A method, system, device and storage medium for automatically recommending primers

Abstract

The application relates to the technical field of computational biology, and discloses a method, a system, a device and a storage medium for automatically recommending primers, which solve the technical problems of poor user experience of primer design and high failure rate of primer combination in the prior art, and the method comprises the following steps: obtaining a double-stranded structure of a gene reference sequence; selecting a target fragment on the double-stranded structure; automatically searching for primers based on the target fragment; respectively predicting the secondary structures of the primers; combining the primers into forward primers and reverse primers, and scoring the primer combinations; and visually outputting the scores of the primer combinations, a primer parameter table and optimization suggestions. According to the above technical scheme, a user only needs to frame a target fragment needing amplification on a displayed double-stranded structure of a gene in a software on a display by using a mouse cursor, and the subsequent primer recommendation process is completely automatic, so that the user experience is improved, and the failure rate of a gene amplification experiment is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of computational biology, and more specifically, to a method, system, apparatus, and storage medium for automated primer recommendation. Background Technology

[0002] Primers are short, single-stranded DNA or RNA fragments used in polymerase chain reactions (PCRs) to guide the amplification of a target gene fragment. They bind specifically to the single strand of the target gene fragment at a specific location, thus achieving specific amplification of the target fragment. The quality of primer design directly affects the specificity, efficiency, reliability, and success of the amplification experiment.

[0003] Currently, primer design is primarily achieved using biomolecular design software. However, the overall automation level of this process is low, with many steps requiring manual intervention. Furthermore, biomolecular design software lacks a comprehensive evaluation of primer combinations after design. When these unevaluated primer combinations are directly applied to biological experiments, it leads to a high failure rate. Summary of the Invention

[0004] This invention provides a method, system, device, and storage medium for automated primer recommendation, in order to solve the technical problems mentioned in the background art, such as low automation in primer combination design and high experimental failure rate caused by primer combinations not being comprehensively evaluated.

[0005] A method for automated primer recommendation includes:

[0006] Obtain the double-stranded structure of the gene reference sequence;

[0007] Select a target segment on the double-chain structure;

[0008] Based on the target fragment, primers that meet the preset primer search conditions are automatically found;

[0009] Predict the secondary structure of each primer separately, and screen out primers that meet the preset secondary structure screening criteria;

[0010] Primers that meet the preset secondary structure screening criteria are combined as forward and reverse primers, and the resulting primer combinations are scored.

[0011] The output includes a visual score of the primer combination, a primer parameter table, and optimization suggestions.

[0012] Furthermore, the source of the gene reference sequence in this invention includes artificially designed and synthesized gene reference sequences, gene reference sequences in gene banks, new gene reference sequences obtained through sequencing, and custom or randomly generated DNA sequences.

[0013] The process of obtaining the double-stranded structure of the gene reference sequence refers to uploading the gene reference sequence to a software program, and then displaying the double-stranded structure of the gene reference sequence on a monitor.

[0014] Preferred,

[0015] The sequence information of the double-stranded structure is symmetrically displayed in the interface. The symmetrically displayed content also includes annotation information, which includes any one or more of the following:

[0016] Sequence scale, including start and end points;

[0017] Defined feature information, including feature name, feature type, and interval information;

[0018] Defined ORF information;

[0019] Predicted ORF information;

[0020] ORF-translated protein amino acid sequence;

[0021] The software automatically labels the restriction endonuclease recognition sites and cleavage sites;

[0022] Information on the designed primers.

[0023] Furthermore, in this invention, selecting the target segment on the double-stranded structure means:

[0024] In a dual-chain structure, select the target segment by moving the mouse;

[0025] Preferably, after selecting the target segment using a box selection operation on the double-chain structure,

[0026] A pop-up window appears on the interface, allowing users to input the preset primer search conditions.

[0027] Furthermore, the preset primer search conditions of the present invention include pre-setting any one or more of the following primer parameters:

[0028] Primer length range;

[0029] The range of depolymerization temperatures Tm;

[0030] The number of bases matched at the 3' end;

[0031] Range of GC content;

[0032] The number of allowed binding sites;

[0033] Length range of PCR products;

[0034] Preferably, the primer length or melting temperature in the preset primer search conditions is determined first.

[0035] Furthermore, in this invention, the automatic search for primers that meet the preset primer search conditions based on the target fragment refers to:

[0036] Based on a single strand of the target fragment, a forward primer that meets the preset primer search conditions is automatically searched.

