Multi-PCR primer design method and device based on simulated annealing algorithm

Abstract

The invention provides a multi-PCR (Polymerase Chain Reaction) primer design method and device based on a simulated annealing algorithm, a set of novel multi-PCR primer design architecture is created by combing multi-primer influence factors and related characteristics and optimizing a multi-primer design process, and specifically, the simulated annealing algorithm is combined on the basis of a primer pool of a parallel architecture, so that the design efficiency is improved. According to the method, the multiple PCR primers are combined with other aspects of further optimization setting, so that the difficulties of complicated design process of the multiple PCR primers, difficulty in non-specific detection, difficult problem of primer dimer, large calculation amount in each link, low automation degree and the like in the traditional technology are overcome, and the design requirements of the high-quality multiple PCR primers are met.

Description

Technical Field

[0001] The present application relates to the field of detection, and in particular to a multiplex PCR primer design method and device based on a simulated annealing algorithm. Background Art

[0002] With the development of biomedical engineering, multiplex polymerase chain reaction (PCR) technology has been increasingly used in species detection, disease diagnosis, genetic disease screening, pathogenic microorganism detection and other fields.

[0003] Multiplex PCR technology refers to the simultaneous amplification of multiple DNA or RNA templates by adding multiple pairs of specific primers into the same reaction system, thereby greatly improving detection efficiency and accuracy.

[0004] Primer design is a key step in multiplex PCR technology. To ensure amplification efficiency, factors such as primer specificity, length, melting temperature (Tm), and GC (i.e., guanine (G) and cytosine (C)) content need to be considered for each target amplification region. In addition, the interaction between primers needs to be considered to avoid the formation of primer dimers.

[0005] Under the influence of the above factors, the design of multiplex PCR primers is extremely complex. In particular, as the number of target amplification regions increases, the amount of calculation increases exponentially, making the design of multiplex PCR primers even more difficult.

[0006] Current multiplex PCR amplification experiments have evolved from a few targets to the stage of simultaneous amplification of hundreds of targets. The number of primers required is constantly increasing, and most software and its design concepts can no longer meet the needs of such large-scale primer design. Summary of the Invention

[0007] This application provides a multiplex PCR primer design method and device based on a simulated annealing algorithm. By sorting out the influencing factors and related characteristics of multiple primers and optimizing the multiple primer design process, a novel multiplex PCR primer design architecture is created. Specifically, based on a parallel architecture primer pool, a simulated annealing algorithm is combined with other further optimization settings to overcome the difficulties associated with traditional technologies such as the complex multiplex PCR primer design process, difficult nonspecific detection, the intractable primer dimer problem, the large amount of computational effort in each link, and the low degree of automation, thereby meeting the needs of high-quality multiplex PCR primer design.

[0008] In a first aspect, the present application provides a multiplex PCR primer design method based on a simulated annealing algorithm, the method comprising:

[0009] Obtain the task of designing multiple PCR primers for the target object;

[0010] Obtaining genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided;

[0011] Based on the genome reference sequence data, target site data and SNV site data, the data input for primer pool generation is obtained;

[0012] Acquire primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as independent subtasks. Different subtasks are processed in parallel on multiple cores of the CPU, and primer designs for each locus on each chromosome are also configured to be independent.

[0013] generating a primer pool based on the data input and primer pool configuration data;

[0014] Based on the primer pool, the preset simulated annealing algorithm is combined to design multiple PCR primers for the target object.

[0015] In a second aspect, the present application provides a multiplex PCR primer design device based on a simulated annealing algorithm, the device comprising:

[0016] An acquisition unit, used for acquiring a multiplex PCR primer design task for a target object;

[0017] The acquisition unit is further used to obtain genome reference sequence data, target site data and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided;

[0018] A processing unit is used to process the data input for primer pool generation based on the genome reference sequence data, target site data and SNV site data;

[0019] The acquisition unit is further configured to acquire primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as independent subtasks, and the different subtasks are processed in parallel on multiple cores of the CPU. The primer designs for each site on each chromosome are also configured to be independent of each other;

[0020] A generating unit, configured to generate a primer pool based on the data input and the primer pool configuration data;

[0021] The processing unit is used to design multiple PCR primers for the target object based on the primer pool in combination with a preset simulated annealing algorithm.

[0022] In a third aspect, the present application provides a processing device comprising a processor and a memory, wherein a computer program is stored in the memory, and when the processor calls the computer program in the memory, the method provided in the first aspect of the present application or any possible implementation of the first aspect of the present application is executed.

[0023] In a fourth aspect, the present application provides a computer-readable storage medium, which stores multiple instructions, and the instructions are suitable for a processor to load to execute the method provided in the first aspect of the present application or any possible implementation of the first aspect of the present application.

