Sequencing simulation method and device, computer equipment, storage medium and sequencing system

Abstract

The invention discloses a sequencing simulation method and device, computer equipment, a storage medium and a sequencing system.In the sequencing simulation method, a configuration template for a sequencing platform can be generated, a corresponding test module is called based on the configuration template, a target sequencing process is simulated through the corresponding test module, and a simulation test result is obtained. In the embodiment of the invention, a test module for configuring template calling can be a simulation sequencing module for the whole process from sample library building to sequencing fluorescence picture processing in the sequencing process, and the influence of different influence factors on sequencing is evaluated in a controllable manner by simulating the sequencing process; according to the method, the influence of different sequencing conditions and sequencing parameters on the sequencing result can be determined, the simulated sequencing data under extreme test conditions can be generated, all sequencing steps are not required to be completed through a real sequencing test through the simulated sequencing process, and the time and the cost are reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a sequencing simulation method, apparatus, computer equipment, storage medium and sequencing system. Background Art

[0002] The sequencing platform is a high-throughput automated multi-omics analysis technology. By combining DNA barcodes and bioinformatics analysis methods, it is an automated multi-omics research tool with high throughput and high resolution, mainly for sample characterization, manipulation and processing. The platform includes multiple throughput cell separation and screening, sample extraction, library construction and high-throughput / single-molecule sequencing systems, serving multi-dimensional omics analysis research and providing important technical support for basic research in life sciences, medical diagnosis and personalized medicine.

[0003] The quality of the sequencing method applied to the sequencing platform will affect the accuracy of the sequencing results obtained by the sequencing platform. In the process of developing the sequencing method, it is necessary to try different conditions and processes and evaluate the sequencing results. If all tests are completed through real experiments, on the one hand, the cost is high, and on the other hand, the research and development time is long, which affects the application of the sequencing platform. Summary of the invention

[0004] In view of this, this application provides the following technical solutions:

[0005] In a first aspect of the embodiments of the present application, a sequencing simulation method is provided, comprising:

[0006] Generate configuration templates for sequencing platforms;

[0007] Calling a corresponding test module based on the configuration template, wherein the test module simulates a target sequencing process to output a simulated test result;

[0008] The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

[0009] A second aspect of the embodiments of the present application provides a sequencing simulation device, comprising:

[0010] A generation unit, used to generate a configuration template for a sequencing platform;

[0011] A calling unit, configured to call a corresponding test module based on the configuration template, wherein the test module simulates a target sequencing process to output a simulated test result;

[0012] The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

[0013] According to a third aspect of the embodiments of the present application, a computer device is provided, including:

[0014] Memory, used to store programs;

[0015] A processor is used to execute the program, and the program is specifically used to implement the sequencing simulation method as described in any one of the above.

[0016] In a fourth aspect of an embodiment of the present application, a storage medium is further provided, wherein the storage medium stores executable instructions, and when the instructions are executed by a processor, a sequencing simulation method as described above is implemented.

[0017] In a fifth aspect of an embodiment of the present application, a sequencing system is also provided, and the sequencing system includes the computer device described above.

[0018] It can be known from the above technical scheme that the present application provides a sequencing simulation method, device, computer equipment, storage medium and sequencing system, in which a configuration template for a sequencing platform can be generated, and a corresponding test module is called based on the configuration template, and the target sequencing process is simulated by the corresponding test module to obtain a simulated test result. In the embodiment of the present application, the test module called by the configuration template can be a simulation sequencing module for the whole process of the sequencing process from sample library building to processing sequencing fluorescence images. By simulating the sequencing process, the influence of different influencing factors on sequencing can be evaluated in a controllable manner, and the influence of different sequencing conditions and sequencing parameters on the sequencing results can be clearly defined, solving the problem of being unable to determine the factors that have a major impact on the sequencing results among many influencing factors during actual sequencing. In addition, the present application can generate a simulated sequencing process with controllable sequencing parameters and sequencing processes, and can generate simulated sequencing data under extreme experimental conditions, solving the problem of being unable to controllably evaluate the performance of various aspects of the sequencing method; by simulating the sequencing process, all sequencing steps do not need to be completed by real sequencing experiments, reducing time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0020] Figure 1 A schematic diagram of a sequencing simulation method provided in an embodiment of the present application;

[0021] Figure 2 A schematic diagram of a system architecture for performing a simulation sequencing method provided in an embodiment of the present application;

[0022] Figure 3 A schematic diagram of a configuration file corresponding to a configuration template provided in an embodiment of the present application;

[0023] Figure 4 A schematic diagram of a configuration file of simulation sequencing parameters for a simulation sequencing module provided in an embodiment of the present application;

[0024] Figure 5 A schematic diagram of the structure of a sequencing simulation device provided in an embodiment of the present application. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. The terms "including" and "having" in this application and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not set to the listed steps or units, but may include steps or units that are not listed.

[0026] In the present application embodiment, a sequencing simulation method is provided, which can simulate the whole process from sample library building to processing sequencing fluorescence images in the sequencing process, because the relevant processes in each sequencing process of sequencing simulation have the needs of continuous modification and customization in the actual application process. Therefore, in the present application embodiment, each execution module or step in the simulation sequencing process is set to a test module that can be called independently, and each test module is called by calling the main program of each test module based on the configuration template. This setting is not only convenient for the user to simulate any processing flow in the sequencing process of the demand, but also convenient for upgrading and optimizing the relevant test modules separately, and also convenient for the functional expansion of the relevant flow in the sequencing process. Among them, each test module can be single-threaded when executing, and when multi-threaded parallel calculation is required, the calling main program corresponding to the configuration template can call multiple test modules for parallel calculation according to the parameter information of the test module to be called at the same time, thereby realizing the flexible configuration in the simulation sequencing process and the purpose of calling according to actual needs, and improving the application effect.

[0027] See also Figure 1 , is a flow chart of a sequencing simulation method provided in an embodiment of the present application, the method may include the following steps:

[0028] S101. Generate a configuration template for a sequencing platform.

[0029] S102. Calling a corresponding test module based on the configuration template, the test module simulates the target sequencing process to output a simulated test result.

[0030] In step S101, a configuration template corresponding to the actual sequencing platform used in the actual sequencing process can be generated. The sequencing platform is a key tool for DNA or RNA sequencing, which can be a platform using a high-throughput sequencing mode, such as a sequencing platform that uses microbeads or high-density chips for sequencing while synthesis. Correspondingly, the sequencing platform can also be a platform based on single-molecule sequencing technology, such as DNA sequencing, which does not require PCR amplification, and achieves individual sequencing of each DNA molecule.

[0031] The configuration template for the sequencing platform can be used to configure the sequencing parameters, sequencing process and other information required in the simulation sequencing process. For example, the simulation sequencing demander can configure the external service connection information in the simulation sequencing process through the configuration template. The external service can be a service platform for external information called in the simulation sequencing process, such as a data platform that provides data storage or import. Specifically, the data input interface and data output interface of each test module can be determined by the configuration template. Among them, the data input interface can be connected to the local data import storage area, the data output interface can be connected to the data receiving end, and can also be connected to the local disk or external storage device, that is, the data storage path parameters of the corresponding test module can be set on the configuration template, and the data output by the test module can be stored in the storage area corresponding to the data storage path. Other interface information can also be configured through the configuration template, such as configuring the configuration interface of the connection display or transmission function, the display information in the simulation sequencing process can be transmitted to the corresponding display device for display, and the relevant simulation test results in the simulation sequencing process can also be transmitted to the specified receiving end through the configuration of the transmission interface. The current existing test module can also be modified or operated through the configuration template. Specifically, the configuration template can provide a modifiable configuration file, in which a plurality of modifiable fields can be included so that the user can modify the parameter value under the corresponding field. In order to accurately assist the user in providing the configuration data of the corresponding parameters, the configuration module can also provide reference information of the corresponding configuration service, such as the format requirements of the modified field, the parameter range of the relevant configurable parameters, the upper limit of the parallel thread application, and other information. For example, when the configuration file is applied to the simulation amplification module, the corresponding field can include the save path of the simulation amplification result, and the user can enter the corresponding save path information in the input area of ​​the field; it can also include the range of the amplification step distribution, and the user can enter the minimum and maximum values ​​of the amplification step distribution under the field; when the configuration file is applied to the simulation sequencing module, the field included can be the simulation sequencing round number, and the user can enter the corresponding simulation sequencing round number value in the input area of ​​the field; it can also include the reaction dissociation constant field, and the user can enter the corresponding reaction dissociation constant value in the input area of ​​the field. The above is only an example of the optional fields of the configuration file in the configuration template, and the relevant fields can be supplemented and adjusted according to the actual application scenario and the simulation sequencing method adopted, and the embodiment of the present application is not limited to this.

[0032] In one implementation of the present application, generating a configuration template for a sequencing platform includes:

[0033] Based on the characteristic information of the sequencing platform, the configuration information of the simulated sequencing is determined; based on the configuration information, a configuration template is generated, wherein the configuration template at least includes the calling information of the test module and the parameter information based on the test module.

[0034] The characteristic information of the sequencing platform can characterize the platform information of the sequencing platform for simulated sequencing, such as the type of sequencing platform, the sequencing technology applied by the sequencing platform, the model of the sequencing platform, the sequencing precautions of the sequencing platform, and the applicable scenario information of the sequencing platform. Thus, the configuration information of the simulated sequencing can be determined according to the characteristic information of the sequencing platform, and the configuration information can characterize the relevant usage information when simulating the sequencing of the sequencing platform. For example, the configuration information may include the interface call information of different test modules in the simulated sequencing process, and may specifically include the data input interface information, data output interface information, etc. of each test module, the association information of each test module, and the format information of the data input to each test module, the condition information of the simulated test of each test module, etc. Specifically, after the configuration template is generated based on the configuration information, the configuration template at least includes the call information of the test module and the parameter information based on the test module. Among them, the call information of the test module can characterize the call conditions, application scenarios, or the type, data volume, and data format of the data that can be input to the test module, and may also include the number of parallel threads that can be executed by the test module. The parameter information based on the test module refers to the relevant limiting parameters of the sequencing process that the test module needs to simulate, or the parameter range and parameter conditions that affect the test result of the test module, and the influencing parameter values ​​that need to be simulated. For example, when the test module includes a simulation library building module, the parameter information based on the simulation library building module may include library length distribution information, library total information, etc. Specifically, different test modules have different corresponding parameter information, which will be described one by one in the relevant implementation of the embodiment of the present application later. In order to facilitate the configuration of the call information of the test module in the configuration template and the parameters based on the test module, a configuration file for storing the relevant information of the configuration template can be configured, and the configuration file is used to store the interface call information and parameter information related to each configuration, so that the user can export and modify the configuration file. Further, the configuration file can also include multiple subfiles, for example, the first subfile is used to set the call information of the test module, and the second subfile is used to set the parameter information of different test modules. Specifically, each test module can also correspond to a subfile, such as the parameter information of the simulation library building module can be set in the subfile of the simulation library building module, and the parameter information of the simulation hybridization module can be set in the subfile of the simulation hybridization module. In order to facilitate the call of relevant information, each sub-file can be stored in the same file storage area, such as storing it in the same folder. When the sequencing simulation process is automatically executed, the corresponding sub-file in the folder can be automatically called to configure the relevant parameters, thereby improving the efficiency of simulated sequencing. By generating a configuration template for the sequencing platform, it is possible to flexibly configure the relevant information, parameters, and corresponding sequencing processes used in the simulated sequencing process, facilitate the simulation of sequencing processes with different requirements, and improve the accuracy and practicality of simulated sequencing.