[0037] And based on the target fragment, another single strand automatically searches for a reverse primer that meets the preset primer search conditions;

[0038] Preferably, the primer sequence, binding site, and related primer parameters of the forward primer are stored accordingly, and the primer sequence, binding site, and related primer parameters of the reverse primer are stored accordingly.

[0039] The automated search here refers to:

[0040] Within the preset primer search conditions, the primer length is determined, and the search proceeds by moving from a preset start position to an end position.

[0041] The starting position and the ending position are the endpoint positions at both ends of the target segment, or,

[0042] The starting position and the ending position are any pre-set positions near both ends of the target segment.

[0043] Furthermore, in this invention, the step of combining primers that meet the preset secondary structure screening conditions into forward and reverse primers and scoring the resulting primer combinations refers to scoring each primer combination using any one or more of the following indicators:

[0044] Calculate the number of mismatches for both the forward and reverse primers, and then score each mismatch individually.

[0045] Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually.

[0046] Calculate the probability of forming a secondary hairpin structure with both the forward and reverse primers. Then, assign a score based on the combined probabilities of both primers forming a secondary hairpin structure.

[0047] Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and then give a combined score based on the probabilities of both primers forming a primer dimer.

[0048] Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually.

[0049] Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually.

[0050] Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually;

[0051] Preferably, a comprehensive score is calculated based on at least two individual scores, and the top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended on the interface in the form of primer combinations; and / or

[0052] The visualization output includes any one or more of the following information: individual score, comprehensive score, and optimization suggestions for the recommended primer combination. Preferably, the visualization displays the primer parameter table for each of the recommended primer combinations, along with the comprehensive score and optimization suggestions. More preferably, the primer parameters displayed in the visualization refer to any one or more of the following primer parameters:

[0053] Primer length;

[0054] The number of bases matched at the 3' end, and whether a single mismatch is allowed at the 3' end;

[0055] The number of bases matched at the 5' end;

[0056] Depolymerization temperature Tm;

[0057] GC count;

[0058] GC content;

[0059] Primer binding site;

[0060] Primer secondary structure;

[0061] Total free energy modifier parameter;

[0062] The corresponding PCR product range and length;

[0063] The corresponding melting temperature Tm value of the PCR product.

[0064] Furthermore, the present invention specifically compares the primers in the recommended primer combination with gene sequences of the same species in existing databases and displays the comparison results;

[0065] The alignment result is the binding site information of the primers in the primer combination on the gene sequence of the same species;

[0066] The existing database can be any one or more of the public databases; and / or the existing database can be a local public feature library built by downloading data from the public database and classifying the downloaded data according to the platform's custom classification; and / or the existing database can be a local feature library built by downloading data from the local public feature library and classifying the downloaded data according to the user's custom classification.

[0067] A system for automatically recommending primers, comprising:

[0068] The sequence information acquisition module is used to acquire and visualize the double-stranded structure of the gene reference sequence;

[0069] The primer selection module is used to select target fragments on a double-stranded structure;

[0070] The primer search module is used to find primers that meet the preset primer search conditions based on the target fragment;

[0071] The structure prediction module is used to predict the secondary structure of each primer and select primers that meet the preset secondary structure selection criteria.

[0072] The scoring module is used to combine primers that meet the preset secondary structure screening conditions into forward and reverse primers and score the resulting primer combinations.

[0073] The visualization module is used to visualize and output the score of the primer combination, the primer parameter table, and optimization suggestions.

[0074] The primer combination editing module allows users to edit and add notes to primers in recommended primer combinations through a visual display interface;

[0075] The primer combination output module allows users to output the determined primer combination in multiple formats.

[0076] Furthermore, the primer selection module is used to select target fragments on the double-stranded structure by dragging and selecting.

[0077] Preferably, after selecting the target fragment on the double-stranded structure,

[0078] A pop-up window appears on the interface, allowing users to input the preset primer search conditions.

[0079] Furthermore, the scoring module of the present invention is used to score based on any one or more of the following indicators:

[0080] Calculate the number of mismatches for both the forward and reverse primers, and then score each mismatch individually.

[0081] Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually.

[0082] Calculate the probability of forming a secondary hairpin structure with both the forward and reverse primers. Then, assign a score based on the combined probabilities of both primers forming a secondary hairpin structure.

[0083] Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and then give a combined score based on the probabilities of both primers forming a primer dimer.

[0084] Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually.