[0024] From the above content, it can be concluded that this application has the following beneficial effects:

[0025] Aiming at the goal of multiplex PCR primer design, this application has created a novel multiplex PCR primer design architecture by sorting out the influencing factors and related characteristics of multiple primers, optimizing the multiplex primer design process, and specifically combining a simulated annealing algorithm with a primer pool of a parallel architecture, supplemented by further optimization settings in other aspects, to overcome the difficulties associated with traditional technologies such as the complex multiplex PCR primer design process, difficult non-specific testing, tricky primer dimer problems, large computational complexity in each link, and low degree of automation, thereby meeting the needs of high-quality multiplex PCR primer design. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 A schematic diagram of a process for designing multiplex PCR primers based on a simulated annealing algorithm in this application;

[0028] Figure 2 A schematic diagram of a two-stage nonspecific test process for this application;

[0029] Figure 3 A logical diagram for the design of multiplex PCR primers for this application;

[0030] Figure 4 A schematic diagram of the structure of a multiplex PCR primer design device based on a simulated annealing algorithm in this application;

[0031] Figure 5 This is a structural diagram of the processing equipment for this application. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of this application.

[0033] The terms "first", "second", etc. in the specification and claims of this application and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate so that the embodiments described herein can be implemented in a sequence other than that illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or modules is not necessarily limited to those steps or modules clearly listed, but may include other steps or modules that are not clearly listed or that are inherent to these processes, methods, products or devices. The naming or numbering of steps in this application does not mean that the steps in the method flow must be executed in the time / logical sequence indicated by the naming or numbering. The process steps that have been named or numbered can be changed in the execution order according to the technical purpose to be achieved, as long as the same or similar technical effects can be achieved.

[0034] The division of modules in this application is a logical division. In actual application, there may be other division methods. For example, multiple modules can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, and the indirect coupling or communication connection between modules can be electrical or other similar forms, which are not limited in this application. Moreover, the modules or submodules described as separate components may or may not be physically separated, may or may not be physical modules, or may be distributed into multiple circuit modules. Some or all of the modules can be selected according to actual needs to achieve the purpose of the solution of this application.

[0035] Before introducing the multiplex PCR primer design method based on simulated annealing algorithm provided by this application, the background content involved in this application is first introduced.

[0036] The multiplex PCR primer design method, device and computer-readable storage medium based on simulated annealing algorithm provided in this application can be applied to processing equipment for use.

[0037] The multiplex PCR primer design method based on a simulated annealing algorithm mentioned in this application can be executed by a multiplex PCR primer design device based on a simulated annealing algorithm, or a server, physical host, or user equipment (UE) of different types of processing devices that integrate the multiplex PCR primer design device based on a simulated annealing algorithm. The multiplex PCR primer design device based on a simulated annealing algorithm can be implemented using hardware or software. The UE can specifically be a terminal device such as a smartphone, tablet computer, laptop computer, desktop computer, or personal digital assistant (PDA). The multiplex PCR primer design processing device based on a simulated annealing algorithm can also be configured as a device cluster.

[0038] It is understandable that the focus of the present application is on the data processing based on the existing data. Therefore, the processing equipment that executes the multiplex PCR primer design method based on the simulated annealing algorithm of the present application or is equipped with the corresponding application service of the multiplex PCR primer design method based on the simulated annealing algorithm of the present application usually only needs to meet the required data processing capabilities, and the specific equipment type and equipment deployment form are relatively flexible.

[0039] If the direct data collection capability or the display of data processing results is also involved, further adaptive configuration of the processing equipment is required.

[0040] Taking the ability to display data processing results as an example, the processing device can display the data processing results, i.e., the multiplex PCR primer design results, through its own configured display screen (including a touch screen). In addition, the processing device can also display through an external display screen device or other device with a display screen, which is also possible in actual situations.

[0041] Next, the multiplex PCR primer design method based on simulated annealing algorithm provided by this application is introduced.

[0042] First, see Figure 1 , Figure 1 A schematic flow chart of the multiplex PCR primer design method based on the simulated annealing algorithm of the present application is shown. The multiplex PCR primer design method based on the simulated annealing algorithm provided by the present application may specifically include the following steps S101 to S106:

[0043] Step S101, obtaining a multiplex PCR primer design task for a target object;

[0044] It can be understood that the multiplex PCR primer design processing involved in the present application scheme is carried out in actual application according to the detection needs of specific objects, and is usually carried out in the form of tasks. For the convenience of explanation, the object targeted by the current multiplex PCR primer design task is recorded as the target object.

[0045] It is easy to understand that, corresponding to the flexible and changeable detection requirements in actual situations, the target objects targeted by the multiplex PCR primer design task can be flexibly adjusted according to actual needs, and this application does not make specific limitations.