[0035] In step S102, the relevant test module can be called based on the call information of the configuration template, wherein the test module can be a module for a certain sequencing process in the sequencing process, or it can be all modules for the entire sequencing process. For example, the test module includes one or more of a simulated library building module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module, and a simulated image processing module, and each module in the test module is an independently callable module. Different test modules simulate different sequencing processes, such as the surface amplification process of the simulated amplification module simulating the sequencing process. The simulated sequencing demander can select the corresponding test module according to the current demand. If the library building and hybridization processes need to be simulated, the interface configuration information of the simulated library building module and the simulated hybridization module can be called in the configuration template, and the two modules are called to complete the corresponding target simulation sequencing process and output the simulation test results. It should be noted that, in the embodiment of the present application, the data to be simulated sequencing can be directly input into the corresponding sequencing module to be simulated. For example, when the sequencing module to be simulated is a simulation library building module, the demander can input the prepared sample to be built into the simulation library building module, or only set the library building parameters corresponding to the simulation library building module, call the sample to be built in the configuration template and input it into the simulation library building module, that is, the demander does not need to prepare sample data, and can directly determine the degree of influence of different parameters on the library building results according to the input of each parameter information. This implementation method is adopted to facilitate the demander to know the influence of different parameter information on the simulation library building results, so as to assist the demander in selecting the optimal library building parameters when performing sequencing experiments on a real sequencing platform. It can also be that the output simulation test result of the previous test module is input into the corresponding next test module as the input information of the next test module. For example, the sequence information of the simulation library corresponding to the simulation library result output by the simulation library building module is input into the simulation hybridization module, so that the sequence information is processed by the simulation hybridization module to simulate the hybridization process, and the simulation hybridization result is obtained. Specifically, the corresponding test module can be selected according to the sequencing simulation requirements, and this application does not limit which test module to use.

[0036] Each test module is configured with a corresponding target sequencing process. When the corresponding test module is called, the test module will automatically execute the corresponding target sequencing process according to the input parameter information and the data to be simulated and processed, so as to output the simulation test results corresponding to the test module. The data to be simulated and processed can be the simulation results of sequencing output by a test module in the current configuration template, or the data to be simulated and processed prepared by the user in advance, such as the samples to be sequenced for simulated sequencing, etc.

[0037] In the actual sequencing process, it is necessary to manually participate in multiple stages, such as the preparation of samples to be sequenced, in the actual sequencing library construction process, it is necessary to fragment DNA or RNA, and to perform processing steps such as adding adapters and barcodes, purification and concentration determination, and the state of the monitoring instrument and the setting of the reaction are also required in the sequencing process. When the sequencing simulation method is performed in the embodiment of the present application, it is only necessary to determine the test module, that is, it is necessary to determine the sequencing link to be simulated according to the actual application requirements, and then input the parameter information corresponding to the test module, and the computer device automatically executes the corresponding target sequencing process according to the input parameter information, and outputs the simulation test result. Specifically, in the embodiment of the present application, the target sequencing process corresponding to each test module can be stored in the mode of a computer application program, and when the corresponding test module needs to be called, the corresponding computer application program can be automatically executed based on the parameter information corresponding to the test module currently input, and the simulation test result corresponding to the test module is output. In the test simulation process, it is only necessary to input the corresponding parameter information or the sample to be simulated, without participating in or operating the sequencing process. In the embodiments of the present application, the target sequencing process corresponding to each test module is described in a certain process. For other optional processes, please refer to the description of the process, which will not be described in detail one by one.

[0038] In the process of sequencing using a real sequencing platform, since there are many indicators for evaluating the quality of sequencing results, these indicators are often affected by the common influence of various conditions such as instruments, biochemistry, algorithms, and the influence mechanism of these complex factors on the evaluation indicators is usually unclear. In order to clarify the possible impact of different sequencing conditions, sequencing parameters, etc. on the sequencing results, a sequencing simulation method is provided in the embodiment of the present application, which can simulate the sequencing process, and can include multiple test modules that characterize the entire sequencing process or a certain sequencing process, and evaluate the impact of different influencing factors on the sequencing index in a controllable manner by inputting parameter information to the corresponding test module. If only real sequencing test data is used for testing, since the real sequencing test data does not have a known standard answer, its experimental data will be affected by the experimental conditions, and therefore the performance and effect of various aspects of the sequencing algorithm cannot be controlled. In addition, it is often difficult to obtain data in extreme cases from real experimental data, which is not conducive to the evaluation of the effect of the algorithm in extreme cases. Through the sequencing simulation method of the present application, it is possible to generate simulated sequencing data with known conditions and controllable processes by setting different parameter information, and it is possible to simulate the generation of simulated sequencing data under extreme experimental conditions, which is conducive to the optimization and development of sequencing algorithms. In the development process of sequencing algorithms, different sequencing conditions and processes are needed to obtain the corresponding sequencing results. If all sequencing conditions and processes are completed through real sequencing experiments, the sequencing time will be longer and the corresponding cost will be higher. The sequencing simulation method of the embodiment of the present application can simulate different sequencing conditions, sequencing parameters and sequencing processes, and can quickly evaluate the possible impact of different optimization conditions on the sequencing results in principle, thereby optimizing the corresponding sequencing experiment and reducing time and cost.

[0039] In combination with actual application scenarios, the sequencing simulation method of the embodiment of the present application is described for different types of test modules. It should be noted that each module in the test module in the embodiment of the present application is an independently callable module, and each module will be mainly described in the following description of the implementation method. If the actual demand requires the selection of at least two modules to complete the simulation sequencing process, the corresponding simulation demand can be achieved in combination with the simulation sequencing process of the corresponding test module.

[0040] In one implementation of the embodiments of the present application, the test module includes a simulated library building module, and the configuration template includes call information of the simulated library building module and library building parameter information based on the simulated library building module for samples to be built, wherein the test module simulates the target test process to output simulated test results, including: the simulated library building module obtains and outputs the simulated library results based on the library building parameter information.

[0041] The simulation library building module is a process of simulating the conversion of DNA or RNA molecules in the sample to be built into a library that can be read by the test platform. In the application process of the simulation library building module, the user can input the library building parameter information corresponding to the simulation library building module, and then the simulation library building module automatically executes the corresponding simulation library building process based on the library building parameter information, and outputs the simulation library result. Specifically, the data input interface and data output interface of the simulation library building module can be obtained through the call information of the simulation library building module, or the thread that can be currently called by the simulation library building module. For example, the simulation library building module can simulate multiple tasks to be built, and the currently applicable thread can be clarified through the call information to avoid adding more library building tasks on the same thread, which affects the efficiency of the simulation library building. Among them, the simulation library building module obtains and outputs the simulation library result based on the library building parameter information, including: the simulation library building module determines the sample interruption parameter information on the reference genome corresponding to the sample to be built based on the library building parameter information; according to the reference genome and the sample interruption parameter information, the sequence information of the simulation library is obtained and output.

[0042] The sample interruption parameter information can be determined according to the characteristic information of the sequencing platform and the corresponding sample characteristic information of the sample to be constructed. The sample to be constructed is interrupted into a set of a certain length by the sample interruption parameter information and the reference genome corresponding to the sample to be constructed, so as to facilitate the subsequent simulation sequencing process. Among them, the processing program corresponding to the simulation library construction module can automatically process and analyze the sample interruption parameter information on the reference genome corresponding to the sample to be constructed according to the library construction parameter information, and obtain and output the sequence information of the simulation library according to the reference genome and the sample interruption parameter information.

[0043] Further, in the embodiment of the present application, the library construction parameter information includes library length distribution information and library total number information, wherein the simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information, including: the simulation library construction module calculates the number of libraries under different lengths based on the library length distribution information and the total number of libraries information; based on the total number of libraries information and the number of libraries of different lengths, the sample interruption parameter information is determined on the reference genome corresponding to the sample. The library length distribution information can be determined based on the sequencing platform currently simulated. This parameter can ensure that the DNA fragments in the library are uniform in size. The user can set the specific value of the parameter according to the actual needs to be simulated. The general length range is between 150-700bp, so that it can be divided into multiple length intervals according to the set length. The total number of libraries information refers to the total number of DNA fragments successfully constructed during the simulation library construction process. This parameter will affect the subsequent sequencing depth and coverage. The number of libraries under different lengths is obtained by calculating the number of fragments in each length interval by multiplying the total number value corresponding to the total number of library information by the ratio of the corresponding length interval. After obtaining the total number of libraries and the number of libraries of different lengths, the sequencing depth and coverage can be simulated, and then the current optimal sample interruption parameter information can be obtained according to the size and structural characteristics of the reference genome. In the embodiment of the present application, different library length distribution information and total number of libraries can be simulated based on the actual simulation sequencing requirements to obtain the corresponding sample interruption parameter information, so that there is no need to perform the real sample interruption operation every time, and the library construction can be simulated based on different parameter information to obtain the optimal sample interruption parameter information and improve the accuracy of subsequent simulation sequencing.

[0044] Further, based on the total number of library information and the number of libraries of different lengths, the sample interruption parameter information is determined on the reference genome corresponding to the sample, including: based on the total number of library information, the 5' end position information of the simulated library is determined on the reference genome corresponding to the sample, and the forward and reverse strand information of the simulated library is determined based on the 5' end position information; based on the number of libraries under different lengths, the library length information of the simulated library is determined, and based on the 5' end position information, the forward and reverse strand information and the library length information, the 3' end position information of the simulated library is determined. Correspondingly, according to the reference genome and the sample interruption parameter information, the sequence information of the simulated library is obtained and output, including: according to the reference genome, the 5' end position information, the forward and reverse strand information, the library length information and the 3' end position information, the sequence information of the simulated library is obtained and output. It should be noted that the above-mentioned 5' end position information, forward and reverse strand information, library length information and 3' end position information and other related information are all related information generated by the computer program that automatically executes the corresponding process through the simulation library construction module, and are different from the information that actually exists in the actual sequencing process. That is, the intermediate process information generated by each simulation test module in the embodiment of the present application is simulation information obtained by analyzing and processing the computer program of the corresponding processing flow.

[0045] The role of the reference genome of the sample is to provide a standard or template for comparison and analysis of sequencing data, so as to accurately interpret the gene sequence information. The 5' end position information and 3' end position information of the simulated library are the starting point and the ending point of the simulated library. Therefore, the forward and reverse strand information of the simulated library can be determined based on the 5' end position information, that is, whether each simulated library is a forward strand or a reverse strand, which can be identified by executing a simulated sequencing primer or a specific sequencing simulation method. The computer processing flow, for example, the probability of the simulated library being a forward strand or a reverse strand can be 50% respectively. Then, according to the reference genome, the 5' end position information, the forward and reverse strand information, the library length information and the 3' end position information, the sequence information of the simulated library of the sample to be built that is finally intercepted is determined and stored. Thereby, the simulated library building module can execute the corresponding processing flow according to the input different library length distribution information and the total number of libraries information, calculate the library number information under different lengths, and determine the sample interruption parameters, so as to simulate the process of library establishment, output the sequence information of the simulated library, and facilitate the simulation and regulation of the library building process in the sequencing process.