[0085] Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually.

[0086] Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually.

[0087] Preferably, a comprehensive score is calculated based on at least two individual scores, and the top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended on the interface in the form of primer combinations; and / or

[0088] The visualization output includes any one or more of the following information: individual score, comprehensive score, and optimization suggestions for the recommended primer combination. Preferably, the visualization displays the primer parameter table for each of the recommended primer combinations, along with the comprehensive score and optimization suggestions. More preferably, the primer parameters displayed in the visualization refer to any one or more of the following primer parameters:

[0089] Primer length;

[0090] The number of bases matched at the 3' end, and whether a single mismatch is allowed at the 3' end;

[0091] The number of bases matched at the 5' end;

[0092] Depolymerization temperature Tm;

[0093] GC count;

[0094] GC content;

[0095] Primer binding site;

[0096] Primer secondary structure;

[0097] Total free energy modifier parameter;

[0098] The corresponding PCR product range and length;

[0099] The Tm value corresponding to the PCR product.

[0100] A computer program for automatically recommending primers, which, when executed, performs the method described above.

[0101] An automated primer recommendation device includes a processor coupled to a memory storing a computer program that, when executed by the processor, causes the electronic device to perform the method described above.

[0102] A computer-readable storage medium includes instructions that, when executed, cause the method described above to be implemented.

[0103] In summary, the present invention has the following beneficial effects:

[0104] The core of this invention lies in providing a method, system, device, and storage medium for automated primer recommendation. The automated primer recommendation method relies on the execution of a computer program, which is stored in a storage medium installed in a computer device with a processor. Therefore, the computer device can execute the computer program stored in the storage medium through its processor to implement the automated primer recommendation method. Furthermore, the overall computer program can be broken down into multiple subroutines that jointly implement the overall method. Each subroutine together constitutes a system, systematically executing the automated primer recommendation method to achieve the function of automated primer recommendation.

[0105] Using the automated primer recommendation method of this invention, when using the software, the user only needs to select a target fragment to be amplified on the double-stranded gene structure displayed on the software interface. The subsequent primer recommendation process is completely automated. After the software runs automatically, it provides a visual output of primer combination scores and optimization suggestions. This primer design software has a high degree of automation, improves the user experience, and greatly reduces the failure rate of gene amplification experiments.

[0106] The automated software operation process is roughly as follows: the computer program automatically finds primers that meet the preset primer search conditions in the target fragment, and after finding the primers, it continues to predict the secondary structure of each primer, selects primers that meet the preset secondary structure screening conditions, and then combines the primers that meet the preset secondary structure screening conditions into forward and reverse primers. Finally, the primer combinations are scored, and the scores and optimization suggestions of the primer combinations are visualized and output in descending order of comprehensive scores. Attached Figure Description

[0107] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0108] Figure 1 A schematic diagram illustrating the double-stranded interface of a gene reference sequence;

[0109] Figure 2 A schematic diagram of an interface with added annotations to display gene reference sequences;

[0110] Figure 3 This is a schematic diagram illustrating the interface for selecting a gene reference sequence.

[0111] Figure 4 A schematic diagram of the pop-up interface to demonstrate primer search conditions;

[0112] Figure 5 This is a schematic diagram of the primer's secondary structure;

[0113] Figure 6 A schematic diagram of the free energy modification parameters;

[0114] Figure 7 A flowchart of a method for automating primer recommendation. Detailed Implementation

[0115] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0116] This invention aims to disclose in exchange for protection a method for automated primer recommendation, the main steps of which are as follows:

[0117] Step A100: Obtain the double-stranded structure of the gene reference sequence.

[0118] In this method, obtaining the double-stranded structure of the gene reference sequence means that the operator manually uploads the gene reference sequence to the software program, and the monitor displays the double-stranded structure of the gene reference sequence. Combined with... Figure 1 ,exist Figure 1 The software displays the double-stranded structure of the gene reference sequence.

[0119] In one embodiment, the double-stranded structure of the gene reference sequence (the primary structure of the DNA double helix (sequence information)) is symmetrically displayed in the interface, and the symmetrically displayed content also includes the annotation information of the gene reference sequence. Combined with... Figure 2 , Figure 2 The double-stranded structure of the gene reference sequence in the image displays the annotation information of the gene reference sequence. The purpose of displaying this annotation information is to facilitate operator identification and selection operations of the gene reference sequence. This annotation information includes, but is not limited to, sequence scales (including start and end points); defined feature information (including feature name, feature type, and interval information); defined and predicted ORF information; the amino acid sequence of the protein translated by the ORF; designed primer information; and software-annotated restriction endonuclease recognition and cleavage sites.