[0046] In addition, the acquisition and processing of multiplex PCR primer design tasks here also have flexible and changeable characteristics. For example, the multiplex PCR primer design tasks can be manually configured, or they can be generated independently, or pre-generated multiplex PCR primer design tasks can be called, or multiplex PCR primer design tasks sent / forwarded by other devices can be received. These are all possible in actual situations.

[0047] As an example, in the present application, the target object may specifically be an object such as the plant cocoa.

[0048] Step S102, obtaining genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided;

[0049] It can be understood that in the present application scheme, the generation and processing of the primer pool is involved, which corresponds to data input and parallel primer design. These two aspects are reflected through the data input obtained by processing steps S102 and S103, and the primer pool configuration data obtained by processing step S104.

[0050] It can be understood that in terms of data input, it specifically involves three aspects: genome reference sequence, target point and SNV site related to the target object, which corresponds to the three aspects of genome reference sequence data, target point data and SNV site data.

[0051] For genomic reference sequence data, it can be entered locally or retrieved / used from a third-party ready-made gene library, such as an online international gene database. The genomic reference sequence file generally contains all chromosome reference sequences. After reading the file, the gene sequence of each chromosome can be extracted.

[0052] For target site data, the acquisition method is similar to that of genome reference sequence data, which describes the target sites on different chromosomes that need to be processed.

[0053] For single nucleotide variant (SNV) site data, the acquisition method is similar to that of genome reference sequence data. It describes single nucleotide variant sites, and such sites need to be avoided when designing multiplex PCR primers.

[0054] Step S103, processing the data input for primer pool generation based on the genome reference sequence data, target site data, and SNV site data;

[0055] It can be understood that after obtaining the genome reference sequence data, target point data and SNV site data, the sites involved can be fused and processed to determine the site processing range, forming data input that is convenient for subsequent primer pool generation and processing to use and call.

[0056] As one of the preferred implementation solutions, the process of processing data input may specifically include the following processing contents:

[0057] 1) Sort the target sites of each chromosome in the sites described by the target site data, and remove the target sites that are not within the site range of the genome reference sequence data;

[0058] It can be understood that here, by checking whether the target site is within the point range of the genome reference sequence data, the site fusion processing of the target point data and the genome reference sequence data is performed, and the target site within the point range of the genome reference sequence data, that is, the current genome reference sequence range, is retained.

[0059] In addition, it also involves sorting processing based on chromosomes, laying the foundation for subsequent data processing.

[0060] 2) For the retained target sites, the adjacent sites are merged according to the distance threshold to form a target area processed by regional units, and the update processing of the sites described by the target point data is completed.

[0061] It can be understood that here, for the target sites retained in the previous step, the proximity principle (based on the adjustable distance threshold N for determination, and the distance can be specifically quantified by the number of sites) is involved in the merging processing of a small number of sites to form one or more corresponding target regions. Under this setting, more convenient and efficient processing effects can be achieved in the subsequent generation and use of primer pools.

[0062] Furthermore, as one of the preferred implementation solutions, the process of processing data input may also include the following processing content:

[0063] Classify and sort the sites described by the SNV site data according to chromosomes;

[0064] Using the classification and sorting results as a reference, filter out the sites that need to be avoided.

[0065] It can be understood that for the sites that need to be avoided as described by the SNV site data, filtering processing can also be carried out on a chromosome basis, which further involves sorting processing, which helps to better carry out data processing and also corresponds to the processing of the previous target sites.

[0066] In this way, the configuration of the sites to be processed is completed.

[0067] At the same time, corresponding to the generation or configuration of the primer pool, and corresponding to the subsequent use of the primer pool, the present application may also involve the content of relevant parameters in the input data.

[0068] Correspondingly, as a preferred implementation solution, the method of the present application may further include:

[0069] Obtaining a reference file, wherein the reference file describes key parameters, including optimal primer length, maximum primer length, minimum primer length, number of return primers, amplification primer length, optimal primer melting temperature, and maximum GC content;

[0070] The key parameters described in the reference document are combined with the default parameters as the content of another part of the data input, wherein the default parameters are basic parameters that have been screened in the past time period and have performed well for multiplex PCR primer design.

[0071] It can be understood that starting from the parameters that can be involved at the detailed level, the addition of key parameters corresponding to the current real-time situation / demand and default parameters that have performed well in the past / history will help promote the subsequent generation / configuration and use of the primer pool, and achieve more delicate and higher-performance processing effects.

[0072] Among them, the default parameters can be related to the key parameters, including primer length, number of returned primers, length of amplified primers, primer dissolution temperature (or primer melting temperature, which can be recorded as Tm) and GC content. Alternatively, they can only involve some of these parameters, or they can involve other parameters based on some of these parameters, or they can directly involve parameters other than these parameters. All of these are possible.