[0046] In one embodiment of the present application, the test module includes a simulated hybridization module, and the configuration template includes call information of the simulated hybridization module and pixel information based on the simulated hybridization module for simulating the original fluorescent image, wherein the test module simulates the target sequencing process to output the simulated test result, including: the simulated hybridization module obtains and outputs the simulated hybridization result based on the pixel information. The simulated hybridization module simulates the processing process of the hybridization library on the chip surface, wherein the term "chip" used in the embodiment of the present application refers to the sequencing chip used in the sequencing platform. However, in the simulated sequencing process of the embodiment of the present application, it is not necessary to use the actual sequencing chip, only some processing processes in the actual sequencing chip or related parameters corresponding to the sequencing chip will be simulated in the corresponding simulated sequencing process, so that the simulated sequencing process can be matched with the actual sequencing process, and the obtained simulated test results can better reflect the relevant information of the actual sequencing, which is convenient for adjusting the relevant processes or parameters in the actual sequencing based on the simulated test results, which can improve the efficiency of the actual sequencing and reduce the cost of the actual sequencing process. The data input interface, data output interface, and calling interface of the corresponding processing flow of the simulated hybridization module can be determined by configuring the calling information of the simulated hybridization module in the template, and the configuration file can also be called based on the calling information of the configuration file in the calling information, so that the pixel information corresponding to the simulated hybridization module for the simulated original fluorescence image is configured in the configuration file. When executing the simulated hybridization processing flow based on the simulated hybridization module, the corresponding parameter information can be pre-configured, that is, based on the pixel information of the simulated hybridization module for the simulated original fluorescence image, the pixel information includes one or more of the following: the number of pixels of the horizontal and vertical axes of the simulated original fluorescence image, the object size corresponding to each pixel, and the density of hybridization points on the chip surface. Correspondingly, the simulated hybridization module obtains and outputs the simulated hybridization result based on the pixel information, including: the simulated hybridization module calculates the total number of hybridization libraries of the simulated original fluorescence image based on the pixel information; according to the total number of hybridization libraries, the process of random hybridization of the simulated library on the chip surface in the simulated original fluorescence image is simulated to obtain and output the sequence information of the library after the simulated hybridization. In the embodiment of the present application, when the simulated hybridization module executes the target sequencing process corresponding to the simulated hybridization, the high-precision pixel information of the simulated original fluorescence image can be processed. That is, in the embodiment of the present application, the pixel information for simulating the original fluorescent image may refer to the pixel information in the usual sense, or may refer to the sub-pixel information corresponding to the smaller segmentation unit corresponding to the pixel, so that the sub-pixel information of the simulated original fluorescent image can be simulated to achieve higher-precision image processing of the simulated original fluorescent image. For example, in the process of obtaining and outputting the simulated hybridization result based on the pixel information, the simulated hybridization position can be made accurate to the level of "0.00x" pixels, and when the relevant information in the simulated original fluorescent image is subsequently merged, it can be merged into the pixel unit, thereby improving the processing accuracy.Correspondingly, if the pixel information refers to sub-pixel information, the unit of the number of pixels on the horizontal and vertical axes of the simulated original fluorescent image, for example, may also be 0.01 pixels. Specifically, when setting the pixel information for simulating the original fluorescent image, the user may set it to pixel information in the usual sense according to actual needs, or may set it to sub-pixel information with higher precision, and this application does not limit this.

[0047] In this process, the user can input the corresponding pixel information based on the actual simulated hybridization needs, that is, the pixel information can be flexibly configured according to the actual needs in the simulated hybridization module. For example, if the user needs to simulate the corresponding hybridization results under the density of different chip surface hybridization points, the density information of the corresponding chip surface hybridization points can be input when calling the simulated hybridization module. After completing this hybridization simulation, the density information of other chip surface hybridization points can be re-entered to complete the process of simulating the density of different chip surface hybridization points to obtain hybridization results. In this way, the influence of the density of the chip surface hybridization points on the final hybridization result can be simulated. In this embodiment, the simulated original fluorescence picture can be the fluorescence picture corresponding to the sequence information of the simulated library output by the simulated library building module in the embodiment of the present application, that is, after executing the simulated library building process, the sequence information of the simulated library generated based on the simulated library building process is processed according to the target sequencing process corresponding to the simulated hybridization; the simulated original fluorescence picture can also be the fluorescence picture corresponding to the simulated library input in advance by the user, and the present application does not limit this.

[0048] Among them, the object space size corresponding to each pixel represents the real size of the image corresponding to each pixel, so that the area of ​​the simulated original fluorescence image can be obtained according to the number of pixels on the horizontal and vertical axes of the simulated original fluorescence image and the object space size corresponding to each pixel. Then, according to the density of hybridization points on the chip surface and the area of ​​the simulated original fluorescence image, the total number of hybridization libraries of the simulated original fluorescence image is calculated, that is, the total number of hybridization libraries = the density of hybridization points on the chip surface * the area of ​​the simulated original fluorescence image. According to the above total number of hybridization libraries, the process of random hybridization of the library on the chip surface is simulated in the simulated original fluorescence image to obtain and output the sequence information of the library after simulated hybridization.

[0049] The simulated hybridization module can simulate the hybridization process based on different pixel information to obtain simulated hybridization results. In this way, the module can be used to determine the influence of different pixel information on the hybridization process. For example, by simulating the different horizontal and vertical axis pixel numbers of the original fluorescent image to determine the corresponding hybridization results, the influence of the horizontal and vertical axis pixel numbers on the hybridization results can be determined, thereby providing corresponding quantitative indicators for the optimization of the sequencing method.

[0050] In one implementation of the present application, the test module may also include a simulation amplification module, and the configuration template includes call information of the simulation amplification module and amplification information based on the simulation amplification module for the hybridized library, wherein the call information of the simulation amplification module may include interface information for calling the simulation amplification module, such as a data input interface, a data output interface, etc. for the simulation amplification module, and may also include call information for calling a configuration file of the simulation amplification module, and the configuration file is used to configure the amplification information of the amplification module. For example, the amplification information of the simulation amplification module may be determined by calling the configuration file. The use scenario information of the simulation amplification module may also be determined by the call information. The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information. The amplification information represents information that can affect the simulation amplification result, that is, the user can input the amplification information corresponding to the current simulation scenario according to the actual simulation sequencing requirements, so that the main indicator information affecting the simulation amplification result can be determined according to different amplification information.

[0051] Further, the amplification information includes one or more of the following: the number of amplification rounds, the amplification efficiency of each round, and the distribution of the amplification step length of each round. It should be noted that the selection of the amplification information can be determined by the user based on the actual simulation demand. For example, the user can only limit the number of amplification rounds in the simulation amplification process. In order to accurately complete the simulation amplification process, corresponding default parameters are also provided in the embodiment of the present application. When the user does not clearly define the amplification efficiency of each round and the distribution of the amplification step length of each round, the fixed parameters corresponding to the amplification efficiency of each round and the distribution of the amplification step length of each round of the simulation amplification module can be selected. These fixed parameters can be displayed when the simulation amplification result is output or the user inputs the amplification information, so that the user can know the amplification information mainly targeted by the current simulation amplification process, which is convenient for the user to optimize and adjust the simulation amplification process. Wherein, the number of amplification rounds refers to the number of times the DNA fragment is copied during the amplification process. When the user determines the specific numerical value corresponding to the number of amplification rounds, it can be determined according to the simulation demand and the sample situation to avoid excessive amplification and cause the problem of affecting the accuracy of the simulation amplification result. Amplification efficiency refers to the efficiency of DNA replication in each round of amplification, which is 100% ideally. However, in actual applications, amplification efficiency may be affected by many factors, such as primer design, reaction conditions, enzyme activity, etc. Therefore, users can set the amplification efficiency of each round based on the conditions of the sequencing experiment to be simulated. The amplification step distribution of each round involves the specific conditions of each step in the amplification, which affects the amplification efficiency. Users can determine the corresponding amplification efficiency by entering different amplification step distributions.

[0052] Specifically, the simulated amplification module obtains and outputs the simulated amplification result based on the amplification information, including: the simulated amplification module determines the library to be simulated amplified in the hybridized library based on the amplification efficiency and the amplification step distribution, and simulates the amplification of the library to be simulated amplified according to the number of amplification rounds, and obtains and outputs the sequence information of the template after the simulated amplification. Among them, in the embodiment of the present application, the hybridized library targeted by the simulated amplification module can be a hybridized library obtained through a real hybridization processing flow, or it can be a hybridized library obtained by simulating the hybridization process using the simulated hybridization module provided in the embodiment of the present application.

[0053] When the processing process of simulating amplification based on the simulation amplification module, the user can call the simulation amplification module based on the call information of the simulation amplification module in the configuration template, and the call information can characterize the call interface, application conditions and corresponding amplification methods that can be simulated of the simulation amplification module. The user can also input the corresponding amplification information to adjust the processing process of the simulation amplification through the amplification information. For example, the user can set the number of amplification rounds, and can also set the information such as the amplification efficiency and amplification step distribution of each round. For example, the user can input the corresponding number of amplification rounds before each simulation amplification processing process, so that the corresponding simulation amplification results under different amplification rounds can be obtained, so that an optimal number of amplification rounds can be selected based on the above-mentioned simulation amplification results in the actual sequencing experiment to complete the sequencing experiment, avoiding the high cost and high time problems caused by repeated experiments. The process of amplification is amplified round by round, that is, it is necessary to complete the corresponding multiple rounds of amplification according to the currently set number of amplification rounds, that is, the current round continues to amplify at the end of the previous round.

[0054] Furthermore, the simulated amplification module simulates amplification of the library to be simulated amplified according to the number of amplification rounds, including: performing a simulated amplification process on the library to be simulated amplified in the first amplification round to obtain a template after simulated amplification in the first amplification round; performing a simulated amplification process on the template after simulated amplification in the first amplification round to obtain a template after simulated amplification in the second amplification round; until performing a simulated amplification process on the template after simulated amplification in the k-1th amplification round to obtain a template after simulated amplification in the kth amplification round, wherein the number of amplification rounds is k. In this simulated amplification process, a corresponding number of amplification rounds can be performed according to the input number of simulated amplification rounds. For example, when k=5, 5 rounds of simulated amplification need to be performed. The simulated amplification module automatically executes the computer program corresponding to the simulated amplification process according to the input amplification information, and outputs the amplified template sequence information.

[0055] According to the input amplification efficiency of each round (for the convenience of description, p% is taken as an example), p% of the libraries are randomly selected from the hybridized libraries as the libraries to be simulated amplified, and these libraries are amplified. At the same time, according to the amplification step distribution, an amplification step distribution is randomly determined for each amplifiable simulated template, and the simulated amplification result is recorded, and the simulated amplification result includes the simulated template sequence and the simulated template position corresponding to the simulated template sequence, and the simulated template position represents the coordinates on the picture in pixels. At this time, the operation of the first round of simulated amplification can be completed. Then, based on the number of amplification rounds, in the second round of simulated amplification, the simulated amplification process of the second amplification round is performed according to the template after the simulated amplification of the first amplification round, that is, the p% library of the simulated template already present on the chip surface in the template after the simulated amplification of the first amplification round is selected as the library to be simulated amplified at present, and after the simulated amplification of the second amplification round is performed, the simulated template sequence in the simulated amplification process of the second amplification round and the simulated template position corresponding to the simulated template sequence are recorded, and the above operation is continued to be repeated, and the simulated amplification process of all amplification rounds is performed to obtain all the simulated template sequences and the simulated template positions corresponding to the template sequences. At this time, the simulated template positions corresponding to all template sequences on the chip and the sequence information of each simulated template can be recorded, so as to obtain and output the sequence information of the template after simulated amplification.