[0120] Step A200: Select the target segment on the double-chain structure.

[0121] In one embodiment, selecting a target segment on the bichain structure means selecting the target segment by moving a mouse cursor over the bichain structure, combined with... Figure 3 When selecting a target fragment, place the mouse cursor at the top left or bottom right corner and drag towards the opposite bottom right or bottom left corner. Alternatively, place the mouse cursor at the left or right endpoint of the target fragment and drag towards the opposite right or left endpoint to select the fragment. When determining the target fragment, if the double-stranded structure of the gene reference sequence displays annotation information, the operator can usually determine which segment of the gene base sequence on the double-stranded structure should be selected as the target fragment for amplification based on the annotation information.

[0122] After the operator selects the target fragment on the double-stranded structure by moving the mouse cursor, a pop-up window appears in the software interface where the user can enter primer search criteria, such as... Figure 4 As shown, primer search criteria can be pre-entered in the pop-up window. The computer program can then automatically search for primers that match these pre-defined criteria on the target fragment.

[0123] The aforementioned preset primer search conditions include, but are not limited to, the following primer search conditions:

[0124] (1) Range of primer length.

[0125] In one embodiment, the primer length is set to range from 18nt to 30nt.

[0126] (2) Deconvolution temperature (Tm) range.

[0127] In one embodiment, the melting temperature Tm is set to a range of 60±1℃.

[0128] (3) The number of bases matched at the 3' end.

[0129] In one embodiment, the minimum number of bases matched at the 3' end is set to 8, meaning that the number of bases matched at the 3' end of the primers found is at least 8, and a single base is allowed to be in a mismatch state.

[0130] (4) GC number and GC content.

[0131] In one embodiment, the GC content of the primers is set between 6 and 18, and the GC content of the primers is set between 40% and 60%.

[0132] (5) Number of binding sites.

[0133] In one embodiment, the number of binding sites is limited to only one. This is because if the primer found, whether forward or reverse, has multiple binding sites on the target fragment, it will affect the quality of subsequent specific amplification experiments of that target fragment.

[0134] (6) Length range of PCR products.

[0135] The length range of the PCR product is preset according to the length of the target gene sequence to be amplified. In one embodiment, the length range of the PCR product is set to 200nt-1000nt.

[0136] For the six primer search conditions mentioned above, primer length or melting temperature is usually prioritized. For example, regarding melting temperature, since longer primers have higher melting temperatures, using melting temperature as the primary search condition can exclude most long primers, as well as some primers with suitable lengths but whose melting temperatures do not meet the preset values. After ensuring the melting temperature is met, primer length is then further selected to exclude shorter primers. Finally, other preset search conditions are checked to ultimately find primers that satisfy all preset search conditions.

[0137] Step A300: Based on the target fragment, primers that meet the preset primer search conditions are automatically found.

[0138] It should be noted that, in one embodiment, the automated search process can employ a computer program with the following search logic: The computer program moves along a preset start position to an end position on the gene fragment, based on the primer length. The preset start and end positions can be the endpoints of the target fragment, or they can be any pre-defined positions near the ends of the target fragment.

[0139] After determining the logic for automated primer finding, the computer program moves from a starting position to an ending position according to preset primer search conditions, aiming to find primers that meet the search criteria. The search process includes searching both strands of the target fragment. The search on one strand aims to find a forward primer, i.e., automatically searching for a forward primer that meets the search criteria based on one single strand of the target fragment; the search on the other strand aims to find a reverse primer, i.e., automatically searching for a reverse primer that meets the search criteria based on the other single strand of the target fragment. After a suitable primer is found, the primer sequence, binding site, and related primer parameters of the forward primer are stored accordingly; the primer sequence, binding site, and related primer parameters of the reverse primer are also stored accordingly.

[0140] Step A400: Predict the secondary structure of each primer and select primers that meet the preset secondary structure screening criteria.

[0141] After the primer search process, in general, many forward and reverse primers that meet the primer search conditions have been found. Then, it is necessary to predict the secondary structure of each primer. Predicting the secondary structure of primers is a relatively complex process. This invention aims to improve the efficiency of secondary structure prediction. The secondary structure prediction method of this invention is as follows.