[0073] In addition, it can be understood that the parameters of primer length, number of return primers, extension primer length, primer melting temperature and GC content also correspond to the primer length, primer melting temperature and GC content that can be involved in the design of single PCR primers. In addition, the design of single PCR primers can also involve the primer melting temperature difference, annealing temperature and 3' end GC.

[0074] Step S104: Acquire primer pool configuration data. The primer pool configuration data configures primer designs for different chromosomes as independent subtasks. These subtasks are processed in parallel on multiple CPU cores, and the primer designs for each locus on each chromosome are also independent.

[0075] It is understandable that the present application has a special design for the primer pool to be automatically and autonomously generated, which can be reflected and implemented through the primer pool configuration data involved herein.

[0076] Specifically, in multiplex PCR primer design, when the number of target sites reaches hundreds or even thousands, the generation of the primer pool takes a lot of time.

[0077] In serial mode, taking the multiplex PCR primer design task for the target object cocoa as an example, it takes more than an hour to design a primer pool for approximately 600 target sites. In addition, as the object of subsequent optimization operations, the primer pool also needs to be frequently fine-tuned and the primer pool regenerated.

[0078] Therefore, the inventors of this application believe that parallel processing can be configured to reduce the time it takes to generate the primer pool. Since the primer design for each chromosome in the primer pool is independent, the primer design for each site thereon is also independent. In this way, tasks can be grouped by chromosome, and the primer design for the same chromosome belongs to the same subtask. Multiple design tasks run in parallel on multiple cores of the CPU. When all subtasks are completed, all results will be aggregated into a primer pool for output.

[0079] After actual testing, under the design of this application, the parallelized primer pool can generate a primer pool containing hundreds of target sites within a few minutes, which is 9-10 times faster than the traditional, original serial mode.

[0080] Step S105, generating a primer pool based on the data input and primer pool configuration data;

[0081] As previously described, after processing the obtained data input and primer pool configuration data, an efficient and high-quality primer pool parallel generation process can be carried out.

[0082] Step S106 , based on the primer pool, a preset simulated annealing algorithm is used to design multiplex PCR primers for the target object.

[0083] The parallelized primer pool generated in parallel can be understood to continue to promote efficient and high-quality multiplex PCR primer design processing for each target site under its parallel architecture that can support large-scale primer design with high performance.

[0084] Specifically, the present application introduces a simulated annealing algorithm to perform better data analysis and processing in the specific multiplex PCR primer design process.

[0085] As for the simulated annealing algorithm (SA), it is a probability-based global optimization algorithm that simulates the physical phenomena of the solid annealing process to find an approximate optimal solution in a large-scale search space. It has the advantages of simple algorithm, easy code implementation, easy to escape from local minimum points, high stability and high reliability.

[0086] In addition, it is understandable that, for PCR primer design processing, in actual situations, it usually involves an adaptive computing environment, and the computing environment can usually adopt existing products, such as Primer3 (a primer design online software) can be integrated in a mixed programming mode. Of course, a self-built computing environment can also be used, which can be adjusted according to actual needs. It should be noted that the optimization settings of the present application scheme cannot be brought by existing products of the computing environment.

[0087] In this way, under the above highly concise and easy-to-operate solution processing architecture, multiplex PCR primer design can be completed efficiently and with high quality.

[0088] At the same time, the present application scheme may also involve the processing of nonspecific tests.

[0089] Specifically, PCR primer nonspecificity refers to the primer amplifying non-target sites, which is a common problem in primer design. For gene sequencing, the large number of invalid gene fragments produced by primer nonspecific amplification increases the sequencing cost.

[0090] For quantitative PCR applications, nonspecific amplification will lead to erroneous test results. For example, in the new coronavirus nucleic acid detection technology based on fluorescent quantitative PCR, if nonspecific primers are used, the analysis results will be false positives; similarly, for detection technology that sequences PCR amplification products, if the primers are consumed in large quantities by nonspecific amplification, resulting in a low content of the target sequence in the PCR amplification product, it will ultimately lead to a false negative.

[0091] Since there are a large number of candidate primers in the primer pool, in order to accelerate the non-specific detection of primers, this application also establishes a two-level non-specific detection process.

[0092] refer to Figure 2 The schematic flow chart of the two-stage nonspecific test of the present application is shown as a preferred implementation scheme herein. The following processing contents may be included in the process of multiplex PCR primer design processing:

[0093] To accelerate the non-specific verification of primers in the primer pool, a gene sequence hash table (or gene sequence hash table) is pre-constructed in a targeted two-level non-specific test architecture. In the primary non-specific test, when the primers are completely matched, the primer sequence is searched through the gene sequence hash table to filter out primers that cause non-specific amplification due to complete match. Then, in the secondary non-specific test, a short sequence similarity evaluation is performed on the remaining primers to find the genomic positions and corresponding primers whose similarity exceeds the threshold.

