High-efficiency and low-cost virus RNA direct sequencing method

Abstract

The invention relates to the technical field of gene sequencing, and discloses a high-efficiency and low-cost virus RNA direct sequencing method which comprises the following steps: extracting the molecular integrity and purification degree of virus RNA, and configuring a nanopore sensor of the virus RNA; identifying the aperture size of the nanopore sensor, extracting the molecular length of the virus RNA, and setting a multi-aperture molecular detection mechanism of the virus RNA in the nanopore sensor; identifying the hole end position of the nanopore sensor, and setting an electrical signal acquisition unit of the nanopore sensor; collecting a current change signal of the virus RNA in real time, and constructing a sequence analysis model of the virus RNA; setting an automatic parameter adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length and the modification characteristics; in combination with a multi-aperture molecular detection mechanism, an electrical signal acquisition unit and an automatic parameter adjustment mode, direct sequencing processing of virus RNA is executed. According to the method, the sequencing efficiency of the virus RNA can be improved while the sequencing cost is reduced.

Description

Technical Field

[0001] The present invention relates to a method for directly sequencing viral RNA with high efficiency and low cost, and belongs to the technical field of gene sequencing. Background Art

[0002] Viral RNA refers to a class of viruses with ribonucleic acid (RNA) as the genetic material, including influenza virus, coronavirus, human immunodeficiency virus (HIV), rabies virus, etc. As the carrier of viral genetic information, the sequence characteristics of viral RNA are directly related to the replication, transmission, pathogenicity and mutation ability of the virus. Therefore, efficient and accurate sequencing of viral RNA has become an important means to analyze the genomic characteristics of the virus, understand the virus replication mechanism, track the virus mutation law, and develop targeted antiviral drugs and vaccines.

[0003] Traditional viral RAN sequencing methods, such as Sanger sequencing, are mainly based on the dideoxynucleotide chain termination method, and sequence by electrophoretically separating DNA fragments of different lengths. Although this method can provide high-precision long sequence information, it is inefficient and costly, and is not suitable for large-scale sequencing.

[0004] Therefore, there is an urgent need for a solution that can improve the sequencing efficiency of viral RNA while reducing the sequencing cost. Summary of the Invention

[0005] The present invention provides a method for directly sequencing viral RNA with high efficiency and low cost, and its main purpose is to improve the sequencing efficiency of viral RNA while reducing the sequencing cost.

[0006] To achieve the above object, a method for directly sequencing viral RNA with high efficiency and low cost provided by the present invention includes:

[0007] Collect a virus sample to be detected, extract the viral RNA in the virus sample, perform a quality test on the viral RNA to obtain a quality test result, and when the quality test result meets a preset effect, extract the molecular integrity and purification degree of the viral RNA;

[0008] Based on the molecular integrity and the purification degree, configure a nanopore sensor for the viral RNA, identify the pore size of the nanopore sensor, and extract the molecular length of the viral RNA. According to the pore size and the molecular length, set a multi-pore molecular detection mechanism of the viral RNA in the nanopore sensor;

[0009] Identify the pore end position of the nanopore sensor, and based on the pore end position, set the electrical signal acquisition unit of the nanopore sensor. Based on the multi-aperture molecular detection mechanism and the electrical signal acquisition unit, collect the current change signal of the viral RNA in real time;

[0010] Based on the current change signal, extract the current signal characteristics of the viral RNA. Based on the current signal characteristics, construct a sequence analysis model of the viral RNA;

[0011] According to the sequence analysis model, identify the characteristic sequence of the viral RNA. Based on the characteristic sequence, extract the modification characteristics of the viral RNA. According to the molecular integrity, the purification degree, the molecular length, and the modification characteristics, set the parameter automatic adjustment mode of the nanopore sensor;

[0012] Combined with the multi-aperture molecular detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode, perform direct sequencing processing of the viral RNA to obtain a direct sequencing result.

[0013] Optionally, configuring the nanopore sensor for the viral RNA based on the molecular integrity and the purification degree includes:

[0014] Based on the molecular integrity, extract the structural characteristics of the viral RNA;

[0015] According to the structural characteristics, determine the sequence length and the structural complexity of the viral RNA;

[0016] Based on the sequence length and the structural complexity, identify the molecular movement speed of the viral RNA;

[0017] According to the purification degree, analyze the surface adsorption ability of the viral RNA;

[0018] Combined with the structural characteristics, the surface adsorption ability, and the molecular movement speed, configure the nanopore sensor for the viral RNA.

[0019] Optionally, setting the multi-aperture molecular detection mechanism of the viral RNA in the nanopore sensor according to the pore size and the molecular length includes:

[0020] According to the molecular length, define the sequencing molecular type of the nanopore sensor;

[0021] Calculate the fitting coefficient between the sequencing molecular type and the pore size;

[0022] Based on the fitting coefficient, set the intelligent matching mode between the pore size and the sequencing molecular type;

[0023] Create the porous-aperture sequencing channel of the viral RNA in the nanopore sensor according to the intelligent matching mode;

[0024] Identify the molecular characteristics of the viral RNA, and set the dynamic sequencing parameters of the porous-aperture sequencing channel according to the molecular characteristics;

[0025] Combine the porous-aperture sequencing channel, the intelligent matching mode and the dynamic sequencing parameters to set up the porous-aperture molecular detection mechanism of the viral RNA in the nanopore sensor.

[0026] Optionally, setting the electrical signal acquisition unit of the nanopore sensor according to the pore end position includes:

[0027] Set the molecular driving electric field of the nanopore sensor according to the pore end position;

[0028] Based on the molecular driving electric field, identify the nanopore sensing molecule of the nanopore sensor;

[0029] Configure the signal extraction device of the nanopore sensing molecule, and collect the analog current signal of the nanopore sensing molecule according to the signal extraction device;

[0030] Identify the signal quality of the analog current signal, and set the signal gain component of the analog current signal based on the signal quality;

[0031] Analyze the output characteristics of the signal gain component, and set the analog-to-digital converter of the analog current signal according to the output characteristics;

[0032] Combine the molecular driving electric field, the signal gain component, the signal extraction device and the analog-to-digital converter to set up the electrical signal acquisition unit of the nanopore sensor.