[0142] When calling the found primer sequences, to predict the secondary structure of one of the primer sequences, it is first necessary to calculate all possible secondary structures of that primer sequence. Then, the possible secondary structure morphologies are calculated and analyzed to determine which morphology is the most likely actual secondary structure generated by that primer sequence. Figure 5 As shown, it is usually sufficient to provide the top three most likely actual secondary structures (the specific number can be adjusted according to the actual situation), and finally generate the secondary structure diagrams corresponding to the top three.

[0143] In this invention, to improve the efficiency of secondary structure prediction, preset secondary structure screening conditions are provided. During the energy calculation and analysis of each secondary structure, if a secondary structure does not meet any of the screening conditions, the energy calculation of that secondary structure is immediately terminated, and the energy calculation of other secondary structures continues.

[0144] The secondary structure screening criteria include:

[0145] (1) Ring length ≤ predetermined ring length;

[0146] In one embodiment, the primer loop length is limited to 3 nt or less, i.e., loop length ≤ 3 nt.

[0147] (2) The number of GCs at the 3' end is less than the predetermined number of GCs; this is used to optimize the accessibility of the 3' end.

[0148] In one embodiment, the GC number at the 3' end of the primer is limited to 3 or less, i.e., the GC number at the 3' end is ≤3, in order to optimize the accessibility of the 3' end.

[0149] (3) The maximum number of consecutive pairs within a single primer is less than or equal to the predetermined maximum number of consecutive pairs; this is used to limit the formation of dimers within a single primer.

[0150] In one embodiment, the maximum number of consecutive pairs within a single primer is limited to 2, i.e., the maximum number of consecutive pairs within a single primer is ≤2, in order to limit the formation of dimers within a single primer.

[0151] When using the above secondary structure filtering criteria for filtering, the specific filtering method is as follows:

[0152] Based on all possible secondary structures of each primer, the computer calculates the free energy modification parameter for each base in each secondary structure. During this calculation, the base pairing status is determined based on the free energy modification parameter value, and the loop length and the maximum number of consecutive base pairs within the primer are accumulated according to the pairing status. The accumulated results from the free energy modification parameter calculation are used for judgment and screening. If a secondary structure that does not meet the screening criteria is found, the energy calculation for the remaining bases of that secondary structure is terminated, and the energy calculation for other secondary structures continues. If all possible secondary structures of the primer are eliminated, the primer is deleted.

[0153] When a primer has a secondary structure that meets all the secondary structure screening criteria, the total free energy modification parameter of each secondary structure is calculated and sorted according to the value of the total free energy modification parameter. In one embodiment, the top 10 optimal solutions are selected.

[0154] The computer program process for selecting the top 10 optimal solutions involves determining whether the total free energy modification parameter is among the top 10 optimal solutions. If so, the secondary structure and the total free energy modification parameter are stored accordingly. Simultaneously, the total free energy modification parameter is used as one of the primer parameters, stored, and subsequently displayed on the interface.

[0155] Among them, combined Figure 6 The rightmost parameter is the total free energy modification parameter. The total free energy modification parameter is calculated by summing the free energy modification parameters of all bases in the secondary structure on the left.

[0156] Step A500: Combine primers that meet the preset secondary structure screening criteria into forward and reverse primers and score the resulting primer combinations.

[0157] Combining primers that meet the preset secondary structure screening criteria into forward and reverse primers and scoring the resulting primer combinations involves evaluating each primer combination using one or more of the following indicators:

[0158] Calculate the number of mismatches for the forward primer and the number of mismatches for the reverse primer, and then give a comprehensive score for each item.

[0159] Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually.

[0160] Calculate the probability of forming a secondary hairpin structure with both the forward and reverse primers. Then, assign a score based on the combined probabilities of both primers forming a secondary hairpin structure.

[0161] Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and give a comprehensive score based on the combined probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer;

[0162] Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually;

[0163] Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually.

[0164] Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually;

[0165] If the above multiple scores are evaluated, a comprehensive score is then calculated based on these scores. The top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended on the interface in the form of primer combinations.

[0166] The comprehensive scoring method can be customized according to the needs of biologists, and can be based on existing mathematical scoring methods such as the mean method and weighted average.

[0167] Step A600: Visualize the output of the primer combination's score, primer parameter table, and optimization suggestions.