[0094] Among them, performing short sequence similarity evaluation on the remaining primers in the secondary nonspecific test may specifically involve the application of the Blast tool (a basic local alignment search tool). Of course, other existing search tools or even self-developed search tools can also be used here.

[0095] Thus, through the above-mentioned setting, the accelerated primer non-specific test can be completed well, overcoming the technical difficulties of primer non-specific verification in the existing technology. At the same time, it also lays a better foundation for the dimer optimization that can be involved next.

[0096] Specifically, when designing PCR primers, interactions between primers must be considered to avoid primer-dimer formation. Given the large number of primers used in multiplex PCR amplification, the potential for primer-dimer formation cannot be ignored. Therefore, after specificity testing, a primer pool must be selected to minimize the likelihood of dimer formation.

[0097] In this regard, as a preferred implementation scheme, the multiplex PCR primer design process may further include the following processing contents:

[0098] A pre-built mathematical model for primer dimers is used to select a primer set with the lowest probability of dimer formation from the primer pool. The objective function configured in the dimer mathematical model considers not only the tendency of dimer formation between primers, but also the effect of the dispersion of the melting temperature of the primer set on dimer formation.

[0099] The evaluation of the dimer formation tendency of the primer set was calculated using a thermodynamic method. The dispersion of the melting temperature of the primer set was characterized by the sum of the differences between the melting temperature of each pair of primers and the optimal melting temperature.

[0100] The calculation formula of the thermal writing method can be obtained from Primer3, or from other places or built by yourself.

[0101] Thus, under this setting, by applying a very detailed primer dimer mathematical model, the dimer problem can be effectively minimized as much as possible, overcoming the difficult dimer problem existing in the prior art.

[0102] As for the optimization algorithm configured based on the simulated annealing algorithm in this application, as a preferred implementation scheme, the optimization algorithm of the simulated annealing algorithm may specifically include the following processing contents:

[0103] 1) Select an initial primer set X0 from the primer pool;

[0104] 2) Evaluate the initial primer set X0 using the objective function F(X);

[0105] 3) Based on the g-th generation primer set Xg, calculate the penalty value SP for each primer pair, and replace the first y primer pairs according to the sorted penalty value SP to generate a temporary primer set T;

[0106] 4) Calculate the objective function F(T) of the temporary primer set T, and calculate the probability based on the calculated result and the annealing temperature to decide whether to use the g-th generation primer set Xg+1, maintain the g-th generation primer set Xg, or evolve to the temporary primer set T;

[0107] 5) Repeat 3) and 4) until an accepted primer set Xfinal (satisfying the processing completion conditions) is obtained and output.

[0108] It can be understood that this is an optimized application of the simulated annealing algorithm of this application, and a very specific implementation solution is provided. Based on the high adaptability to the multiple PCR primer design process of this application, it has very good application value.

[0109] Furthermore, for the above solution content, you can also combine Figure 3 A logical schematic diagram of the multiplex PCR primer design of the present application is shown for a more vivid understanding.

[0110] At the same time, we can also use the following set of examples with the target object being the plant cocoa for a more vivid understanding.

[0111] Specifically, in practical applications, the plant cocoa can be used as the processing object for multiplex PCR primer design. Its reference genome size is 4.5*107bp (BP length refers to the number of base pairs in a DNA molecule), the number of input target sites is 596, and the successfully generated primer pool contains candidate primers for 558 targets. The simulated annealing algorithm is used for optimal PCR primer design.

[0112] All properties of the final primer set met experimental expectations. The primer lengths were distributed between 18 and 33, and primers with the set optimal length accounted for approximately 60% of the total primers. The primer Tm values were near the optimal value (61.5°C ± 1°C) accounted for 85.3%. 300 pairs of primers from the optimal primer set were randomly selected to carry out multiplex PCR amplification experiments and second-generation high-throughput sequencing experiments. The analysis results showed that the primer set had a detection rate of as low as 95.4%, and the utilization rate was higher than 95.5%, indicating that the primer set has good specific amplification of the target region, further proving that the multiplex PCR primer design scheme of this application can be reliably applied to large-scale primer design.

[0113] At the same time, after completing the multiplex PCR primer design for the current target object, it is understandable that the multiplex PCR primer design results may also involve corresponding data application links in practical applications.

[0114] In the data application stage, the multiplex PCR primer design results can be stored locally, stored remotely, displayed, prompted to complete the design, pushed results, or further data analysis and processing can be performed.

[0115] It is easy to understand that specific data application processing can be carried out according to pre-configured and real-time configured data application policies / rules, which is relatively flexible.