[0033] Optionally, setting the molecular driving electric field of the nanopore sensor according to the pore end position includes:

[0034] Determine the nanopore entrance and exit corresponding to the nanopore sensor according to the pore end position;

[0035] Collect the sequencing molecules at the nanopore entrance and exit, and analyze the charge characteristics of the sequencing molecules;

[0036] Configure the external electric field of the nanopore sensor according to the charge characteristics;

[0037] Measure the electric field intensity of the external electric field, and extract the buffer medium where the sequencing molecule is located when passing through the nanopore entrance and exit;

[0038] Analyze the conductivity characteristics of the buffer medium;

[0039] Based on the electric field strength, the charge characteristics, and the conductivity characteristics, determine the driving conditions for the sequencing molecules;

[0040] Combine the sequencing molecules, the applied electric field, and the driving conditions to set the molecular driving electric field of the nanopore sensor.

[0041] Optionally, constructing the sequence analysis model of the viral RNA based on the current signal characteristics includes:

[0042] Based on the current signal characteristics, identify the base sequence of the viral RNA;

[0043] Collect the current signals corresponding to the base sequences and query the time series of the current signals;

[0044] Perform alignment processing on the time series and the base sequences to obtain an alignment result;

[0045] Based on the current signal characteristics, analyze the sequence characteristics of the base sequences;

[0046] According to the alignment result and the sequence characteristics, set the mapping relationship between the current signal characteristics and the base sequences;

[0047] Based on the mapping relationship, construct the sequence analysis model of the viral RNA.

[0048] Optionally, identifying the characteristic sequence of the viral RNA according to the sequence analysis model includes:

[0049] According to the sequence analysis model, extract the functional elements and repetitive sequences of the viral RNA;

[0050] Identify the conserved sequence regions of the functional elements and extract the modification sites of the conserved sequence regions;

[0051] According to the modification sites, analyze the activity degree of the functional elements;

[0052] Based on the activity degree, identify the conserved sequence of the viral RNA;

[0053] Combine the functional elements, the repetitive sequences, the modification sites, and the conserved sequence to identify the characteristic sequence of the viral RNA.

[0054] Optionally, extracting the modification characteristics of the viral RNA based on the characteristic sequence includes:

[0055] Identify the current signal characteristics corresponding to the characteristic sequence;

[0056] Extract the current signal intensity and signal duration of the viral RNA from the current signal characteristics;

[0057] Identify the chemical modification sites of the viral RNA based on the current signal intensity and the duration;

[0058] Analyze the sequence type of the characteristic sequence based on the current signal characteristics;

[0059] Analyze the modification type of the viral RNA based on the chemical modification sites;

[0060] Identify the modification intensity of the viral RNA based on the modification type;

[0061] Combine the modification type, the modification intensity and the chemical modification sites to extract the modification characteristics of the viral RNA.

[0062] Optionally, setting the parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length and the modification characteristics includes:

[0063] Collect the electrical data of the nanopore sensor and extract the voltage and current of the nanopore sensor from the electrical data;

[0064] Set an automatic threshold adjustment mechanism for the current and the voltage according to the molecular integrity and the purification degree;

[0065] Identify the data acquisition rate of the nanopore sensor based on the molecular length;

[0066] Analyze the current change mode of the nanopore sensor according to the modification characteristics;

[0067] Construct a real-time feedback network for the data acquisition rate and the current change mode;

[0068] Identify the multi-parameter change trend of the nanopore sensor based on the real-time feedback network;

[0069] Extract the key turning points of the multi-parameter change trend and set the trigger conditions for the automatic threshold adjustment mechanism according to the key turning points;

[0070] Combine the automatic threshold adjustment mechanism, the real-time feedback network and the trigger conditions to set the parameter automatic adjustment mode of the nanopore sensor.

[0071] Compared with the problems described in the background art, in the embodiments of the present invention, by performing quality detection on the viral RNA to obtain a quality detection result, it is possible to ensure that the purity, integrity, and concentration of the RNA meet the requirements of viral RNA sequencing. And by designing a suitable sequencing platform based on the integrity and purity of the molecules, the sequencing efficiency and data quality of viral RNA are improved. Further, in the embodiments of the present invention, by configuring a nanopore sensor for the viral RNA based on the molecular integrity and the purification degree, the efficiency of the sequencing process and the reliability of the data can be ensured, which helps to accurately identify the modifications and structural features of the RNA. And in the embodiments of the present invention, by setting a multi-aperture molecular detection mechanism for the viral RNA in the nanopore sensor according to the pore size and the molecular length, an appropriate pore size can be selected according to the length of the RNA molecule, thereby optimizing the sequencing efficiency and data quality, and improving the accuracy and stability of the sequencing. In the embodiments of the present invention, by identifying the pore end position of the nanopore sensor and setting an electrical signal acquisition unit of the nanopore sensor according to the pore end position, the current change when the viral RNA molecule passes through the nanopore can be monitored in real time, and then the sequence information of the RNA molecule can be inferred, improving the accuracy of viral RNA molecule sequencing. Further, in the embodiments of the present invention, by collecting the current change signal of the viral RNA in real time based on the multi-aperture molecular detection mechanism and the electrical signal acquisition unit, the base sequence of the viral RNA can be directly read without complex preprocessing steps, significantly improving the sequencing speed of the viral RNA. In the embodiments of the present invention, by constructing a sequence analysis model of the viral RNA based on the current signal characteristics, the interpretation of the current signal can be further optimized, reducing errors and improving the sequencing accuracy. Further, in the embodiments of the present invention, by setting a parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length, and the modification characteristics, the sensor parameters can be quickly adjusted according to the real-time situation of the viral RNA without manual intervention, thereby greatly improving the sequencing efficiency. At the same time, it can ensure that the sensor can accurately capture the characteristic signals of different length molecules, thereby improving the accuracy and reliability of the sequencing. In the embodiments of the present invention, by combining the multi-aperture molecular detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode, direct sequencing processing of the viral RNA is performed to obtain a direct sequencing result, which can simultaneously detect different gene fragments of the virus, provide more comprehensive virus information, and can dynamically optimize the detection conditions according to the characteristic sequence of the viral RNA, improving the accuracy and efficiency of the detection, providing strong support for rapid and accurate virus detection. Therefore, an efficient and low-cost direct sequencing method for viral RNA provided by the embodiments of the present invention can improve the sequencing efficiency of viral RNA while reducing the sequencing cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1Schematic flowchart of a high - efficiency and low - cost method for direct sequencing of viral RNA provided by an embodiment of the present invention;

[0073] Figure 2 Schematic diagram of modules for implementing the method for direct sequencing of viral RNA with high efficiency and low cost provided by an embodiment of the present invention.