[0056] The above-mentioned is only a processing process of a kind of simulated amplification of the simulated amplification module in the embodiment of the present application, according to actual application requirements and application scenarios, if there is a multiplexing scenario to the sequencing simulation method, it is also possible to configure at least one target sequencing process for the corresponding test module. As the actual sequencing platform, different amplification methods can also be configured, including but not limited to PCR (polymerase chain reaction), MDA (multiple displacement amplification) and LAMP (loop-mediated isothermal amplification) etc. Therefore, different amplification processing processes can be configured for the simulated amplification module, that is, the simulated amplification module can be configured with different amplification processing programs, but the user only needs to input the amplification information into the simulated amplification module, and then the information of which amplification processing process is called can be input, so that the simulated amplification module can be based on these input information, automatically call the corresponding simulated amplification computer program, meet the user verification of the same amplification information corresponding to different simulated amplification processing modes, and whether the obtained simulated amplification results are the same. In this way, the user can not only flexibly set the corresponding amplification information according to the characteristics of the sequencing platform to be simulated, but also select the corresponding amplification processing mode, so that it can be matched with the real sequencing platform, so that the accuracy of the simulated amplification result is higher.

[0057] In the embodiment of the present application, the simulation amplification module obtains and outputs the process of the simulation amplification result based on the amplification information, and can also be an amplification process of simulating isothermal amplification performed by the simulation amplification module. In the process of simulating isothermal amplification, the user can input the amplification information corresponding to the simulated isothermal amplification process. For example, at this time, the corresponding amplification information can at least include the isothermal amplification temperature, the isothermal amplification reaction time and the amplification efficiency, that is, the simulation amplification module can simulate and obtain the corresponding simulation amplification results under different isothermal amplification temperatures, so that the user can determine the isothermal amplification temperature used in the actual sequencing process based on the output results of the simulation amplification module. Correspondingly, the simulation amplification module can also simulate and obtain the simulation amplification results under different isothermal amplification reaction times. Specifically, the simulation amplification module obtains and outputs the simulation amplification results based on the amplification information, including the following two methods:

[0058] Method 1: The simulation amplification module obtains the simulated amplified library for the hybridized library corresponding to the time tT based on the isothermal amplification temperature and the isothermal amplification time; and performs the simulated amplification processing corresponding to the time t based on the simulated amplified library for the hybridized library corresponding to the time tT, until the sequence information of the template after the simulated amplification that meets the amplification efficiency is obtained and output. The isothermal amplification process simulated by the simulation amplification module in this method is hybridization first and then amplification, that is, the process of performing isothermal amplification is to amplify a hybridized library. Among them, t represents the time to be performed for the current simulated amplification, and T represents the interval duration corresponding to the isothermal amplification reaction time. For example, T can be the same as the isothermal amplification time, or it can be a certain time added to the isothermal amplification time. For example, in the simulation process of performing isothermal amplification by the simulation amplification module, the user can select the isothermal amplification temperature of 65-70°C and the isothermal amplification time of 0.5-1 hour. At this time, T can be the same as the selected isothermal amplification time, or a certain termination reaction time can be added on the basis of the time. For example, if the isothermal amplification time input by the user is 50 minutes, T can be set to 60 minutes. Therefore, the isothermal amplification process corresponding to different isothermal amplification temperatures and isothermal amplification times can be simulated by the simulation amplification module. The difference in the corresponding isothermal amplification temperature and isothermal amplification time will affect the yield and stability of the amplified product. In the process of performing the simulated isothermal amplification by the simulation amplification module, it can be performed only once, or the number of times the simulated isothermal amplification process is performed can be determined based on the amplification efficiency. For example, if the amplification efficiency is lower than expected or the number of templates after the simulated amplification is small, multiple amplifications can be performed to achieve the purpose of meeting the preset amplification efficiency. Specifically, in the process of the simulated isothermal amplification, the setting of the relevant parameters can be set by the user according to the actual simulation requirements, and this application does not limit this.

[0059] Mode 2: The simulated amplification module performs simulated amplification processing for time t based on the simulated amplification library corresponding to time tT, and obtains and outputs the sequence information of the template after simulated amplification that meets the amplification efficiency. At this time, the simulated amplification module performs the processing flow of simulated isothermal amplification. The difference between mode 2 and mode 1 is that mode 2 adopts the simulated isothermal amplification process of hybridization and amplification, that is, in this mode, the simulated hybridization module and the simulated library increase module can be performed simultaneously. At this time, the simulated hybridization module can perform simulated hybridization processing for time t based on the simulated hybridization result corresponding to time tT, and obtain the simulated hybridization result corresponding to time t. Therefore, in mode 2, the simulated amplification library corresponding to time tT based on the simulated amplification module is a library that has not been hybridized, but the process of simulated hybridization and amplification is performed simultaneously. In the actual application process, if the user needs to simulate hybridization and amplification, mode 2 can be selected to perform the processing flow of simulated hybridization and simulated amplification through the simulated hybridization module and the simulated amplification module at the same time, thereby improving the simulation efficiency.

[0060] In one embodiment of the present application, the test module also includes a simulation sequencing module, and the configuration template includes call information of the simulation sequencing module and simulation sequencing parameters based on the simulation sequencing module for the template to be sequenced, wherein the test module simulates the target sequencing process to output simulation test results, including: the simulation sequencing module obtains and outputs simulation sequencing results based on the simulation sequencing parameters.

[0061] Among them, the simulated sequencing module is used to simulate the sequencing process, and the calling interface, application scenario, usage conditions, and simulated sequencing platform or simulated sequencing method of the simulated sequencing module can be determined by configuring the calling information of the simulated sequencing module in the template. The simulated sequencing parameters based on the simulated sequencing module mainly refer to the parameters that can affect the final simulated sequencing results. When calling the simulated sequencing module, the relevant simulated sequencing parameters used in the simulated sequencing process can be input to obtain the influence of the simulated sequencing parameters on the simulated sequencing results.

[0062] Specifically, in the embodiment of the present application, the sequencing method used by the simulation sequencing module is a fluorescent sequencing method, that is, a technique for DNA sequence determination using fluorescently labeled nucleotides. If the simulated sequencing platform uses a sequencing-by-synthesis technique, the target sequencing process corresponding to the corresponding simulation sequencing module can be a synthetic sequencing process using a dNTP with a removable azide group and a fluorescent label, and each synthesis can only add one base and determine the type of the base by a fluorescent signal. The processing flow is a fixed computer program automatically executed by the simulation sequencing module. During the simulation sequencing process, the user does not need to monitor or regulate the simulation sequencing process, and only needs to enter the corresponding simulation sequencing parameters before the simulation sequencing is executed. Specifically, in one embodiment, the simulation sequencing parameters may include one or more of the following: the reaction dissociation constants of the four AGCT bases corresponding to the fluorescent and non-fluorescent AGCT bases, the reaction concentrations corresponding to the various different bases, the number of simulation sequencing rounds, the efficiency of the fluorescent group removal of different bases, and the probability of being unable to perform subsequent simulation sequencing. The reaction dissociation constant describes the stability of the current base to be reacted and its ligand binding. The user can determine the sequence information of the simulated sequencing under different stability levels of the current base to be reacted and its ligand set according to the different reaction dissociation constants set, so as to determine the reaction dissociation constant that meets the actual real sequencing test. The AGCT bases with fluorescence and without fluorescence refer to whether the corresponding bases are fluorescently labeled, that is, in the simulation sequencing process in the embodiment of the present application, the reaction dissociation constant of the base with fluorescence label can be selected at the corresponding template, or the base without fluorescence label can be selected, so that the application scenario is more extensive. The reaction concentration corresponding to various different bases can be the concentration of bases in different reagents, such as the concentration of hot bases in hot-mix reagents, the concentration of cold bases in hot-mix reagents, etc. The user can set the reaction concentration corresponding to different bases according to the actual simulated reagents and the corresponding simulation sequencing method. If the simulation sequencing module simulates a sequencing process of synthesis and sequencing, the user can also set the probability of failure to perform subsequent simulation sequencing, that is, the probability of the template chain fixed on the chip to be simulated sequencing being washed away or denatured during each round of sequencing. The parameter setting can be used to determine the probability parameter that meets the actual sequencing requirements, so as to improve the accuracy of subsequent applications in the actual sequencing process.

[0063] There are also multiple rounds of simulated sequencing in the simulated sequencing process. The simulated sequencing module obtains and outputs simulated sequencing results based on the simulated sequencing parameters, including: the simulated sequencing module performs the first round of simulated sequencing processing on the template to be sequenced based on the simulated sequencing parameters to obtain the first base simulated sequencing result of the template to be sequenced; performs the second round of simulated sequencing processing based on the first base simulated sequencing result to obtain the second base simulated sequencing result of the template to be sequenced; until the nth round of simulated sequencing processing is performed based on the simulated sequencing result of the n-1th base to obtain the sequence information of the simulated sequencing, wherein the number of simulated sequencing rounds is n. Among them, the template to be sequenced can be a template that needs to complete the simulated sequencing process prepared in advance by the user, or it can be a template to be sequenced that has been simulated and processed by the simulated library construction module, simulated hybridization module, simulated amplification module and other modules provided in the embodiments of the present application.

[0064] Specifically, the simulation sequencing process of each simulation sequencing round is similar, except that the current round of simulation sequencing uses the base simulation sequencing results obtained in the previous round of simulation sequencing. Taking the first round of simulation sequencing as an example, the simulation sequencing process includes: determining the first base of all templates to be sequenced as the base to be reacted; randomly generating base reaction information of the base to be reacted according to the reaction dissociation parameters corresponding to the template to be sequenced; if the base reaction information indicates that the base to be reacted successfully binds to the corresponding base, recording the base type of the base to be reacted; if the base reaction information indicates that the base to be reacted is prevented from binding to the corresponding base, determining the next base of the first base as the base to be reacted, and continuing to determine the base type of the base to be reacted until the base types of all bases to be reacted corresponding to the template to be sequenced are obtained; and counting the total number of different types of bases corresponding to each pixel in the fluorescent image corresponding to the template to be sequenced to obtain the simulation sequencing result.