[0116] For example, in the case of species detection, the design of multiplex PCR primers may be specifically related to the detection / identification / comparison / identification of the DNA of the cocoa plant. This may then lead to the species detection process for the cocoa plant, and further to the generation of the corresponding test report.

[0117] Finally, regarding the above scheme content, in general, for the goal of multiplex PCR primer design, this application has created a novel multiplex PCR primer design architecture by sorting out the influencing factors and related characteristics of multiple primers, optimizing the multiple primer design process, and specifically combining the simulated annealing algorithm on the basis of the primer pool of the parallel architecture, and supplemented by further optimization settings in other aspects, to overcome the difficulties of the traditional technology such as the complex multiplex PCR primer design process, the difficulty of non-specific testing, the tricky primer dimer problem, the large amount of calculation in each link and the low degree of automation, so as to meet the needs of high-quality multiplex PCR primer design.

[0118] The above is an introduction to the multiplex PCR primer design method based on the simulated annealing algorithm provided in this application. In order to facilitate better implementation of the multiplex PCR primer design method based on the simulated annealing algorithm provided in this application, this application also provides a multiplex PCR primer design device based on the simulated annealing algorithm from the perspective of functional modules.

[0119] See Figure 4 , Figure 4 Schematic diagram of a multiplex PCR primer design device based on a simulated annealing algorithm in this application. In this application, the multiplex PCR primer design device 400 based on a simulated annealing algorithm may specifically include the following structure:

[0120] An acquisition unit 401 is used to acquire a multiplex PCR primer design task for a target object;

[0121] The acquisition unit 401 is further configured to acquire genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided;

[0122] A processing unit 402 is used to process the data input for primer pool generation based on the genome reference sequence data, target site data and SNV site data;

[0123] The acquisition unit 401 is further configured to acquire primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as independent subtasks, with the different subtasks being processed in parallel on multiple cores of the CPU, and the primer designs for each locus on each chromosome are also configured to be independent of each other;

[0124] A generating unit 403 is configured to generate a primer pool based on the data input and the primer pool configuration data;

[0125] The processing unit 404 is used to design multiplex PCR primers for the target object based on the primer pool in combination with a preset simulated annealing algorithm.

[0126] In a preferred implementation, the process of processing data input includes the following processing contents:

[0127] Among the points described by the target point data, the target sites of each chromosome are sorted, and the target sites that are not within the point range of the genome reference sequence data are eliminated;

[0128] For the retained target sites, the adjacent sites are merged according to the distance threshold to form a target area processed by regional units, completing the update processing of the sites described by the target point data.

[0129] In another preferred implementation, the process of processing data input further includes the following processing:

[0130] Classify and sort the sites described by the SNV site data according to chromosomes;

[0131] Using the classification and sorting results as a reference, filter out the sites that need to be avoided.

[0132] In another preferred implementation, the acquisition unit 401 is further configured to acquire a reference file, wherein the reference file describes key parameters, the key parameters including optimal primer length, maximum primer length, minimum primer length, number of return primers, amplification primer length, optimal primer melting temperature, optimal primer melting temperature, and maximum GC content;

[0133] The processing unit 403 is also used to merge the key parameters described in the reference file with the default parameters as the content of another part of the data input, wherein the default parameters are basic parameters that have been screened in the past time period and have good performance for multiple PCR primer design.

[0134] In another preferred implementation, the multiplex PCR primer design process includes the following processing:

[0135] To accelerate the non-specific verification of primers in the primer pool, a gene sequence hash table is pre-constructed in a specially designed two-level non-specific test architecture. In the primary non-specific test, when the primers are completely matched, the primer sequences are searched through the gene sequence hash table to filter out primers that cause non-specific amplification due to complete match. Then, in the secondary non-specific test, a short sequence similarity evaluation is performed on the remaining primers to find the genomic positions and corresponding primers whose similarity exceeds the threshold.

[0136] In another preferred implementation, the multiplex PCR primer design process further includes the following processing:

[0137] A pre-built mathematical model for primer dimers is used to select a primer set with the lowest probability of dimer formation from the primer pool. The objective function configured in the dimer mathematical model considers not only the tendency of dimer formation between primers, but also the effect of the dispersion of the melting temperature of the primer set on dimer formation.

[0138] The evaluation of the dimer formation tendency of the primer set was calculated using a thermodynamic method. The dispersion of the melting temperature of the primer set was characterized by the sum of the differences between the melting temperature of each pair of primers and the optimal melting temperature.