[0074] The implementation, functional characteristics, and advantages of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed implementation manners

[0075] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0076] The embodiments of the present application provide a method for direct sequencing of viral RNA with high efficiency and low cost. The execution subject of the method for direct sequencing of viral RNA with high efficiency and low cost includes, but is not limited to, at least one of electronic devices such as a server, a terminal, etc. that can be configured to execute the method provided by the embodiments of the present application. In other words, the method for direct sequencing of viral RNA with high efficiency and low cost can be executed by software or hardware installed on a terminal device or a server device. The server includes, but is not limited to: a single server, a server cluster, a cloud server, or a cloud server cluster, etc.

[0077] Embodiment 1:

[0078] Refer to Figure 1 As shown, it is a schematic flowchart of a method for direct sequencing of viral RNA with high efficiency and low cost provided by an embodiment of the present invention. In this embodiment, the method for direct sequencing of viral RNA with high efficiency and low cost includes:

[0079] S1. Collect a virus sample to be detected, extract the viral RNA in the virus sample, perform a quality test on the viral RNA to obtain a quality test result, and when the quality test result meets a preset effect, extract the molecular integrity and purification degree of the viral RNA.

[0080] In the embodiment of the present invention, by collecting a virus sample to be detected and extracting the viral RNA in the virus sample, data support can be provided for the subsequent sequencing process. The virus sample refers to the material collected from a virus - containing substance for subsequent detection and analysis, such as the blood or throat swab of an infected person.

[0081] Optionally, the collection of the virus sample to be detected can be obtained through laboratory tests, and the extraction of the viral RNA in the virus sample can be achieved by using a FastPure Cell kit.

[0082] Furthermore, in the embodiments of the present invention, by performing quality detection on the viral RNA to obtain a quality detection result, it can be ensured that the purity, integrity, and concentration of the RNA meet the requirements of viral RNA sequencing. The quality detection result refers to the specific data and conclusions obtained after detecting and evaluating the integrity, purity, concentration, etc. of the viral RNA.

[0083] Optionally, the quality detection of the viral RNA can be evaluated by a microfluidic electrophoresis analysis method.

[0084] In the embodiments of the present invention, when the quality detection result meets the preset effect, the molecular integrity and purification degree of the viral RNA are extracted. An appropriate sequencing platform can be designed based on the molecular integrity and purity, improving the sequencing efficiency and data quality of the viral RNA. The preset effect refers to the standard for judging whether the extracted viral RNA meets the requirements of subsequent experiments. For example, the RNA concentration should reach the minimum value required by the experiment. The molecular integrity refers to the integrity and degradation degree of the RNA molecule, and the purification degree refers to the ratio of the content of the viral RNA molecule to other impurities.

[0085] Optionally, when the quality detection result meets the preset effect, the extraction of the molecular integrity and purification degree of the viral RNA can be achieved through an electrophoresis pattern generated by agarose gel electrophoresis.

[0086] S2. Based on the molecular integrity and the purification degree, configure a nanopore sensor for the viral RNA, identify the pore size of the nanopore sensor, and extract the molecular length of the viral RNA. According to the pore size and the molecular length, set a multi-pore molecular detection mechanism for the viral RNA in the nanopore sensor.

[0087] In the embodiments of the present invention, by configuring a nanopore sensor for the viral RNA based on the molecular integrity and the purification degree, the efficiency of the sequencing process and the reliability of the data can be ensured, which helps to accurately identify the modifications and structural features of the RNA. The nanopore sensor refers to a single-molecule detection device based on nanotechnology, such as the Oxford Nanopore nanopore sequencing device.

[0088] As an embodiment of the present invention, the nanopore sensor for the viral RNA configured based on the molecular integrity and the purification degree includes: extracting the structural features of the viral RNA based on the molecular integrity; determining the sequence length and the structural complexity of the viral RNA according to the structural features; identifying the molecular movement speed of the viral RNA based on the sequence length and the structural complexity; analyzing the surface adsorption ability of the viral RNA according to the purification degree; and configuring the nanopore sensor for the viral RNA by combining the structural features, the surface adsorption ability and the molecular movement speed.

[0089] Wherein, the structural features refer to the arrangement mode and composition characteristics of viral RNA molecules in space, including the single-stranded or double-stranded nature of the RNA chain, the nucleotide sequence and arrangement mode, etc.; the sequence length refers to the number of nucleotides in the RNA molecule; the structural complexity refers to the complexity of the internal structure of the RNA molecule, such as the presence of multiple hairpin structures and stem-loop structures in the RNA molecule; the molecular movement speed refers to the movement speed of the RNA molecule during the sequencing process, such as the speed when passing through the nanopore; and the surface adsorption ability refers to the adsorption ability of the RNA molecule during the sequencing process, such as the adsorption ability of the RNA molecule on the surface of the nanopore.

[0090] Optionally, the extraction of the structural features of the viral RNA based on the molecular integrity can be realized by using the molecular dynamics simulation method; the sequence length of the viral RNA can be obtained by using the high-throughput sequencing technology according to the structural features; and the structural complexity of the viral RNA can be determined by using the RNAfold thermodynamic model according to the structural features.

[0091] Furthermore, in the embodiment of the present invention, by identifying the pore size of the nanopore sensor and extracting the molecular length of the viral RNA, the sequencing parameters of the nanopore sensor can be optimized. The pore size refers to the diameter or width of the nanopore in the nanopore sensor, and the molecular length refers to the physical size of the RNA molecule.

[0092] Optionally, the identification of the pore size of the nanopore sensor can be obtained by using a transmission electron microscope, and the extraction of the molecular length of the viral RNA can be realized by using bioinformatics tools, such as the BioPython tool.

[0093] In an embodiment of the present invention, by setting the porous aperture molecular detection mechanism of the viral RNA in the nanopore sensor according to the aperture size and the molecular length, an appropriate aperture can be selected according to the length of the RNA molecule, thereby optimizing the sequencing efficiency and data quality, and improving the accuracy and stability of sequencing. The porous aperture molecular detection mechanism refers to a system that simultaneously performs biomolecular detection using multiple nanopores with different apertures.