[0168] The system calculates and outputs one or more of the following information: individual score, overall score, and optimization suggestions for the recommended primer combination. In one embodiment, after calculating the individual score, overall score, and optimization suggestions, a software interface is also used to visually display the individual score, overall score, primer parameter table, and optimization suggestions for the recommended primer combination.

[0169] When visually displaying recommended primer combinations, users can edit and add notes on the recommended primer combinations in the visualization interface. Based on the individual scores, comprehensive scores, primer parameter tables, and optimization suggestions of the recommended primer combinations, users can select the primer combinations they want according to their experimental needs. Users can also output the selected primer combinations in multiple formats.

[0170] Primer parameters refer to the primer parameters that have been calculated and stored in steps A100-A500 above. The primer parameter table can be used to visually display any one or more of the following primer parameters:

[0171] Primer length.

[0172] The number of bases matched at the 3' end, and whether a single mismatch is allowed at the 3' end.

[0173] The number of bases matched at the 5' end.

[0174] Depolymerization temperature Tm.

[0175] GC count.

[0176] GC content.

[0177] Primer binding site.

[0178] Secondary structure of primers.

[0179] Total free energy modifier parameter.

[0180] The corresponding PCR product range and length.

[0181] The corresponding melting temperature Tm value of the PCR product.

[0182] Furthermore, this invention specifically compares the primers in the recommended primer combinations with gene sequences of the same species in existing databases and displays the comparison results. Specific comparison refers to searching for binding site information of the primers in the primer combinations on gene sequences of the same species, determining the location and number of binding sites. This allows for the assessment of the specificity between the primers in the primer combinations and gene sequences of the same species.

[0183] The existing databases are public databases, such as any one or more of the NCBI database, Ensembl database, and UniProt database.

[0184] Alternatively, the existing database can be a platform database, which refers to a platform-customized database that downloads data from any one or more public databases (NCBI database, Ensembl database, UniProt database) and establishes the data based on the platform's custom classification.

[0185] Alternatively, the existing database can also be used to download data from the platform's custom database and build a user database based on the downloaded data through user-defined categories.

[0186] In summary, combining Figure 7 The steps of the method for automatically recommending primers according to the present invention are as follows:

[0187] Step A100: Obtain the double-stranded structure of the gene reference sequence;

[0188] Step A200: Select the target segment on the double-chain structure;

[0189] Step A300: Based on the target fragment, primers that meet the preset primer search conditions are automatically found;

[0190] Step A400: Predict the secondary structure of each primer and select primers that meet the preset secondary structure screening criteria;

[0191] Step A500: Combine primers that meet the preset secondary structure screening criteria into forward and reverse primers and score the resulting primer combinations.

[0192] Step A600: Visualize and output the score of the primer combination, primer parameter table, and optimization suggestions.

[0193] Based on the above method for automated primer recommendation, the overall computer program implementing this method is broken down into multiple subroutines. These subroutines work together to run the overall method, and each subroutine contributes to the complete system. The following is a detailed description of this system.

[0194] An automated primer recommendation system includes: a sequence information acquisition module, a primer selection module, a primer search module, a structure prediction module, a scoring module, and a visualization module.

[0195] The sequence information acquisition module is used to acquire and visualize the double-stranded structure of the gene reference sequence.

[0196] After the operator manually uploads the gene reference sequence to the software program, the structure acquisition module controls the software interface on the monitor to display the double-stranded structure of the gene reference sequence. Combined with... Figure 1 ,exist Figure 1 The software displays the double-stranded structure of the gene reference sequence.

[0197] The primer selection module is used to select target fragments on a double-stranded structure.

[0198] The primer selection module can be used for manual selection or automated selection via a computer program. Automated selection via a computer program could involve the user clicking on either end of the target fragment, and the primer selection module automatically selecting the target fragment region between those two endpoints.

[0199] The primer search module is used to find primers that meet the preset primer search conditions based on the target fragment.

[0200] In the primer search module program, some parameters can be manually set by the user. These manually set parameters are the preset primer search conditions.

[0201] After the user manually sets the preset primer search conditions, the primer search module moves within the target fragment to search for primers according to the program. During the search, based on one single strand of the target fragment, it automatically searches for a forward primer that meets the preset primer search conditions; based on the other single strand of the target fragment, it automatically searches for a reverse primer that meets the preset primer search conditions. Finally, the primer sequence, binding site, and related primer parameters of the forward primer are stored accordingly; the primer sequence, binding site, and related primer parameters of the reverse primer are also stored accordingly.