[0065] In the embodiment of the present application, the simulation sequencing method adopted by the simulation sequencing module is a fluorescent sequencing method, and its goal is to obtain the base situation of each simulation template "reacted" in each round of sequencing, that is, whether the base is synthesized, how many bases are further synthesized, and what type of bases are respectively, such as bases with or without fluorescence. Therefore, when the simulation sequencing module executes the corresponding target sequencing process, the total number of different types of bases corresponding to each pixel in the fluorescent picture corresponding to the template to be sequenced can be determined by executing. Specifically, the target sequencing process corresponding to the simulation sequencing module is to simulate the fluorescence scanner to scan each pixel on the chip in different fluorescence channels, and record the fluorescence brightness under each channel, and determine the base sequencing result of the template sequence corresponding to the template to be sequenced based on the fluorescence brightness, that is, to encapsulate the process into an automatically executable computer program and store it in the simulation sequencing module. When the user enters the simulation sequencing parameters, the simulation sequencing module will automatically execute the computer program based on the simulation sequencing parameters to obtain the base sequencing results. For each template sequence of the template to be sequenced, according to the reaction dissociation constants of the eight bases corresponding to the template to be sequenced, the base reaction information at the template of the current simulation sequencing round is randomly generated, and the base reaction information specifically includes whether the current base to be reacted is combined with a base, and if it is combined with a base, it is recorded which of the eight bases is, that is, the base type of the base to be reacted is recorded. The blocking situation will also be recorded. For the case of failure of blocking, the next base corresponding to the template is directly exposed as the base to be reacted, and the above steps are repeated until the base types of all bases to be reacted corresponding to the template to be sequenced are obtained. The total number of different types of bases corresponding to each pixel in the fluorescence picture corresponding to the chip is counted to obtain the simulation sequencing result. Among them, the target sequencing process corresponding to the simulation sequencing module can also include processing processes such as correction between channels, and finally determine the base sequencing result of the template sequence corresponding to the template to be sequenced. It should be noted that in the embodiment of the present application, the calculation of the fluorescence brightness corresponding to different pixels in different fluorescence channels is based on the total number of different types of bases corresponding to the fluorescence brightness distribution and each pixel. Therefore, the random fluctuation of base fluorescence brightness is taken into account. In addition, the computational simulation process simulates the fluorescence brightness corresponding to each pixel position under ideal conditions without optical diffraction limit. If it is necessary to simulate actual conditions, the simulation sequencing process can be optimized according to the actual conditions, or the correction parameters of the fluorescence brightness corresponding to each pixel position can be set to achieve simulation sequencing under actual conditions.

[0066] Correspondingly, in the actual sequencing process, there will be a process of excising the fluorescent group and terminator. In order to make the simulation sequencing result more accurate, when executing the simulation sequencing process based on the simulation sequencing module, a calculation function can be set to represent the excision of the fluorescent group. The calculation function can include the intensity and duration of the simulated fluorescent signal, and the state after excision. After the simulation excision, the sequence state is updated to remove the detected fluorescent marker. The excision of the corresponding simulation terminator can be realized by setting a conditional judgment, such as when a termination base is added, the extension of the chain is stopped by simulation. That is, any process in the simulation actual sequencing process in the embodiment of the present application can be realized by setting a corresponding computer program, without the need for user execution. The user only needs to input the corresponding relevant parameters to be simulated. Specifically, when executing the simulated sequencing process, if the base reaction information indicates that the base to be reacted has matched the corresponding base, the next base of the current base to be reacted is determined as the base to be reacted; if the base reaction information indicates that the base to be reacted does not match the corresponding base, the current base to be reacted is determined as the base to be reacted; if the terminator of the template to be sequenced is not successfully removed and the base to be reacted does not match the corresponding base, a prompt message is output that the next simulated sequencing round of the current simulated sequencing round cannot react. That is, when executing the target process corresponding to the simulated sequencing, the next base will be determined according to the current simulated sequencing result, so as to continue processing.

[0067] The simulated sequencing module can be used to simulate the sequencing process. Users can adjust and update different simulated sequencing parameters according to actual simulation requirements to obtain simulated sequencing results that meet the sequencing requirements, and can determine the simulated sequencing parameters for real sequencing experiments based on the simulated sequencing results, thereby improving the accuracy and efficiency of sequencing.

[0068] In one embodiment of the present application, the test module includes a simulated image processing module, and the configuration template includes calling information of the simulated image processing module and image processing parameters for fluorescent images based on the simulated image processing module, wherein the test module simulates the target sequencing process to output simulated test results, including: the simulated image processing module obtains and outputs simulated sequencing fluorescent images based on the image processing parameters.

[0069] The simulated image processing module is used to process the test result data obtained by each test module, and processes the test result data according to the image processing parameters in the configuration template, and obtains and outputs the simulated sequencing fluorescence image. Among them, the call information of the simulated image processing module in the configuration template may include the call interface information of the simulated image processing module, and the corresponding processing thread information of the simulated image, and may also include its call condition information, such as whether multi-threaded parallel processing is possible, and the application scenarios and application conditions for its processing. The image processing parameters of the simulated image processing module are adjustable parameters set based on the processing flow of the current simulated image, and the image processing parameters can affect the image effect of the final simulated sequencing fluorescence image.

[0070] Furthermore, the image processing parameters include the point spread function in the simulated imaging system, the fluorescence brightness probability distribution corresponding to the fluorescent AGCT bases in different fluorescence channels, the background brightness spatial distribution corresponding to different fluorescence channels, and the random brightness noise characteristics corresponding to different fluorescence channels. The simulated image processing module obtains and outputs the simulated sequencing fluorescence image based on the image processing parameters, including:

[0071] According to the fluorescence brightness probability distribution of the fluorescent AGCT bases in different fluorescence channels and the total number of different types of fluorescently labeled bases corresponding to each pixel, the base fluorescence brightness information corresponding to different pixels in different fluorescence channels is calculated; according to the base fluorescence brightness information of each pixel in the four fluorescence channels in the fluorescence image corresponding to each round of sequencing, the first simulated fluorescence image corresponding to the four fluorescence channels of each round of sequencing is generated; the first simulated fluorescence image is processed based on the point spread function to output the second simulated fluorescence image; according to the background brightness spatial distribution corresponding to different fluorescence channels and the random brightness noise characteristics corresponding to different fluorescence channels, the background and noise brightness corresponding to different pixels in different fluorescence channels are calculated; the background and noise brightness corresponding to different pixels in different fluorescence channels are superimposed on the second simulated fluorescence image to obtain a simulated sequencing fluorescence image.

[0072] Specifically, according to the fluorescence brightness distribution corresponding to the fluorescent AGCT base in different fluorescence channels, and according to the background brightness distribution corresponding to different fluorescence channels, after removing the background noise, the fluorescence brightness corresponding to different pixels in different fluorescence channels is calculated, and the fluorescence intensity difference between different channels can be compared. Among them, the fluorescence picture corresponding to each round of sequencing can be a fluorescence picture generated based on a real sequencing platform, or it can be a fluorescence picture corresponding to the sequence information of each simulated sequencing round generated based on the simulated sequencing module in the embodiment of the present application. Among them, the point spread function can characterize how an ideal point light source in the imaging system is expanded into a light spot of a certain shape, for example, the point spread function can be set to a Gaussian distribution function. First, the fluorescence picture can be preprocessed, such as removing noise, background correction and contrast adjustment, and then according to the base fluorescence brightness information of each pixel in the fluorescence picture corresponding to each round of sequencing in the four fluorescence channels, the first simulated fluorescence picture corresponding to each round of sequencing is generated, and then the point spread function is applied to each pixel of the first simulated fluorescence picture, which is usually implemented by convolution operation, that is, the point spread function is multiplied by each local area of ​​the first simulated fluorescence picture, and the pixel value after diffusion is obtained by summing, and the second simulated fluorescence picture is generated and output according to the pixel value after diffusion of each pixel point. According to the spatial distribution of background brightness corresponding to different fluorescence channels and the random brightness noise characteristics corresponding to different fluorescence channels, the background and noise brightness corresponding to different pixels in different fluorescence channels are calculated; the background and noise brightness corresponding to different pixels in different fluorescence channels are superimposed on the second simulated fluorescence image to obtain a simulated sequencing fluorescence image, that is, the simulated sequencing fluorescence image is the final image output by the simulated image processing module.

[0073] In this embodiment, the processing process of the point spread function in different imaging systems can be simulated by the simulated image processing module, thereby obtaining a simulated sequencing fluorescence image, and further obtaining a simulated sequencing image, thereby ensuring the processing of image information in the simulated sequencing process.

[0074] It should be noted that in the test module called based on the configuration template in the embodiment of the present application, each test module can be called separately, that is, the simulation processing of a certain process in the sequencing process can be realized, or all test modules can be called as a whole to complete the simulation of the whole sequencing process, or multiple test modules can be called in combination with actual application requirements to complete the simulation of the process in the relevant sequencing process, thereby realizing the ability to flexibly simulate different processing processes in the sequencing process, making the scope of application wider. Further, in order to ensure that the user can trace back the relevant result information of the sequencing simulation process, the output results of each test module can be saved separately in the embodiment of the present application. Even if the simulation of the whole sequencing process is selected, the simulated library results of the simulated library construction module, the simulated hybridization results of the simulated hybridization module, the simulated amplification results of the simulated amplification module, the simulated sequencing results of the simulated sequencing module, and the simulated sequencing fluorescence images of the simulated image processing module can be saved separately, so that the user can adjust the parameter information of the relevant test modules according to the output results of different test modules, so that there is no need to re-execute the whole sequencing simulation process, which reduces the time of simulated sequencing.

[0075] Furthermore, the calling information for each test module and the parameter information based on the test module can be saved in the same file storage area. In this way, when the computer program that executes the sequencing simulation method is running, the relevant configuration file in the file storage area can be directly called to automatically configure the parameter information of each test module. The user does not need to wait for the current test module to be completed before configuring the next test module, which improves the efficiency of the automated execution of the sequencing simulation method.

[0076] See also Figure 2 , which is a schematic diagram of a system architecture for executing a sequencing simulation method provided in an embodiment of the present application, the system architecture includes a main program for executing the sequencing simulation method, that is, the main program can automatically execute a test module selected by a user based on the calling information of the test module included in the configuration template and the parameter information based on the test module, or automatically execute the entire simulation sequencing process, that is, execute the processing process of all test modules.

[0077] exist Figure 2 In the architecture shown, firstly, the configuration file corresponding to the configuration template is imported into the main program, wherein the configuration file corresponding to the configuration template can be a file generated in the format of an Excel table. Figure 3 As shown, the configuration file may include configurable parameter information and information automatically generated based on the parameter information. Figure 3As shown in the figure, if the user can input the number of pixels on the Y axis of the simulated original fluorescence image, if the user inputs 1024, then the parameter field that can be recognized by the main program, such as fig_size_y, can be automatically calculated based on the value; the user can also enter the probability of each round of amplification in the hydrogel, if the user enters 0.3, then the parameter field that can be recognized by the main program is automatically calculated as p_amplification. The corresponding parameter information in the specific configuration file can be determined based on the actual application scenario. It should be noted that in Figure 3 Only some example field information is shown in the figure. For example, it can also include the maximum and minimum values ​​of the amplification step distribution when the chip surface is amplified, the pixel value of the Gaussian function when the imaging system point spread function is a Gaussian function, etc. Specifically, the configuration field can be set according to the actual simulated sequencing platform and the sequencing method used by the sequencing platform, and is not limited to Figure 3 Some of the fields are shown.

[0078] Correspondingly, a parameter information configuration file corresponding to a specific test module can also be generated, such as Figure 4 The configuration file of the simulated sequencing parameters for the simulated sequencing module shown in the figure may also include configurable parameter information, that is, parameter information that can be input by the user, and a parameter part that is automatically calculated based on the parameter information. Figure 4 As shown, users can input the base concentrations of different bases in the corresponding reagents, such as the concentration of hot bases in the Hot-mix reagent. This can automatically calculate the probability of adding a hot T base, the probability of adding a hot G base, the probability of adding a hot C base, and the probability of adding a hot A base at the position where an A base should be added. It should be noted that Figure 4 This is only an exemplary partial display, for example, it can also include the fluctuation information of the fluorescence brightness of a single molecule T hot base in the G fluorescence channel, etc. Figure 3 and Figure 4 The gray part shows the relevant parameter information that users can input and adjust, and the white part is the parameter information automatically calculated. Figure 3 and Figure 4 The configurable parameters shown in are only examples. The relevant parameters that the user can input and adjust can be determined according to the actual application scenario, and this application does not limit this.