[0094] As an embodiment of the present invention, the setting of the porous aperture molecular detection mechanism of the viral RNA in the nanopore sensor according to the aperture size and the molecular length includes: defining the sequencing molecular type of the nanopore sensor according to the molecular length; calculating the fitting coefficient between the sequencing molecular type and the aperture size; setting an intelligent matching mode between the aperture size and the sequencing molecular type based on the fitting coefficient; creating a porous aperture sequencing channel for the viral RNA in the nanopore sensor according to the intelligent matching mode; identifying the molecular characteristics of the viral RNA, and setting dynamic sequencing parameters for the porous aperture sequencing channel according to the molecular characteristics; and setting the porous aperture molecular detection mechanism of the viral RNA in the nanopore sensor by combining the porous aperture sequencing channel, the intelligent matching mode, and the dynamic sequencing parameters.

[0095] Among them, the sequencing molecular type refers to the molecular types divided according to the molecular length during sequencing, such as long RNA molecules, short RNA molecules, etc. The fitting coefficient refers to a parameter that measures the matching degree between the sequencing molecule and the aperture size of the nanopore sensor. The intelligent matching mode refers to an automated algorithm that automatically matches the aperture size of the nanopore sensor according to the type of sequencing molecule. The porous aperture sequencing channel refers to different aperture channels in the nanopore sensor that can perform sequencing simultaneously. The molecular characteristics refer to the physical and chemical properties of the sequencing molecule, such as the length, diameter, charge distribution, base composition, etc. of the molecule. The dynamic sequencing parameters refer to the parameters that can be adjusted in real time according to the molecular characteristics during sequencing, such as voltage, current, sequencing speed, etc.

[0096] Optionally, the setting of the intelligent matching mode between the aperture size and the sequencing molecular type based on the fitting coefficient can be implemented using a machine learning model. For example, a model is trained using the collected RNA molecule feature data, with the features of the RNA molecule as the input and the most suitable nanopore aperture as the output. The creation of the porous aperture sequencing channel for the viral RNA in the nanopore sensor according to the intelligent matching mode can be achieved using a porous aperture nanopore sensor. The identification of the molecular characteristics of the viral RNA can be determined by an electron microscope. The setting of the dynamic sequencing parameters for the porous aperture sequencing channel according to the molecular characteristics can be achieved by implementing sequencing control software, such as MinKNOW software.

[0097] In an alternative embodiment of the present invention, the fitting coefficient between the sequencing molecule type and the pore size is calculated using the following formula:

[0098]

[0099] where r represents the fitting coefficient between the sequencing molecule type and the pore size, and L e represents the molecular length corresponding to the e-th sequencing molecule type, P represents the pore size, and D e represents the molecular diameter corresponding to the e-th sequencing molecule type, n represents the total number of molecules corresponding to the sequencing molecule type, and e represents the molecular serial number corresponding to the sequencing molecule type.

[0100] S3. Identify the pore end positions of the nanopore sensor, and based on the pore end positions, set the electrical signal acquisition unit of the nanopore sensor. Based on the multi-pore-size molecule detection mechanism and the electrical signal acquisition unit, collect the current change signals of the viral RNA in real time.

[0101] In the embodiment of the present invention, by identifying the pore end positions of the nanopore sensor and setting the electrical signal acquisition unit of the nanopore sensor based on the pore end positions, the current changes when viral RNA molecules pass through the nanopores can be monitored in real time, and then the sequence information of the RNA molecules can be inferred, improving the accuracy of viral RNA molecule sequencing. The pore end positions refer to the two regions where the entrances and exits of the nanopores are located, and the electrical signal acquisition unit refers to the device used to detect the electrical signals generated when RNA molecules pass through the nanopores.

[0102] As an embodiment of the present invention, setting the electrical signal acquisition unit of the nanopore sensor according to the pore end positions includes: setting the molecular driving electric field of the nanopore sensor according to the pore end positions; identifying the nanopore sensing molecules of the nanopore sensor based on the molecular driving electric field; configuring the signal extraction device of the nanopore sensing molecules, and collecting the analog current signals of the nanopore sensing molecules according to the signal extraction device; identifying the signal quality of the analog current signals, and setting the signal gain component of the analog current signals based on the signal quality; analyzing the output characteristics of the signal gain component, and setting the analog-to-digital converter of the analog current signals according to the output characteristics; and setting the electrical signal acquisition unit of the nanopore sensor by combining the molecular driving electric field, the signal gain component, the signal extraction device, and the analog-to-digital converter.

[0103] Among them, the analysis-driven electric field refers to an externally applied electric field applied at both ends of the nanopore. The nanopore sensing molecule refers to a biomolecule that can be detected when passing through the nanopore, such as RNA, DNA, protein, etc. The signal extraction device refers to a device used to detect and extract the current change generated when the nanopore sensing molecule passes through the nanopore, such as an electrode system. The analog current signal refers to a continuously changing current signal extracted from the nanopore sensor. The signal quality refers to the characteristics of the analog current signal, including signal-to-noise ratio (SNR), signal amplitude, noise level, etc. The signal gain component refers to a device used to amplify the analog current signal, such as a low-noise amplifier. The output characteristics refer to the characteristics of the signal after being amplified by the signal gain component, including the amplitude range, frequency response, bandwidth, etc. of the output signal. The analog-to-digital converter refers to an electronic device that converts an analog signal into a digital signal.

[0104] Optionally, based on the molecule-driven electric field, the identification of the nanopore sensing molecule of the nanopore sensor can be determined by the molecule passing through the nanopore under the drive of the electric field. The configuration of the signal extraction device of the nanopore sensing molecule can be obtained through the electrode system. The identification of the signal quality of the analog current signal can be determined according to the noise level of the signal. The analysis of the output characteristics of the signal gain component can be realized by using an oscilloscope.

[0105] As another embodiment of the present invention, setting the molecule-driven electric field of the nanopore sensor according to the pore end position includes: determining the nanopore entrance and exit corresponding to the nanopore sensor according to the pore end position; collecting the sequencing molecules at the nanopore entrance and exit, and analyzing the charge characteristics of the sequencing molecules; configuring the externally applied electric field of the nanopore sensor according to the charge characteristics; measuring the electric field strength of the externally applied electric field, and extracting the buffer medium where the sequencing molecule is located when passing through the nanopore entrance and exit; analyzing the conductivity characteristics of the buffer medium; determining the driving conditions of the sequencing molecule based on the electric field strength, the charge characteristics and the conductivity characteristics; and setting the molecule-driven electric field of the nanopore sensor in combination with the sequencing molecule, the externally applied electric field and the driving conditions.

