the probability that the base corresponding to each ion current belongs to the base corresponding to the decrease in current for that standard ion. The tunneling current is compared with the standard tunneling current to obtain the probability that the base corresponding to the tunneling current belongs to the base corresponding to the standard tunneling current. The sequencing computing center assigns a time weight to the classification probability corresponding to each ion current based on the time of measurement of each ion current relative to the time of measurement of the tunneling current. The classification probability corresponding to each ion current, the time weight of the classification probability corresponding to each ion current, and the classification probability corresponding to the tunneling current are input into the neural network model to obtain the probability that the base to be identified belongs to each type of base. The type of the base with the highest probability is taken as the type of the base to be identified, thereby realizing the identification of the base type.

7. The base sequencing verification system based on ion current and tunneling current characteristics according to claim 6, characterized in that, The sequencing computing center compares the decrease in ion current with the corresponding decrease in standard ion current in the base standard feature library. If the difference exceeds the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is 0. If the difference does not exceed the measurement range of the standard ion current decrease, the probability that the base corresponding to the ion current belongs to the base corresponding to the standard ion current decrease is obtained based on the proportion of the difference to the measurement range of the standard ion current decrease.

8. The base sequencing verification system based on ion current and tunneling current characteristics according to claim 6 or 7, characterized in that, The sequencing computing center compares the tunneling current with the corresponding standard tunneling current in the standard base feature library. If the difference exceeds the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the category of the base corresponding to the standard tunneling current is considered to be 0. If the difference does not exceed the measurement range of the standard tunneling current, the probability that the base corresponding to the tunneling current belongs to the category of the base corresponding to the standard tunneling current is obtained based on the proportion of the difference relative to the measurement range of the standard tunneling current.

9. The base sequencing verification system based on ion current and tunneling current characteristics according to claim 6 or 7, characterized in that, The sequencing computing center uses the time when the tunneling current is measured as the reference time for the corresponding base. It calculates the time interval between the time when the multiple ion currents corresponding to the base are measured and the reference time. If the time interval is not zero, the reciprocal of the time interval is used as the time weight of the classification probability corresponding to the corresponding ion current. If the time interval is zero, the sum of the reciprocals of the adjacent time intervals is used as the time weight of the classification probability corresponding to the corresponding ion current.

10. The base sequencing verification system based on ion current and tunneling current characteristics according to claim 6 or 7, characterized in that, The expression for the neural network model is as follows: ； in, For the first The probability that a base belongs to base X; This represents the total weight of the classification probabilities corresponding to the ion current. The total weight of the classification probability corresponding to the tunneling current. ; For the first ion current The corresponding number Each base belongs to the standard ion current decrease. The classification probability of the corresponding base X; For the first ion current Corresponding classification probability Time weighting; The quantity of ion current; tunneling current The corresponding number Each base represents a standard tunneling current. The classification probability of the corresponding base X.

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