[0079] After the main program calls the configuration file corresponding to the configuration template, it can automatically determine the test module that needs to be called. For example, if the user needs to rebuild the library, the simulated library building module will be called to obtain the simulated library results. If the simulated library building module does not need to be called, the user can also be allowed to input the corresponding library data. Then it can be determined whether re-hybridization is needed. If it is necessary, the simulated hybridization module will be called to obtain the simulated hybridization results. If the simulated hybridization module does not need to be called, the user can be allowed to input the corresponding hybridization library. If it is determined that a simulated amplification process needs to be performed, the simulated amplification module will be called to obtain the simulated amplification results. If the simulated amplification module does not need to be called, the user can also be allowed to input the corresponding amplification data. If a simulated sequencing process needs to be performed, the simulated sequencing module will be called to obtain the simulated sequencing results. If the simulated image processing module needs to be called, this module will be called to process the fluorescent image and obtain a simulated sequencing fluorescent image. Among them, the fluorescent image can be a fluorescent image generated based on the simulated sequencing result. It should be noted that in Figure 2 In the architecture shown, each module can be used separately, and the data input to the corresponding module can be from the previous test module, or it can be the relevant data input by the user. For example, the data input to the simulated hybridization module can be the simulated library result output by the simulated library construction module, or it can be the library data input by the user. Correspondingly, the data input to the simulated sequencing module can be the simulated amplification result output by the simulated amplification module, or it can be the amplification data input by the user. Specifically, the simulated library result includes the sequence information of the simulated library; the simulated hybridization result includes the sequence information of the library after the simulated hybridization; the simulated amplification result includes the sequence information of the template after the simulated amplification; the simulated sequencing result includes the sequence information of the simulated sequencing.

[0080] Specifically, the corresponding call information and parameter information in the configuration template may also include instrument reagent condition parameters in the simulation sequencing process, such as configuration parameters of the optical imaging system, characteristic parameters of chemical reagents, etc.; configuration parameters of the simulation sequencing software, such as the storage location of the simulation image, the number of threads of the simulation program running, etc. Among them, the file corresponding to the output simulation test result needs to be the same as the file format of the sequencing platform for the purpose, such as the output simulation sequencing fluorescence image, and its storage rules and format are consistent with the storage rules and formats of the real sequencing images of the sequencing platform. In the embodiment of the present application, the test module called by the configuration template can be a simulation sequencing module for the whole process of the sequencing process from sample library building to processing sequencing fluorescence images. By simulating the sequencing process, the influence of different influencing factors on sequencing can be evaluated in a controllable manner, and the influence of different sequencing conditions and sequencing parameters on the sequencing results can be clarified, which solves the problem that the factors that have a major impact on the sequencing results cannot be determined among many influencing factors during actual sequencing. In addition, the present application can generate a simulated sequencing process with controllable sequencing parameters and sequencing processes, and can generate simulated sequencing data under extreme experimental conditions, thus solving the problem of being unable to controllably evaluate the performance of various aspects of the sequencing method. By simulating the sequencing process, it is not necessary for all tests to be completed through real sequencing experiments, thus reducing time and cost.

[0081] In the present application, a sequencing simulation device is also provided. Figure 5 , the device may include:

[0082] A generating unit 501 is used to generate a configuration template for a sequencing platform;

[0083] A calling unit 502 is used to call a corresponding test module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result;

[0084] The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

[0085] Optionally, the generating unit includes:

[0086] A first determination subunit is used to determine configuration information of simulated sequencing based on characteristic information of a sequencing platform;

[0087] The first generating subunit is used to generate a configuration template based on the configuration information, where the configuration template at least includes calling information of the test module and parameter information based on the test module.

[0088] Optionally, the test module includes a simulated library construction module, the configuration template includes call information of the simulated library construction module and library construction parameter information for samples to be constructed based on the simulated library construction module, wherein the call unit includes a first call subunit, the first call subunit is used to call the simulated library construction module based on the configuration module, and the test module simulates the target sequencing process to output a simulated test result, including:

[0089] The simulated library construction module obtains and outputs the simulated library results based on the library construction parameter information.

[0090] Optionally, the simulation library construction module obtains and outputs simulation library results based on the library construction parameter information, including:

[0091] The simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information;

[0092] According to the reference genome and sample segmentation parameter information, the sequence information of the simulated library is obtained and output.

[0093] Optionally, the library construction parameter information includes library length distribution information and total library information, wherein the simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information, including:

[0094] The simulation library construction module calculates the number of libraries of different lengths based on the library length distribution information and the total number of libraries;

[0095] Based on the total number of libraries and the number of libraries of different lengths, the sample interruption parameter information is determined on the reference genome corresponding to the sample.

[0096] Optionally, based on the total number of libraries and the number of libraries of different lengths, sample interruption parameter information is determined on the reference genome corresponding to the sample, including:

[0097] Based on the total number of libraries, the 5' end position information of the simulated library is determined on the reference genome corresponding to the sample, and the forward and reverse strand information of the simulated library is determined based on the 5' end position information;

[0098] Based on the information of the number of libraries at different lengths, the library length information of the simulated library is determined, and based on the 5' end position information, the forward and reverse strand information and the library length information, the 3' end position information of the simulated library is determined;

[0099] According to the reference genome and sample interruption parameter information, the sequence information of the simulated library is obtained and output, including:

[0100] According to the reference genome, 5' end position information, forward and reverse strand information, library length information and 3' end position information, the sequence information of the simulated library is obtained and output.

[0101] Optionally, the test module includes a simulated hybridization module, the configuration template includes call information of the simulated hybridization module and pixel information based on the simulated hybridization module for simulating the original fluorescent image, wherein the call unit includes a second call subunit, a second call subunit, and is used to call the simulated hybridization module based on the configuration information, and the test module simulates the target sequencing process to output a simulated test result, including:

[0102] The simulated hybridization module obtains and outputs simulated hybridization results based on pixel information.

[0103] Optionally, the pixel information includes one or more of the following: the number of pixels on the horizontal and vertical axes of the simulated original fluorescence image, the object space size corresponding to each pixel, and the density of hybridization points on the chip surface. The simulated hybridization module obtains and outputs the simulated hybridization results based on the pixel information, including:

[0104] The simulated hybridization module calculates the total number of hybridization libraries that simulate the original fluorescence image based on pixel information;

[0105] According to the total number of hybridization libraries, the random hybridization process of the libraries on the chip surface is simulated in the simulated original fluorescence image to obtain and output the sequence information of the libraries after simulated hybridization.

[0106] Optionally, the test module includes a simulated amplification module, the configuration template includes call information of the simulated amplification module and amplification information based on the simulated amplification module for a hybridized library, wherein the call unit includes a third call subunit, the third call subunit is used to call the simulated amplification module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including:

[0107] The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information.

[0108] Optionally, the amplification information includes one or more of the following: the number of amplification rounds, the amplification efficiency of each round, and the amplification step distribution of each round. The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information, including:

[0109] The simulated amplification module determines the library to be simulated amplified in the hybridized library based on the amplification efficiency and the amplification step length distribution, performs simulated amplification on the library to be simulated amplified according to the number of amplification rounds, and obtains and outputs the sequence information of the template after the simulated amplification;

[0110] Wherein, performing simulated amplification on the library to be simulated amplified according to the number of amplification rounds includes:

[0111] Performing a first round of simulated amplification on the library to be simulated amplified to obtain a template after the first round of simulated amplification;

[0112] Performing a simulated amplification process for a second amplification round based on the template after simulated amplification in the first amplification round to obtain a template after simulated amplification in the second amplification round;

[0113] Until the kth round of simulated amplification is performed based on the template after the simulated amplification in the k-1th round of amplification, to obtain the template after the simulated amplification in the kth round of amplification, wherein the number of amplification rounds is k.

[0114] Optionally, the amplification information at least includes isothermal amplification temperature, isothermal amplification reaction time and amplification efficiency, wherein the simulation amplification module obtains and outputs the simulation amplification result based on the amplification information, including:

[0115] The simulation amplification module obtains a simulated amplified library for the hybridized library corresponding to the time tT based on the isothermal amplification temperature and the isothermal amplification time; and performs a simulated amplification process corresponding to the time t based on the simulated amplified library for the hybridized library corresponding to the time tT, until the sequence information of the template after the simulated amplification that satisfies the amplification efficiency is obtained and output;

[0116] Alternatively, the simulated amplification module performs simulated amplification processing for time t based on the simulated amplified library corresponding to time tT, and obtains and outputs sequence information of the simulated amplified template that meets the amplification efficiency;

[0117] Wherein, t represents the time for the current simulated amplification to be performed, and T represents the interval duration corresponding to the isothermal amplification reaction time.

[0118] Optionally, the test module includes a simulated sequencing module, the configuration template includes call information of the simulated sequencing module and simulated sequencing parameters for a template to be sequenced based on the simulated sequencing module, wherein the call unit includes a fourth call subunit, the fourth call subunit is used to call the simulated sequencing module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including:

[0119] The simulation sequencing module obtains and outputs simulation sequencing results based on the simulation sequencing parameters.

[0120] Optionally, the simulated sequencing parameters include one or more of the following: reaction dissociation constants of the four AGCT bases corresponding to the fluorescent and non-fluorescent AGCT bases, reaction concentrations corresponding to various bases, the number of simulated sequencing rounds, the removal efficiency of the fluorescent groups of different bases, and the probability of being unable to perform subsequent simulated sequencing. The simulated sequencing module obtains and outputs simulated sequencing results based on the simulated sequencing parameters, including:

[0121] The simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a simulation sequencing result of the first base of the template to be sequenced;

[0122] Performing a second round of simulated sequencing processing based on the first base simulated sequencing result to obtain a second base simulated sequencing result of the template to be sequenced;

[0123] Until the nth round of simulated sequencing is performed based on the simulated sequencing result of the n-1th base, the sequence information of the simulated sequencing is obtained, wherein the number of simulated sequencing rounds is n.

[0124] Optionally, the simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a first base simulation sequencing result of the template to be sequenced, including:

[0125] The first base of all templates to be sequenced is determined as the base to be reacted;

[0126] Randomly generate base reaction information of the base to be reacted according to the reaction dissociation parameters corresponding to the template to be sequenced;

[0127] If the base reaction information indicates that the base to be reacted successfully combines with the corresponding base, the base type of the base to be reacted is recorded;

[0128] If the base reaction information indicates that the base to be reacted is prevented from combining with the corresponding base, the next base of the first base is determined as the base to be reacted, and the base types of the base to be reacted are continuously determined until the base types of all the bases to be reacted corresponding to the template to be sequenced are obtained;

[0129] The total number of different types of bases corresponding to each pixel in the fluorescent image corresponding to the template to be sequenced is counted to obtain a simulated sequencing result.

[0130] Optionally, the device further comprises: a base determination subunit to be reacted for:

[0131] If the base reaction information indicates that the base to be reacted has matched the corresponding base, the next base of the current base to be reacted is determined as the base to be reacted;

[0132] If the base reaction information indicates that the base to be reacted does not match the corresponding base, the current base to be reacted is determined as the base to be reacted;

[0133] If the terminator of the template to be sequenced is not successfully removed and the base to be reacted does not match the corresponding base, a prompt message is output that the next simulated sequencing round of the current simulated sequencing round cannot react.