[0106] Among them, the nanopore entrances and exits refer to the two endpoints in the nanopore sensor where the sequencing molecules enter and leave. The sequencing molecules refer to the target molecules to be sequenced by the nanopore sensor, such as RNA molecules. The charge characteristics refer to the charge distribution and quantity on the surface of the sequencing molecules. The applied external electric field refers to the electric field applied in the nanopore sensor. The electric field strength refers to the magnitude of the applied external electric field. The buffer medium refers to the solution used to maintain the stability of the electrochemical environment in the nanopore sensor, usually containing electrolytes and buffers. The conductivity characteristics refer to the ion conduction ability in the buffer medium. The driving conditions refer to the comprehensive conditions required to ensure the smooth passage of the sequencing molecules through the nanopore in the nanopore sensor.

[0107] Optionally, the analysis of the charge characteristics of the sequencing molecules can be achieved by using quantum chemical calculation methods, such as the density functional theory method. According to the charge characteristics, the configuration of the applied external electric field of the nanopore sensor can be formed by applying a voltage in the electrolyte solutions on both sides of the nanopore through a patch clamp amplifier. The measurement of the electric field strength of the applied external electric field can be realized by using an electric field strength tester. The analysis of the conductivity characteristics of the buffer medium can be obtained through a conductivity meter.

[0108] Furthermore, in the embodiment of the present invention, by based on the multi-aperture molecule detection mechanism and the electrical signal acquisition unit, the current change signal of the viral RNA is collected in real time, and the base sequence of the viral RNA can be directly read without complex preprocessing steps, significantly improving the sequencing speed of the viral RNA. The current change signal refers to the signal that causes the current of the nanopore to change when the viral RNA collected by the electrical signal acquisition unit passes through the nanopore.

[0109] S4. Based on the current change signal, extract the current signal characteristics of the viral RNA, and based on the current signal characteristics, construct a sequence analysis model of the viral RNA.

[0110] In the embodiment of the present invention, by based on the current change signal, extracting the current signal characteristics of the viral RNA, errors that may occur during the sequencing process can be identified and corrected, thereby improving the sequencing accuracy. The signal characteristics refer to the quantitative parameters extracted from the current change signal that can reflect the physical and chemical characteristics when the viral RNA molecule passes through the nanopore, such as the amplitude, frequency, and duration of the current change, etc.

[0111] Optionally, based on the current change signal, the extraction of the current signal characteristics of the viral RNA can be achieved by using MATLAB tools.

[0112] Furthermore, in the embodiment of the present invention, by constructing a sequence analysis model of the viral RNA based on the current signal characteristics, the interpretation of the current signal can be further optimized, errors can be reduced, and the sequencing accuracy can be improved. The sequence analysis model refers to a mathematical model constructed based on the current signal characteristics for analyzing the viral RNA sequence.

[0113] As an embodiment of the present invention, constructing the sequence analysis model of the viral RNA based on the current signal characteristics includes: identifying the base sequence of the viral RNA based on the current signal characteristics; collecting the current signals corresponding to the base sequence and querying the time series of the current signals; performing alignment processing on the time series and the base sequence to obtain an alignment result; analyzing the sequence characteristics of the base sequence based on the current signal characteristics; setting the mapping relationship between the current signal characteristics and the base sequence according to the alignment result and the sequence characteristics; and constructing the sequence analysis model of the viral RNA based on the mapping relationship.

[0114] Among them, the base sequence refers to the arrangement order of bases in the viral RNA molecule, such as adenine A, guanine G, cytosine C, and uracil U. The current signal refers to the electrical signal generated due to the interaction between the base and the nanopore when the viral RNA molecule passes through the nanopore. The time series refers to the time stamp recording the current change when the viral RNA molecule passes through the nanopore. The data alignment processing refers to the time matching and calibration of the time series of the current signal and the base sequence. The docking result refers to the corresponding relationship between the current signal and the base sequence after the data alignment processing is completed. The sequence characteristics refer to the regularities or patterns with biological significance in the base sequence, such as base modifications (such as m6A, m5C), repetitive sequences, etc. The mapping relationship refers to the corresponding relationship between the current signal characteristics and the base sequence, such as current intensity: 100 pA, duration: 10 ms → base: A.

[0115] Optionally, the recognition of the base sequence of the viral RNA based on the current signal characteristics can be achieved through a decoding tool, such as the BaseNet tool. The alignment processing of the current signal and the base sequence can be realized by using an alignment tool, such as the Tombo tool. The setting of the mapping relationship between the current signal characteristics and the base sequence according to the alignment result and the sequence characteristics can be achieved through a self-organizing mapping neural network.

[0116] S5. According to the sequence analysis model, identify the characteristic sequence of the viral RNA, extract the modification characteristics of the viral RNA based on the characteristic sequence, and set the parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length, and the modification characteristics.

[0117] In the embodiments of the present invention, by identifying the characteristic sequence of the viral RNA according to the sequence analysis model, the pathogenic gene or viral sequence can be accurately identified, providing an important reference basis for clinical diagnosis. The characteristic sequence refers to a fragment of the base sequence with specific patterns or characteristics in the viral RNA molecule, such as a sequence that varies among different viral strains.

[0118] As an embodiment of the present invention, the identifying the characteristic sequence of the viral RNA according to the sequence analysis model includes: extracting the functional elements and repetitive sequences of the viral RNA according to the sequence analysis model; identifying the conserved sequence region of the functional elements and extracting the modification sites of the conserved sequence region; analyzing the activity level of the functional elements according to the modification sites; identifying the conserved sequence of the viral RNA based on the activity level; and combining the functional elements, the repetitive sequences, the modification sites and the conserved sequence to identify the characteristic sequence of the viral RNA.

[0119] Among them, the functional element refers to a structure or sequence with a specific function in the viral RNA molecule, such as a promoter, a terminator, a transcriptional regulatory sequence, etc. The repetitive sequence refers to a base sequence that appears multiple times in the viral RNA molecule, such as a direct terminal repeat sequence, an inverted terminal repeat sequence, etc. The conserved sequence region refers to a region of the base sequence that remains highly consistent during viral evolution. The modification site refers to the position of the base in the viral RNA molecule where chemical modification occurs, such as the m6A modification site. The activity level refers to the functional activity level of the functional element in the biological process. For example, a strong promoter can efficiently recruit RNA polymerase, thereby promoting high expression of the gene. The conserved sequence refers to a base sequence that remains highly consistent during viral evolution, such as the ribosomal RNA sequence.

[0120] Optionally, the identification of the conserved sequence region of the functional element can be achieved through a conservation scoring tool, such as the PhastCons tool. The extraction of the modification sites of the conserved sequence region can be realized by using a gene editing tool, such as the CRISPR tool. According to the modification sites, the analysis of the activity level of the functional element can be determined through the DREAM platform.