[0139] In another preferred implementation, the simulated annealing algorithm includes the following processing contents:

[0140] 1) Select an initial primer set X0 from the primer pool;

[0141] 2) Evaluate the initial primer set X0 using the objective function F(X);

[0142] 3) Based on the g-th generation primer set Xg, calculate the penalty value SP for each primer pair, and replace the first y primer pairs according to the sorted penalty value SP to generate a temporary primer set T;

[0143] 4) Calculate the objective function F(T) of the temporary primer set T, and calculate the probability based on the calculated result and the annealing temperature to decide whether to use the g-th generation primer set Xg+1, maintain the g-th generation primer set Xg, or evolve to the temporary primer set T;

[0144] 5) Repeat 3) and 4) until the accepted primer set Xfinal is obtained and output.

[0145] This application also provides a processing device from the perspective of hardware structure, see Figure 5 , Figure 5 The schematic diagram of the structure of the processing device of the present application is shown. Specifically, the processing device of the present application may include a processor 501, a memory 502 and an input / output device 503. The processor 501 is used to execute the computer program stored in the memory 502 to implement the following Figure 1 Each step of the multiplex PCR primer design method based on simulated annealing algorithm in the corresponding embodiment; or, the processor 501 is used to execute the computer program stored in the memory 502 to implement the following Figure 4 The memory 502 is used to store the functions of each unit in the embodiment, and the processor 501 executes the above Figure 1 The computer program required for the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment.

[0146] For example, the computer program may be divided into one or more modules / units, one or more of which are stored in the memory 502 and executed by the processor 501 to complete the present application. One or more modules / units may be a series of computer program instruction segments capable of performing specific functions, and the instruction segments are used to describe the execution process of the computer program in a computer device.

[0147] The processing device may include, but is not limited to, a processor 501, a memory 502, and an input / output device 503. Those skilled in the art will appreciate that the illustrations are merely examples of processing devices and do not limit the processing device. The processing device may include more or fewer components than shown, or a combination of certain components, or different components. For example, the processing device may also include a network access device, a bus, etc., and the processor 501, the memory 502, the input / output device 503, etc. are connected via the bus.

[0148] The processor 501 may be a central processing unit (CPU), or other general-purpose processors, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. A general-purpose processor may be a microprocessor or any conventional processor. The processor is the control center of the processing device and connects various parts of the entire device using various interfaces and lines.

[0149] The memory 502 can be used to store computer programs and / or modules. The processor 501 implements various functions of the computer device by running or executing the computer programs and / or modules stored in the memory 502 and accessing the data stored in the memory 502. The memory 502 may mainly include a program storage area and a data storage area. The program storage area may store an operating system, at least one application required for a function, etc.; the data storage area may store data created based on the use of the processing device, etc. In addition, the memory may include high-speed random access memory and non-volatile memory, such as a hard disk, internal memory, a plug-in hard disk, a smart media card (SMC), a secure digital (SD) card, a flash card, at least one disk storage device, a flash memory device, or other volatile solid-state storage device.

[0150] When the processor 501 is used to execute the computer program stored in the memory 502, it can specifically implement the following functions:

[0151] Obtain the task of designing multiple PCR primers for the target object;

[0152] Obtaining genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided;

[0153] Based on the genome reference sequence data, target site data and SNV site data, the data input for primer pool generation is obtained;

[0154] Acquire primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as independent subtasks. Different subtasks are processed in parallel on multiple cores of the CPU, and primer designs for each locus on each chromosome are also configured to be independent.

[0155] generating a primer pool based on the data input and primer pool configuration data;

[0156] Based on the primer pool, the preset simulated annealing algorithm is combined to design multiple PCR primers for the target object.

[0157] Those skilled in the art will clearly understand that for the convenience and brevity of description, the specific working process of the multiplex PCR primer design device, processing equipment and corresponding units described above based on the simulated annealing algorithm can refer to the following. Figure 1 The description of the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment will not be repeated here.

[0158] Those skilled in the art will appreciate that all or part of the steps in the various methods of the above embodiments may be accomplished by instructions, or by controlling related hardware through instructions. The instructions may be stored in a computer-readable storage medium and loaded and executed by a processor.

[0159] To this end, the present application provides a computer-readable storage medium, which stores a plurality of instructions, which can be loaded by a processor to execute the present application as follows: Figure 1 The steps of the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment, the specific operation can refer to Figure 1 The description of the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment will not be repeated here.

[0160] The computer-readable storage medium may include a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk, etc.

[0161] Due to the instructions stored in the computer readable storage medium, the present application can be executed as follows: Figure 1 The steps of the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment can thus realize the following steps of the present application: Figure 1 The beneficial effects that can be achieved by the multiplex PCR primer design method based on the simulated annealing algorithm in the corresponding embodiment are detailed in the previous description and will not be repeated here.