[0134] Optionally, the test module includes a simulated image processing module, the configuration template includes call information of the simulated image processing module and image processing parameters for the fluorescent image based on the simulated image processing module, wherein the call unit includes a fifth call subunit, the fifth call subunit is used to call the simulated image processing module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including:

[0135] The simulated image processing module obtains and outputs simulated sequencing fluorescence images based on the image processing parameters.

[0136] Optionally, the image processing parameters include a point spread function in a simulated imaging system, a fluorescence brightness probability distribution corresponding to fluorescent AGCT bases in different fluorescence channels, a background brightness spatial distribution corresponding to different fluorescence channels, and a random brightness noise characteristic corresponding to different fluorescence channels. The simulated image processing module obtains and outputs a simulated sequencing fluorescence image based on the image processing parameters, including:

[0137] According to the fluorescence brightness probability distribution of the fluorescent AGCT bases in different fluorescence channels, and the total number of different types of fluorescently labeled bases corresponding to each pixel, the fluorescence brightness information of the bases corresponding to different pixels in different fluorescence channels is calculated;

[0138] Generate a first simulated fluorescence image corresponding to the four fluorescence channels of each round of sequencing according to the base fluorescence brightness information of each pixel in the four fluorescence channels in the fluorescence image corresponding to each round of sequencing;

[0139] Processing the first simulated fluorescence image based on the point spread function, and outputting a second simulated fluorescence image;

[0140] According to the background brightness spatial distribution corresponding to different fluorescence channels and the random brightness noise characteristics corresponding to different fluorescence channels, the background and noise brightness corresponding to different pixels in different fluorescence channels are calculated;

[0141] The background and noise brightness corresponding to different pixels in different fluorescence channels are superimposed on the second simulated fluorescence image to obtain a simulated sequencing fluorescence image.

[0142] In an embodiment of the present application, a computer device is also provided, including:

[0143] Memory, used to store programs;

[0144] A processor is used to execute a program, wherein the program is specifically used to implement the sequencing simulation method as described above, and the sequencing simulation method includes:

[0145] Generate configuration templates for sequencing platforms;

[0146] The corresponding test module is called based on the configuration template, and the test module simulates the target sequencing process to output the simulated test results;

[0147] The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

[0148] Based on the foregoing embodiments, an embodiment of the present application provides a storage medium, which stores executable instructions. When the instructions are executed by a processor, a sequencing simulation method as described above is implemented.

[0149] In an embodiment of the present application, a sequencing system is also provided, and the sequencing system includes the above-mentioned computer device.

[0150] It should be noted that the processor or CPU may be at least one of an Application Specific Integrated Circuit (ASIC), a Digital Signal Processor (DSP), a Digital Signal Processing Device (DSPD), a Programmable Logic Device (PLD), a Field Programmable Gate Array (FPGA), a Central Processing Unit (CPU), a controller, a microcontroller, and a microprocessor. It is understandable that the electronic device that implements the above-mentioned processor function may also be other, and the embodiments of the present application are not specifically limited.

[0151] It should be noted that the above-mentioned computer storage medium / memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), a magnetic random access memory (FRAM), a flash memory (Flash Memory), a magnetic surface memory, an optical disc, or a compact disc read-only memory (CD-ROM) and other memories; it can also be various terminals including one or any combination of the above-mentioned memories, such as mobile phones, computers, tablet devices, personal digital assistants, etc.

[0152] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0153] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0154] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sequencing simulation method, characterized in that: include: Generate configuration templates for sequencing platforms; Calling a corresponding test module based on the configuration template, wherein the test module simulates a target sequencing process to output a simulated test result; The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

2. The sequencing simulation method according to claim 1, characterized in that: The generating of a configuration template for a sequencing platform comprises: Determining configuration information for simulated sequencing based on characteristic information for the sequencing platform; Based on the configuration information, generate a configuration template, wherein the configuration template at least includes calling information of a test module and parameter information based on the test module; Optionally, the test module includes a simulated library construction module, and the configuration template includes call information of the simulated library construction module and library construction parameter information for samples to be constructed based on the simulated library construction module, wherein the test module simulates the target sequencing process to output a simulated test result, including: The simulated library construction module obtains and outputs simulated library results based on the library construction parameter information; Optionally, the simulated library construction module obtains and outputs simulated library results based on the library construction parameter information, including: The library construction simulation module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information; According to the reference genome and the sample interruption parameter information, acquiring and outputting sequence information of the simulated library; Optionally, the library construction parameter information includes library length distribution information and total library information, wherein the simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information, including: The simulation library construction module calculates the number of libraries of different lengths based on the library length distribution information and the total number of libraries information; Based on the total number of libraries information and the number of libraries of different lengths, determining sample interruption parameter information on the reference genome corresponding to the sample; Optionally, determining sample interruption parameter information on a reference genome corresponding to the sample based on the total library number information and the number of libraries of different lengths includes: Based on the total number of libraries, determining the 5' end position information of the simulated library on the reference genome corresponding to the sample, and determining the forward and reverse strand information of the simulated library based on the 5' end position information; Based on the information on the number of libraries at different lengths, the library length information of the simulated library is determined, and based on the 5' end position information, the forward and reverse strand information and the library length information, the 3' end position information of the simulated library is determined; The step of acquiring and outputting sequence information of a simulated library according to the reference genome and the sample interruption parameter information includes: The sequence information of the simulated library is acquired and outputted according to the reference genome, the 5' end position information, the forward and reverse strand information, the library length information and the 3' end position information.

3. The simulation sequencing method according to any one of claims 1 to 2, characterized in that: The test module includes a simulated hybridization module, and the configuration template includes call information of the simulated hybridization module and pixel information of a simulated original fluorescent image based on the simulated hybridization module, wherein the test module simulates a target sequencing process to output a simulated test result, including: The simulated hybridization module obtains and outputs a simulated hybridization result based on the pixel information; Optionally, the pixel information includes one or more of the following: the number of pixels in the horizontal and vertical axes of the simulated original fluorescence image, the object space size corresponding to each pixel, and the density of hybridization points on the chip surface. The simulated hybridization module obtains and outputs the simulated hybridization result based on the pixel information, including: The simulated hybridization module calculates the total number of hybridization libraries of the simulated original fluorescence image based on the pixel information; According to the total number of the hybridization libraries, a random hybridization process of the libraries on the chip surface is simulated in the simulated original fluorescence image to obtain and output sequence information of the libraries after simulated hybridization; Optionally, the test module includes a simulated amplification module, and the configuration template includes call information of the simulated amplification module and amplification information based on the simulated amplification module for the hybridized library, wherein the test module simulates the target sequencing process to output a simulated test result, including: The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information; Optionally, the amplification information includes one or more of the following: the number of amplification rounds, the amplification efficiency of each round, and the amplification step distribution of each round. The simulated amplification module obtains and outputs the simulated amplification result based on the amplification information, including: The simulated amplification module determines a library to be simulated amplified in the hybridized library based on the amplification efficiency and the amplification step length distribution, performs simulated amplification on the library to be simulated amplified according to the number of amplification rounds, and obtains and outputs sequence information of the template after the simulated amplification; Wherein, performing simulated amplification on the library to be simulated amplified according to the number of amplification rounds includes: Performing a first round of simulated amplification on the library to be simulated amplified to obtain a template after the first round of simulated amplification; Performing a second round of simulated amplification based on the template after simulated amplification in the first round of amplification to obtain a template after simulated amplification in the second round of amplification; Until the kth round of simulated amplification is performed based on the template after the simulated amplification in the k-1th round of amplification, to obtain the template after the simulated amplification in the kth round of amplification, wherein the number of amplification rounds is k; Optionally, the amplification information at least includes isothermal amplification temperature, isothermal amplification reaction time and amplification efficiency, wherein the simulation amplification module obtains and outputs the simulation amplification result based on the amplification information, including: The simulated amplification module obtains a simulated amplified library for the hybridized library corresponding to the time tT based on the isothermal amplification temperature and the isothermal amplification time; and performs simulated amplification processing corresponding to the time t based on the simulated amplified library for the hybridized library corresponding to the time tT, until obtaining and outputting sequence information of the template after simulated amplification that meets the amplification efficiency; Alternatively, the simulated amplification module performs simulated amplification processing for time t based on the simulated amplified library corresponding to time tT, and obtains and outputs sequence information of the simulated amplified template that meets the amplification efficiency; Wherein, t represents the time for the current simulated amplification to be performed, and T represents the interval duration corresponding to the isothermal amplification reaction time.

4. The sequencing simulation method according to any one of claims 1 to 3, characterized in that: The test module includes a simulated sequencing module, and the configuration template includes call information of the simulated sequencing module and simulated sequencing parameters for a template to be sequenced based on the simulated sequencing module, wherein the test module simulates a target sequencing process to output a simulated test result, including: The simulation sequencing module obtains and outputs the simulation sequencing results based on the simulation sequencing parameters; Optionally, the simulated sequencing parameters include one or more of the following: reaction dissociation constants of the four AGCT bases corresponding to the fluorescent and non-fluorescent AGCT bases, reaction concentrations corresponding to various bases, the number of simulated sequencing rounds, the excision efficiency of the fluorescent groups of different bases, and the probability of being unable to perform subsequent simulated sequencing. The simulated sequencing module obtains and outputs simulated sequencing results based on the simulated sequencing parameters, including: The simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a first base simulation sequencing result of the template to be sequenced; Performing a second round of simulated sequencing processing based on the first base simulation sequencing result to obtain a second base simulation sequencing result of the template to be sequenced; Until the nth round of simulated sequencing is performed based on the simulated sequencing result of the n-1th base to obtain the sequence information of the simulated sequencing, wherein the number of the simulated sequencing rounds is n; Optionally, the simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a first base simulation sequencing result of the template to be sequenced, including: The first base of all templates to be sequenced is determined as the base to be reacted; Randomly generating base reaction information of the base to be reacted according to the reaction dissociation parameter corresponding to the template to be sequenced; If the base reaction information indicates that the base to be reacted successfully combines with the corresponding base, the base type of the base to be reacted is recorded; If the base reaction information indicates that the base to be reacted is prevented from combining with the corresponding base, the next base of the first base is determined as the base to be reacted, and the base types of the base to be reacted are continuously determined until the base types of all the bases to be reacted corresponding to the template to be sequenced are obtained; Counting the total number of different types of bases corresponding to each pixel in the fluorescent image corresponding to the template to be sequenced, to obtain a simulated sequencing result; Optionally, the method further comprises: If the base reaction information indicates that the base to be reacted has matched the corresponding base, the next base of the current base to be reacted is determined as the base to be reacted; If the base reaction information indicates that the base to be reacted does not match the corresponding base, the current base to be reacted is determined as the base to be reacted; If the terminator of the template to be sequenced is not successfully removed and the base to be reacted does not match the corresponding base, a prompt message is outputted indicating that the next simulated sequencing round of the current simulated sequencing round cannot react.