[0121] Furthermore, in the embodiments of the present invention, by extracting the modification characteristics of the viral RNA based on the characteristic sequence, it is possible to help optimize the sequencing parameters of the viral RNA, reduce the sequencing errors caused by modifications, and improve the accuracy of sequencing. The modification characteristics refer to the chemical modifications that occur on the viral RNA molecule, such as m6A (N6-methyladenosine), m5C (5-methylcytosine), etc.

[0122] As an embodiment of the present invention, extracting the modification features of the viral RNA based on the characteristic sequence includes: identifying the current signal features corresponding to the characteristic sequence; extracting the current signal intensity and signal duration of the viral RNA from the current signal features; identifying the chemical modification sites of the viral RNA according to the current signal intensity and the duration; analyzing the sequence type of the characteristic sequence based on the current signal features; analyzing the modification type of the viral RNA according to the chemical modification sites; identifying the modification intensity of the viral RNA based on the modification type; and extracting the modification features of the viral RNA by combining the modification type, the modification intensity and the chemical modification sites.

[0123] Among them, the current signal intensity refers to the current amplitude generated when the viral RNA molecule passes through the nanopore, the signal duration refers to the residence time when a single base or base fragment in the viral RNA molecule passes through the nanopore, the chemical modification site refers to the specific base position where chemical modification occurs in the viral RNA molecule, the sequence type refers to the classification of sequence regions with specific biological significance in the viral RNA molecule, such as functional elements, repetitive sequences, etc., the modification type refers to the types of chemical modifications that occur in the viral RNA molecule, such as 5-methylcytosine, and the modification intensity refers to the abundance or intensity of a specific modification type in the viral RNA molecule.

[0124] Optionally, the identification of the chemical modification sites of the viral RNA according to the current signal intensity and the duration can be achieved through bioinformatics tools, such as the Mega l odon tool. The analysis of the sequence type of the characteristic sequence based on the current signal features can be obtained through sequence feature extraction tools, such as the MEME Su i te tool. The analysis of the modification type of the viral RNA according to the chemical modification sites can be achieved by using mass spectrometry. The identification of the modification intensity of the viral RNA based on the modification type can be obtained through immunofluorescence detection.

[0125] In the embodiment of the present invention, by setting the parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length and the modification features, the sensor parameters can be quickly adjusted according to the real-time situation of the viral RNA without manual intervention, thus greatly improving the sequencing efficiency. At the same time, it can ensure that the sensor can accurately capture the characteristic signals of molecules with different lengths, thereby improving the accuracy and reliability of sequencing. The parameter automatic adjustment mode refers to a technical mode in which the nanopore sensor automatically adjusts its working parameters according to the molecular characteristics monitored in real time during the working process.

[0126] As an embodiment of the present invention, setting the parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length, and the modification characteristics includes: collecting the electrical data of the nanopore sensor, and extracting the voltage and current of the nanopore sensor from the electrical data; setting an automatic threshold adjustment mechanism for the current and the voltage according to the molecular integrity and the purification degree; identifying the data acquisition rate of the nanopore sensor based on the molecular length; analyzing the current change mode of the nanopore sensor according to the modification characteristics; constructing a real-time feedback network for the data acquisition rate and the current change mode; identifying the multi-parameter change trend of the nanopore sensor based on the real-time feedback network; extracting the key turning points of the multi-parameter change trend, and setting the triggering conditions of the automatic threshold adjustment mechanism according to the key turning points; and combining the automatic threshold adjustment mechanism, the real-time feedback network, and the triggering conditions to set the parameter automatic adjustment mode of the nanopore sensor.

[0127] Among them, the electrical data refers to the data related to current and voltage collected by the nanopore sensor during operation. The voltage refers to the electric field strength applied by the nanopore sensor during operation. The current refers to the current signal generated when the viral RNA molecule passes through the nanopore. The automatic threshold adjustment mechanism refers to a system that automatically adjusts the thresholds of current and voltage according to the real-time monitored electrical data. The data acquisition rate refers to the frequency at which the nanopore sensor collects electrical data during operation. For example, a data acquisition rate of 100 kHz means collecting 100,000 data points per second. The current change mode refers to the specific mode of the current signal generated when the viral RNA molecule passes through the nanopore. The real-time feedback network refers to a network that can monitor the system output in real time and adjust the system input according to the output information. The multi-parameter change trend refers to the change trend of multiple parameters (such as current change amplitude, blocking duration, data acquisition rate, etc.) over time during the detection process. The key turning point refers to the point at which the parameter changes significantly in the multi-parameter change trend, such as the point where the current intensity suddenly increases. The triggering condition refers to the specific condition for triggering parameter adjustment in the automatic threshold adjustment mechanism.

[0128] Optionally, the collection of the electrical data of the nanopore sensor can be obtained by using the real-time monitoring system of the nanopore sensor. The identification of the data acquisition rate of the nanopore sensor based on the molecular length can be realized by using real-time signal processing algorithms such as Fourier transform. The identification of the multi-parameter change trend of the nanopore sensor based on the real-time feedback network can be realized by using statistical methods such as the moving average method. The extraction of the key turning points of the multi-parameter change trend can be achieved by establishing an information map of the multi-parameter change trend.

[0129] S6. Combine the multi-aperture molecule detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode to perform direct sequencing processing on the viral RNA to obtain a direct sequencing result.

[0130] In the embodiment of the present invention, by combining the multi-aperture molecule detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode, direct sequencing processing of the viral RNA is performed to obtain a direct sequencing result, which can simultaneously detect different gene fragments of the virus, provide more comprehensive virus information, and at the same time can dynamically optimize the detection conditions according to the characteristic sequences of the viral RNA, improving the accuracy and efficiency of detection, providing strong support for rapid and accurate virus detection. The direct sequencing processing refers to the process of directly sequencing natural RNA molecules without the need to reverse transcribe RNA into cDNA or perform PCR amplification. The direct sequencing result refers to the RNA sequence information obtained through direct sequencing processing, including the base sequence of the RNA molecule, the position and type of modification sites, etc.