[0202] The structure prediction module is used to predict the secondary structure of each primer and select primers that meet the preset secondary structure selection criteria.

[0203] The structural prediction module includes the following sub-units: secondary structure calculation unit, free energy modification parameter calculation unit, secondary structure screening unit, and secondary structure selection unit.

[0204] The secondary structure calculation unit is used to calculate all possible secondary structures for each primer.

[0205] The free energy modification parameter calculation unit is used to calculate the free energy modification parameter of each base in each secondary structure, and then sum the free energy modification parameters of all bases in the secondary structure to obtain the total free energy modification parameter.

[0206] The secondary structure screening unit is used to screen based on the calculation results of the free energy modification parameters of each base in each secondary structure during the process of calculating the free energy modification parameters one by one. If a secondary structure that does not meet the secondary structure screening conditions is found, the energy calculation of other bases in the secondary structure is terminated, and the energy calculation of other secondary structures is continued. If all possible secondary structures of the primer are screened out, the primer is deleted.

[0207] The secondary structure selection unit is used to sum the free energy modification parameters of all bases in the secondary structure to obtain the total free energy modification parameters, and to determine whether the total free energy modification parameters are among the top N optimal solutions. If so, the secondary structure of the primer, the total free energy modification parameters, and the free energy modification parameters of each base are selected and stored accordingly.

[0208] The scoring module is used to combine primers that meet the preset secondary structure screening criteria into forward and reverse primers and score the resulting primer combinations.

[0209] The scoring module is used to score one or more of the following indicators:

[0210] Calculate the number of mismatches for the forward primer and the number of mismatches for the reverse primer, and then score each item based on the combined results.

[0211] Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually.

[0212] The probability of the forward primer forming a secondary hairpin structure and the probability of the reverse primer forming a secondary hairpin structure are calculated, and a single score is given based on the combined probability of the forward primer forming a secondary hairpin structure and the reverse primer forming a secondary hairpin structure.

[0213] Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and give a comprehensive score based on the combined probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer.

[0214] Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually.

[0215] Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually.

[0216] Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually.

[0217] Preferably, a comprehensive score is calculated based on at least two individual scores, and the top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended in the form of primer combinations on the interface.

[0218] The visualization module is used to visualize the output of the primer combination's score, primer parameter table, and optimization suggestions.

[0219] The visualization module provides visual output of one or more of the following: individual scores, overall scores, and optimization suggestions for the recommended primer combinations. This output is then displayed on the software interface, showing the primer parameter tables for each recommended primer combination, along with the overall score and optimization suggestions.

[0220] The primer combination editing module allows users to edit and add notes to primers in recommended primer combinations through a visual display interface.

[0221] The primer combination output module allows users to output the determined primer combination in multiple formats.

[0222] The specific details of each module / unit in the above system have been described in detail in the implementation method section. For any undisclosed details, please refer to the implementation method section, and therefore will not be repeated here.

[0223] This specific embodiment also provides a computer program for automatically recommending primers, which, when executed, performs the method described above.

[0224] This specific embodiment also provides an automated primer recommendation device, including a processor coupled to a memory storing a computer program. When the processor executes the computer program, the electronic device performs the method described above.

[0225] This specific embodiment also provides a computer-readable storage medium, which includes instructions that, when executed, cause the above-described method to be implemented.

[0226] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for automated primer recommendation, characterized in that, include: Obtain the double-stranded structure of the gene reference sequence; Select a target segment on the double-chain structure; Based on the target fragment, primers that meet the preset primer search conditions are automatically found; Predict the secondary structure of each primer separately, and screen out primers that meet the preset secondary structure screening criteria; Primers that meet the preset secondary structure screening criteria are combined as forward and reverse primers, and the resulting primer combinations are scored. The output includes a visual score of the primer combination, a primer parameter table, and optimization suggestions.

2. The method for automated primer recommendation according to claim 1, characterized in that, The sources of the gene reference sequences mentioned include artificially designed and synthesized gene reference sequences, gene reference sequences in gene banks, new gene reference sequences obtained through sequencing, and custom or randomly generated DNA sequences. The process of obtaining the double-stranded structure of the gene reference sequence refers to uploading the gene reference sequence to a software program, and then displaying the double-stranded structure of the gene reference sequence on a monitor. Preferred, The sequence information of the double-stranded structure is symmetrically displayed in the interface. The symmetrically displayed content also includes annotation information, which includes any one or more of the following: Sequence scale, including start and end points; Defined feature information, including feature name, feature type, and interval information; Defined ORF information; Predicted ORF information; ORF-translated protein amino acid sequence; The software automatically labels the restriction endonuclease recognition sites and cleavage sites; Information on the designed primers.