[0162] The above is a detailed introduction to the multiplex PCR primer design method, device, processing equipment and computer-readable storage medium based on the simulated annealing algorithm provided by the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. At the same time, for those skilled in the art, based on the ideas of the present application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A multiplex PCR primer design method based on simulated annealing algorithm, characterized in that: The method comprises: Obtain the task of designing multiple PCR primers for the target object; Obtaining genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes the sites to be avoided; Based on the genome reference sequence data, target site data and SNV site data, the data input for primer pool generation is obtained; Obtaining primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as independent subtasks, wherein the different subtasks are processed in parallel on multiple cores of a CPU, and the primer designs for each site on each chromosome are also configured to be independent of each other; generating a primer pool based on the data input and the primer pool configuration data; Based on the primer pool, a preset simulated annealing algorithm is used to design multiplex PCR primers for the target object.

2. The method according to claim 1, characterized in that The process of processing the data input includes the following processing: Sorting the target sites of each chromosome in the sites described by the target site data, and removing the target sites that are not within the site range of the genome reference sequence data; For the target sites that are retained, adjacent sites are merged according to the distance threshold to form a target area that is processed in area units, thereby completing the update process of the sites described by the target point data.

3. The method according to claim 2, characterized in that The process of processing the data input also includes the following processing contents: Classify and sort the sites described by the SNV site data according to the chromosome; Using the classification and sorting results as a reference, filter out the sites that need to be avoided.

4. The method according to claim 1, wherein The method further comprises: Obtaining a reference file, wherein the reference file describes key parameters, including optimal primer length, maximum primer length, minimum primer length, number of return primers, amplification primer length, optimal primer melting temperature, optimal primer melting temperature, and maximum GC content; The key parameters described in the reference document are combined with the default parameters as the content of another part of the data input, wherein the default parameters are basic parameters that have been screened in the past time period and have good performance for multiplex PCR primer design.

5. The method according to claim 1, wherein The process of the multiplex PCR primer design process includes the following processing contents: To accelerate the non-specific verification of primers in the primer pool, a gene sequence hash table is pre-constructed in a targeted two-level non-specific test architecture. In the primary non-specific test, when the primers are completely matched, the primer sequences are searched through the gene sequence hash table to filter out primers that cause non-specific amplification due to complete match. Then, in the secondary non-specific test, a short sequence similarity evaluation is performed on the remaining primers to find the genomic positions and corresponding primers whose similarity exceeds the threshold.

6. The method according to claim 5, characterized in that The multiplex PCR primer design process also includes the following processing: A primer set with the lowest dimer formation probability is selected from the primer pool using a pre-constructed primer dimer mathematical model, wherein the objective function configured in the dimer mathematical model considers not only the dimer formation tendency between primers but also the effect of the dispersion of the melting temperature of the primer set on dimer formation; The evaluation of the dimer formation tendency of the primer set is calculated using a thermodynamic method. The dispersion degree of the melting temperature of the primer set is characterized by the sum of the differences between the melting temperature of each pair of primers and the optimal melting temperature.

7. The method according to claim 1, characterized in that The simulated annealing algorithm includes the following processing contents: 1) selecting an initial primer set X0 from the primer pool; 2) Evaluate the initial primer set X0 using the objective function F(X); 3) Based on the g-th generation primer set Xg, calculate the penalty value SP for each primer pair, and replace the first y primer pairs according to the sorted penalty value SP to generate a temporary primer set T; 4) calculating the objective function F(T) of the temporary primer set T, and calculating the probability based on the calculated result and the annealing temperature to decide whether to use the g-th generation primer set Xg+1, maintain the g-th generation primer set Xg, or evolve to the temporary primer set T; 5) Repeat 3) and 4) until the accepted primer set Xfinal is obtained and output.

8. A multiplex PCR primer design device based on simulated annealing algorithm, characterized in that: The device comprises: An acquisition unit, used for acquiring a multiplex PCR primer design task for a target object; The acquisition unit is further configured to acquire genome reference sequence data, target site data, and SNV site data related to the target object, wherein the SNV site data specifically describes sites to be avoided; A processing unit is used to process the data input for primer pool generation based on the genome reference sequence data, target site data and SNV site data; The acquisition unit is further configured to acquire primer pool configuration data, wherein the primer pool configuration data configures primer designs for different chromosomes as mutually independent subtasks, wherein the different subtasks are processed in parallel on multiple cores of the CPU, and the primer designs for each site on each chromosome are also configured to be mutually independent; a generating unit, configured to generate a primer pool based on the data input and the primer pool configuration data; The processing unit is used to design multiple PCR primers for the target object based on the primer pool in combination with a preset simulated annealing algorithm.

9. A processing device, characterized in that: The method comprises a processor and a memory, wherein a computer program is stored in the memory, and when the processor calls the computer program in the memory, the method according to any one of claims 1 to 7 is executed.

10. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a plurality of instructions, and the instructions are suitable for being loaded by a processor to execute the method according to any one of claims 1 to 7.

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