[0131] Compared with the problems described in the background art, in the embodiments of the present invention, by performing quality detection on the viral RNA to obtain a quality detection result, it can be ensured that the purity, integrity, and concentration of the RNA meet the requirements of viral RNA sequencing. And according to the integrity and purity of the molecules, a suitable sequencing platform is designed to improve the sequencing efficiency and data quality of viral RNA. Further, in the embodiments of the present invention, by configuring the nanopore sensor for the viral RNA based on the molecular integrity and the purification degree, it can ensure the high efficiency of the sequencing process and the reliability of the data, help accurately identify the modifications and structural features of the RNA. And in the embodiments of the present invention, by setting the multi-aperture molecular detection mechanism of the viral RNA in the nanopore sensor according to the pore size and the molecular length, a suitable pore size can be selected according to the length of the RNA molecule, thereby optimizing the sequencing efficiency and data quality, and improving the accuracy and stability of the sequencing. In the embodiments of the present invention, by identifying the pore end position of the nanopore sensor and setting the electrical signal acquisition unit of the nanopore sensor according to the pore end position, the current change when the viral RNA molecule passes through the nanopore can be monitored in real time, and then the sequence information of the RNA molecule can be inferred, improving the accuracy of viral RNA molecule sequencing. Further, in the embodiments of the present invention, by based on the multi-aperture molecular detection mechanism and the electrical signal acquisition unit, the current change signal of the viral RNA is collected in real time, the base sequence of the viral RNA can be directly read without complex preprocessing steps, significantly improving the sequencing speed of the viral RNA. In the embodiments of the present invention, by constructing a sequence analysis model of the viral RNA based on the current signal characteristics, the interpretation of the current signal can be further optimized, reducing errors and improving the sequencing accuracy. Further, in the embodiments of the present invention, by setting the parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length, and the modification characteristics, the sensor parameters can be quickly adjusted according to the real-time situation of the viral RNA without manual intervention, thereby greatly improving the sequencing efficiency, and at the same time ensuring that the sensor can accurately capture the characteristic signals of different length molecules, thus improving the accuracy and reliability of the sequencing. In the embodiments of the present invention, by combining the multi-aperture molecular detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode, the direct sequencing process of the viral RNA is performed to obtain a direct sequencing result, different gene fragments of the virus can be detected simultaneously, providing more comprehensive virus information, and at the same time the detection conditions can be dynamically optimized according to the characteristic sequence of the viral RNA, improving the accuracy and efficiency of the detection, providing strong support for rapid and accurate virus detection. Therefore, a high-efficiency and low-cost direct sequencing method for viral RNA provided by the embodiments of the present invention can improve the sequencing efficiency of viral RNA while reducing the sequencing cost.

[0132] Example 2:

[0133] Such asFigure 2 As shown, it is a functional module diagram of an efficient and low-cost direct virus RNA sequencing system of the present invention.

[0134] The efficient and low-cost direct virus RNA sequencing system 200 of the present invention can be installed in an electronic device. According to the functions achieved, the efficient and low-cost direct virus RNA sequencing system may include a molecular mass detection module 201, a sequencing platform preparation module 202, a current signal acquisition module 203, a sequence analysis module 204, a sequencing platform optimization module 205, and a direct sequencing module 206. The modules of the present invention may also be referred to as units, which refer to a series of computer program segments that can be executed by a processor of an electronic device and can complete fixed functions, and are stored in the memory of the electronic device.

[0135] In an embodiment of the present invention, the functions of each module / unit are as follows:

[0136] The molecular mass detection module 201 is used to collect a virus sample to be detected, extract the virus RNA in the virus sample, perform quality detection on the virus RNA, obtain a quality detection result, and when the quality detection result meets a preset effect, extract the molecular integrity and purification degree of the virus RNA;

[0137] The sequencing platform preparation module 202 is used to configure a nanopore sensor for the virus RNA based on the molecular integrity and the purification degree, identify the pore size of the nanopore sensor, extract the molecular length of the virus RNA, and set a multi-pore molecular detection mechanism for the virus RNA in the nanopore sensor according to the pore size and the molecular length.

[0138] The current signal acquisition module 203 is used to identify the pore end position of the nanopore sensor, set an electrical signal acquisition unit of the nanopore sensor according to the pore end position, and collect the current change signal of the virus RNA in real time based on the multi-pore molecular detection mechanism and the electrical signal acquisition unit;

[0139] The sequence analysis module 204 is used to extract the current signal characteristics of the virus RNA based on the current change signal, and construct a sequence analysis model of the virus RNA based on the current signal characteristics;

[0140] The sequencing platform optimization module 205 is used to identify the characteristic sequence of the virus RNA according to the sequence analysis model, extract the modification characteristics of the virus RNA based on the characteristic sequence, and set a parameter automatic adjustment mode of the nanopore sensor according to the molecular integrity, the purification degree, the molecular length, and the modification characteristics;

[0141] The direct sequencing module 206 is configured to perform direct sequencing processing of the viral RNA by combining the multi-aperture molecule detection mechanism, the electrical signal acquisition unit, and the parameter automatic adjustment mode, and obtain a direct sequencing result.

[0142] Specifically, each module in the high-efficiency and low-cost viral RNA direct sequencing system 200 in the embodiments of the present invention adopts the same technical means as those in the Figure 1 high-efficiency and low-cost viral RNA direct sequencing method described above, and can produce the same technical effects, which will not be elaborated here.

[0143] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An efficient and low-cost direct viral RNA sequencing method, characterized in that: The method comprises: Collecting a virus sample to be tested, extracting viral RNA from the virus sample, performing a quality test on the viral RNA to obtain a quality test result, and when the quality test result meets a preset effect, extracting the molecular integrity and degree of purification of the viral RNA; Based on the molecular integrity and the degree of purification, a nanopore sensor for the viral RNA is configured, the pore size of the nanopore sensor is identified, and the molecular length of the viral RNA is extracted, and according to the pore size and the molecular length, a multi-aperture molecular detection mechanism for the viral RNA in the nanopore sensor is set; Identify the hole end position of the nanopore sensor, and according to the hole end position, set the electrical signal acquisition unit of the nanopore sensor, and based on the multi-aperture molecular detection mechanism and the electrical signal acquisition unit, collect the current change signal of the viral RNA in real time; Extracting the current signal characteristics of the viral RNA based on the current change signal, and constructing a sequence analysis model of the viral RNA based on the current signal characteristics; According to the sequence analysis model, the characteristic sequence of the viral RNA is identified, based on the characteristic sequence, the modification characteristics of the viral RNA are extracted, and according to the molecular integrity, the degree of purification, the molecular length and the modification characteristics, the automatic parameter adjustment mode of the nanopore sensor is set; In combination with the multi-aperture molecular detection mechanism, the electrical signal acquisition unit and the parameter automatic adjustment mode, direct sequencing processing of the viral RNA is performed to obtain direct sequencing results.