5. The sequencing simulation method according to any one of claims 1 to 4, characterized in that: The test module includes a simulated image processing module, and the configuration template includes call information of the simulated image processing module and image processing parameters for fluorescent images based on the simulated image processing module, wherein the test module simulates a target sequencing process to output a simulated test result, including: The simulated image processing module acquires and outputs a simulated sequencing fluorescence image based on the image processing parameters; Optionally, the image processing parameters include a point spread function in a simulated imaging system, a fluorescence brightness probability distribution corresponding to fluorescent AGCT bases in different fluorescence channels, a background brightness spatial distribution corresponding to different fluorescence channels, and a random brightness noise characteristic corresponding to different fluorescence channels. The simulated image processing module acquires and outputs a simulated sequencing fluorescence image based on the image processing parameters, including: Calculate the base fluorescence brightness information corresponding to different pixels in different fluorescence channels according to the fluorescence brightness probability distribution of the fluorescent AGCT bases in different fluorescence channels and the total number of different types of fluorescently labeled bases corresponding to each pixel; Generate a first simulated fluorescence image corresponding to the four fluorescence channels of each round of sequencing according to the base fluorescence brightness information of each pixel in the fluorescence image corresponding to each round of sequencing in the four fluorescence channels; Processing the first simulated fluorescence image based on the point spread function, and outputting a second simulated fluorescence image; Calculating the background and noise brightness corresponding to different pixels in different fluorescent channels according to the background brightness spatial distribution corresponding to the different fluorescent channels and the random brightness noise characteristics corresponding to the different fluorescent channels; The background and noise brightness corresponding to the different pixels in different fluorescence channels are superimposed on the second simulated fluorescence image to obtain a simulated sequencing fluorescence image.

6. A sequencing simulation device, characterized in that: include: A generation unit, used to generate a configuration template for a sequencing platform; A calling unit, configured to call a corresponding test module based on the configuration template, wherein the test module simulates a target sequencing process to output a simulated test result; The test module includes one or more of a simulated library construction module, a simulated hybridization module, a simulated amplification module, a simulated sequencing module and a simulated image processing module, and each module in the test module is an independently callable module.

7. The device according to claim 6, characterized in that The generating unit comprises: A first determination subunit is used to determine configuration information of simulated sequencing based on characteristic information of a sequencing platform; A first generating subunit is used to generate a configuration template based on the configuration information, where the configuration template at least includes calling information of the test module and parameter information based on the test module; Optionally, the test module includes a simulated library construction module, the configuration template includes call information of the simulated library construction module and library construction parameter information for samples to be constructed based on the simulated library construction module, wherein the call unit includes a first call subunit, the first call subunit is used to call the simulated library construction module based on the configuration module, and the test module simulates the target sequencing process to output a simulated test result, including: The simulation library construction module obtains and outputs the simulation library results based on the library construction parameter information; Optionally, the simulation library construction module obtains and outputs simulation library results based on the library construction parameter information, including: The simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information; According to the reference genome and sample interruption parameter information, the sequence information of the simulated library is obtained and output; Optionally, the library construction parameter information includes library length distribution information and total library information, wherein the simulation library construction module determines the sample interruption parameter information on the reference genome corresponding to the sample to be constructed based on the library construction parameter information, including: The simulation library construction module calculates the number of libraries of different lengths based on the library length distribution information and the total number of libraries; Based on the total number of libraries and the number of libraries of different lengths, the sample interruption parameter information is determined on the reference genome corresponding to the sample; Optionally, based on the total number of libraries and the number of libraries of different lengths, sample break parameter information is determined on the reference genome corresponding to the sample, including: Based on the total number of libraries, the 5' end position information of the simulated library is determined on the reference genome corresponding to the sample, and the forward and reverse strand information of the simulated library is determined based on the 5' end position information; Based on the information of the number of libraries at different lengths, the library length information of the simulated library is determined, and based on the 5' end position information, the forward and reverse strand information and the library length information, the 3' end position information of the simulated library is determined; According to the reference genome and sample interruption parameter information, the sequence information of the simulated library is obtained and output, including: According to the reference genome, 5' end position information, forward and reverse strand information, library length information and 3' end position information, the sequence information of the simulated library is obtained and output; Optionally, the test module includes a simulated hybridization module, the configuration template includes call information of the simulated hybridization module and pixel information based on the simulated hybridization module for simulating the original fluorescent image, wherein the call unit includes a second call subunit, a second call subunit, and is used to call the simulated hybridization module based on the configuration information, and the test module simulates the target sequencing process to output a simulated test result, including: The simulated hybridization module obtains and outputs simulated hybridization results based on pixel information; Optionally, the pixel information includes one or more of the following: the number of pixels on the horizontal and vertical axes of the simulated original fluorescence image, the object space size corresponding to each pixel, and the density of hybridization points on the chip surface. The simulated hybridization module obtains and outputs the simulated hybridization results based on the pixel information, including: The simulated hybridization module calculates the total number of hybridization libraries that simulate the original fluorescence image based on pixel information; According to the total number of hybridization libraries, the random hybridization process of the libraries on the chip surface is simulated in the simulated original fluorescence image to obtain and output the sequence information of the libraries after simulated hybridization; Optionally, the test module includes a simulated amplification module, the configuration template includes call information of the simulated amplification module and amplification information based on the simulated amplification module for the hybridized library, wherein the call unit includes a third call subunit, the third call subunit is used to call the simulated amplification module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including: The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information; Optionally, the amplification information includes one or more of the following: the number of amplification rounds, the amplification efficiency of each round, and the amplification step distribution of each round. The simulation amplification module obtains and outputs the simulation amplification result based on the amplification information, including: The simulated amplification module determines the library to be simulated amplified in the hybridized library based on the amplification efficiency and the amplification step length distribution, performs simulated amplification on the library to be simulated amplified according to the number of amplification rounds, and obtains and outputs the sequence information of the template after the simulated amplification; Wherein, performing simulated amplification on the library to be simulated amplified according to the number of amplification rounds includes: Performing a first round of simulated amplification on the library to be simulated amplified to obtain a template after the first round of simulated amplification; Performing a simulated amplification process for a second amplification round based on the template after simulated amplification in the first amplification round to obtain a template after simulated amplification in the second amplification round; Until the kth round of simulated amplification is performed based on the template after the simulated amplification in the k-1th round of amplification, to obtain the template after the simulated amplification in the kth round of amplification, wherein the number of amplification rounds is k; Optionally, the amplification information includes at least isothermal amplification temperature, isothermal amplification reaction time and amplification efficiency, wherein the simulation amplification module obtains and outputs the simulation amplification result based on the amplification information, including: The simulation amplification module obtains a simulated amplified library for the hybridized library corresponding to the time tT based on the isothermal amplification temperature and the isothermal amplification time; and performs a simulated amplification process corresponding to the time t based on the simulated amplified library for the hybridized library corresponding to the time tT, until the sequence information of the template after the simulated amplification that satisfies the amplification efficiency is obtained and output; Alternatively, the simulated amplification module performs simulated amplification processing for time t based on the simulated amplified library corresponding to time tT, and obtains and outputs sequence information of the simulated amplified template that meets the amplification efficiency; Wherein, t represents the time of the current simulated amplification to be performed, and T represents the interval time corresponding to the isothermal amplification reaction time; Optionally, the test module includes a simulated sequencing module, the configuration template includes call information of the simulated sequencing module and simulated sequencing parameters for a template to be sequenced based on the simulated sequencing module, wherein the call unit includes a fourth call subunit, the fourth call subunit is used to call the simulated sequencing module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including: The simulation sequencing module obtains and outputs the simulation sequencing results based on the simulation sequencing parameters; Optionally, the simulated sequencing parameters include one or more of the following: reaction dissociation constants of the four AGCT bases corresponding to the fluorescent and non-fluorescent AGCT bases, reaction concentrations corresponding to various bases, the number of simulated sequencing rounds, the removal efficiency of the fluorescent groups of different bases, and the probability of being unable to perform subsequent simulated sequencing. The simulated sequencing module obtains and outputs simulated sequencing results based on the simulated sequencing parameters, including: The simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a simulation sequencing result of the first base of the template to be sequenced; Performing a second round of simulated sequencing processing based on the first base simulated sequencing result to obtain a second base simulated sequencing result of the template to be sequenced; Until the nth round of simulated sequencing is performed based on the simulated sequencing result of the n-1th base, to obtain the sequence information of the simulated sequencing, wherein the number of simulated sequencing rounds is n; Optionally, the simulation sequencing module performs a first round of simulation sequencing processing on the template to be sequenced based on the simulation sequencing parameters to obtain a first base simulation sequencing result of the template to be sequenced, including: The first base of all templates to be sequenced is determined as the base to be reacted; Randomly generate base reaction information of the base to be reacted according to the reaction dissociation parameters corresponding to the template to be sequenced; If the base reaction information indicates that the base to be reacted successfully combines with the corresponding base, the base type of the base to be reacted is recorded; If the base reaction information indicates that the base to be reacted is prevented from combining with the corresponding base, the next base of the first base is determined as the base to be reacted, and the base types of the base to be reacted are continuously determined until the base types of all the bases to be reacted corresponding to the template to be sequenced are obtained; Counting the total number of different types of bases corresponding to each pixel in the fluorescent image corresponding to the template to be sequenced to obtain a simulated sequencing result; Optionally, the device further comprises: a base determination subunit to be reacted for: If the base reaction information indicates that the base to be reacted has matched the corresponding base, the next base of the current base to be reacted is determined as the base to be reacted; If the base reaction information indicates that the base to be reacted does not match the corresponding base, the current base to be reacted is determined as the base to be reacted; If the terminator of the template to be sequenced is not successfully removed and the base to be reacted does not match the corresponding base, a prompt message is output that the next simulated sequencing round of the current simulated sequencing round cannot react; Optionally, the test module includes a simulated image processing module, the configuration template includes call information of the simulated image processing module and image processing parameters for the fluorescent image based on the simulated image processing module, wherein the call unit includes a fifth call subunit, the fifth call subunit is used to call the simulated image processing module based on the configuration template, and the test module simulates the target sequencing process to output a simulated test result, including: The simulated image processing module obtains and outputs simulated sequencing fluorescence images based on the image processing parameters; Optionally, the image processing parameters include a point spread function in a simulated imaging system, a fluorescence brightness probability distribution corresponding to fluorescent AGCT bases in different fluorescence channels, a background brightness spatial distribution corresponding to different fluorescence channels, and a random brightness noise characteristic corresponding to different fluorescence channels. The simulated image processing module obtains and outputs a simulated sequencing fluorescence image based on the image processing parameters, including: According to the fluorescence brightness probability distribution of the fluorescent AGCT bases in different fluorescence channels, and the total number of different types of fluorescently labeled bases corresponding to each pixel, the fluorescence brightness information of the bases corresponding to different pixels in different fluorescence channels is calculated; Generate a first simulated fluorescence image corresponding to the four fluorescence channels of each round of sequencing according to the base fluorescence brightness information of each pixel in the four fluorescence channels in the fluorescence image corresponding to each round of sequencing; Processing the first simulated fluorescence image based on the point spread function, and outputting a second simulated fluorescence image; According to the background brightness spatial distribution corresponding to different fluorescence channels and the random brightness noise characteristics corresponding to different fluorescence channels, the background and noise brightness corresponding to different pixels in different fluorescence channels are calculated; The background and noise brightness corresponding to different pixels in different fluorescence channels are superimposed on the second simulated fluorescence image to obtain a simulated sequencing fluorescence image.

8. A computer device, characterized in that: include: Memory, used to store programs; A processor is used to execute the program, wherein the program is specifically used to implement the sequencing simulation method as described in any one of claims 1-5.

9. A storage medium, characterized in that: The storage medium stores executable instructions, and when the instructions are executed by the processor, the sequencing simulation method according to any one of claims 1 to 5 is implemented.

10. A sequencing system, characterized in that: The sequencing system comprises the computer device of claim 8.

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