2. A highly efficient and low-cost direct viral RNA sequencing method as claimed in claim 1, characterized in that: The nanopore sensor for configuring the viral RNA based on the molecular integrity and the degree of purification comprises: Based on the molecular integrity, extracting structural features of the viral RNA; Determining the sequence length and structural complexity of the viral RNA based on the structural characteristics; Based on the length of the sequence and the complexity of the structure, identifying the molecular movement speed of the viral RNA; Analyzing the surface adsorption capacity of the viral RNA according to the degree of purification; The nanopore sensor of the viral RNA is configured by combining the structural features, the surface adsorption capacity and the molecular movement speed.

3. A highly efficient and low-cost direct viral RNA sequencing method as claimed in claim 1, characterized in that: The multi-aperture molecular detection mechanism of the viral RNA in the nanopore sensor is set according to the pore size and the molecular length, including: Defining the type of sequencing molecules of the nanopore sensor according to the molecular length; Calculating the fit coefficient between the sequencing molecule type and the pore size; Based on the fit coefficient, setting an intelligent matching mode between the pore size and the sequencing molecule type; According to the intelligent matching mode, creating a multi-aperture sequencing channel for the viral RNA in the nanopore sensor; Identifying the molecular characteristics of the viral RNA and setting the dynamic sequencing parameters of the multi-aperture sequencing channel according to the molecular characteristics; In combination with the multi-aperture sequencing channel, the intelligent matching mode and the dynamic sequencing parameters, a multi-aperture molecular detection mechanism of the viral RNA in the nanopore sensor is set.

4. A highly efficient and low-cost direct viral RNA sequencing method as claimed in claim 1, characterized in that: The electrical signal acquisition unit of the nanopore sensor is set according to the pore end position, comprising: According to the position of the pore end, setting the molecular driving electric field of the nanopore sensor; Based on the molecule-driven electric field, identifying the nanopore sensing molecule of the nanopore sensor; Configuring a signal extraction device for the nanopore sensing molecule, and collecting a simulated current signal of the nanopore sensing molecule according to the signal extraction device; identifying a signal quality of the analog current signal and setting a signal gain component of the analog current signal based on the signal quality; Analyzing the output characteristics of the signal gain component, and setting the analog-to-digital converter of the analog current signal according to the output characteristics; The electrical signal acquisition unit of the nanopore sensor is set up in combination with the molecular driving electric field, the signal gain component, the signal extraction device and the analog-to-digital converter.

5. A highly efficient and low-cost direct viral RNA sequencing method as claimed in claim 4, characterized in that: The step of setting the molecular driving electric field of the nanopore sensor according to the pore end position comprises: Determining the nanopore entrance and exit corresponding to the nanopore sensor according to the pore end position; Collecting sequencing molecules at the entrance and exit of the nanopore, and analyzing the charge characteristics of the sequencing molecules; configuring an applied electric field of the nanopore sensor according to the charge characteristic; Measuring the electric field strength of the external electric field, and extracting the buffer medium in which the sequencing molecule is located when passing through the nanopore entrance and exit; analyzing the conductivity characteristics of the buffer medium; Determining driving conditions of the sequencing molecule based on the electric field strength, the charge characteristics, and the conductivity characteristics; The molecular driving electric field of the nanopore sensor is set in combination with the sequencing molecule, the external electric field and the driving condition.

6. The efficient and low-cost direct viral RNA sequencing method according to claim 1, characterized in that: The constructing of the sequence analysis model of the viral RNA based on the current signal characteristics comprises: Based on the current signal characteristics, identifying the base sequence of the viral RNA; collecting current signals corresponding to the base sequence, and querying the time series of the current signals; Performing an alignment process of the time series and the base sequence to obtain an alignment result; Analyzing sequence characteristics of the base sequence based on the current signal characteristics; According to the alignment result and the sequence characteristics, setting a mapping relationship between the current signal characteristics and the base sequence; Based on the mapping relationship, a sequence analysis model of the viral RNA is constructed.

7. The efficient and low-cost direct viral RNA sequencing method according to claim 1, characterized in that: The step of identifying the characteristic sequence of the viral RNA according to the sequence analysis model comprises: Extracting the functional elements and repetitive sequences of the viral RNA according to the sequence analysis model; Identifying the conserved sequence region of the functional element and extracting the modification site of the conserved sequence region; Analyzing the activity level of the functional element according to the modification site; Based on the degree of activity, identifying conserved sequences of the viral RNA; The characteristic sequence of the viral RNA is identified by combining the functional element, the repetitive sequence, the modification site and the conserved sequence.

8. The efficient and low-cost direct viral RNA sequencing method according to claim 1, characterized in that: The step of extracting the modification features of the viral RNA based on the characteristic sequence comprises: Identifying current signal characteristics corresponding to the characteristic sequence; Extracting the current signal intensity and signal duration of the viral RNA from the current signal characteristics; Identifying chemical modification points of the viral RNA based on the current signal intensity and the duration; Analyzing the sequence type of the characteristic sequence based on the current signal characteristics; Analyzing the modification type of the viral RNA according to the chemical modification point; Based on the modification type, identifying the modification intensity of the viral RNA; The modification characteristics of the viral RNA are extracted by combining the modification type, the modification intensity and the chemical modification point.

9. The efficient and low-cost direct viral RNA sequencing method according to claim 1, characterized in that: The automatic parameter adjustment mode of the nanopore sensor is set according to the molecular integrity, the degree of purification, the molecular length and the modification characteristics, including: Collecting electrical data of the nanopore sensor, and extracting the voltage and current of the nanopore sensor from the electrical data; According to the integrity of the molecule and the degree of purification, an automatic threshold adjustment mechanism for the current and the voltage is set; identifying a data acquisition rate of the nanopore sensor based on the molecular length; analyzing a current change pattern of the nanopore sensor according to the modification characteristics; Constructing a real-time feedback network of the data acquisition rate and the current change pattern; Based on the real-time feedback network, identifying the multi-parameter change trend of the nanopore sensor; Extracting key turning points of the multi-parameter change trend, and setting triggering conditions of the automatic threshold adjustment mechanism according to the key turning points; In combination with the automatic threshold adjustment mechanism, the real-time feedback network and the trigger condition, an automatic parameter adjustment mode of the nanopore sensor is set.

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