3. The method for automated primer recommendation according to any one of claims 1-2, characterized in that, The selection of the target segment on the double-stranded structure refers to: In a dual-chain structure, select the target segment by moving the mouse; Preferably, after selecting the target segment using a box selection operation on the double-chain structure, A pop-up window appears on the interface, allowing users to input the preset primer search conditions.

4. A method for automated primer recommendation according to any one of claims 1-3, characterized in that, The preset primer search conditions include presetting any one or more of the following primer parameters: Primer length range; The range of depolymerization temperatures Tm; The number of bases matched at the 3' end; Range of GC content; The number of allowed binding sites; Length range of PCR products; Preferably, the primer length or melting temperature in the preset primer search conditions is determined first.

5. A method for automated primer recommendation according to any one of claims 1-4, characterized in that, The automatic search for primers that meet the preset primer search conditions based on the target fragment refers to: Based on a single strand of the target fragment, a forward primer that meets the preset primer search conditions is automatically searched. And based on the target fragment, another single strand automatically searches for a reverse primer that meets the preset primer search conditions; Preferably, the primer sequence, binding site, and related primer parameters of the forward primer are stored accordingly, and the primer sequence, binding site, and related primer parameters of the reverse primer are stored accordingly. The automated search here refers to: Within the preset primer search conditions, the primer length is determined, and the search proceeds by moving from a preset start position to an end position. The starting position and the ending position are the endpoint positions at both ends of the target segment, or, The starting position and the ending position are any pre-set positions near both ends of the target segment.

6. A method for automated primer recommendation according to any one of claims 1-5, characterized in that, The step of combining primers that meet the preset secondary structure screening criteria into forward and reverse primers and scoring the resulting primer combinations refers to scoring each primer combination using any one or more of the following indicators: Calculate the number of mismatches for both the forward and reverse primers, and then score each mismatch individually. Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually. Calculate the probability of forming a secondary hairpin structure with both the forward and reverse primers. Then, assign a score based on the combined probabilities of both primers forming a secondary hairpin structure. Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and then give a combined score based on the probabilities of both primers forming a primer dimer. Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually. Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually. Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually; Preferably, a comprehensive score is calculated based on at least two individual scores, and the top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended on the interface in the form of primer combinations; and / or The visualization output includes any one or more of the following information: individual score, comprehensive score, and optimization suggestions for the recommended primer combination. Preferably, the visualization displays the primer parameter table for each of the recommended primer combinations, along with the comprehensive score and optimization suggestions. More preferably, the primer parameters displayed in the visualization refer to any one or more of the following primer parameters: Primer length; The number of bases matched at the 3' end, and whether a single mismatch is allowed at the 3' end; The number of bases matched at the 5' end; Depolymerization temperature Tm; GC count; GC content; Primer binding site; Primer secondary structure; Total free energy modifier parameter; The corresponding PCR product range and length; The corresponding melting temperature Tm value of the PCR product.

7. A method for automated primer recommendation according to any one of claims 1-6, characterized in that, It also includes specifically comparing the primers in the recommended primer combination with gene sequences of the same species in an existing database and displaying the comparison results; The alignment result is the binding site information of the primers in the primer combination on the gene sequence of the same species; The existing database can be any one or more of the public databases; and / or the existing database can be a local public feature library built by downloading data from the public database and classifying the downloaded data according to the platform's custom classification; and / or the existing database can be a local feature library built by downloading data from the local public feature library and classifying the downloaded data according to the user's custom classification. and / or It also includes allowing users to edit and add notes to the primers in the recommended primer combinations in a visual display interface; It also allows users to output the determined primer combinations in multiple formats.

8. A system for automatically recommending primers, characterized in that, include: The sequence information acquisition module is used to acquire and visualize the double-stranded structure of the gene reference sequence; The primer selection module is used to select target fragments on a double-stranded structure; The primer search module is used to find primers that meet the preset primer search conditions based on the target fragment; The structure prediction module is used to predict the secondary structure of each primer and select primers that meet the preset secondary structure selection criteria. The scoring module is used to combine primers that meet the preset secondary structure screening conditions into forward and reverse primers and score the resulting primer combinations. The visualization module is used to visualize and output the score of the primer combination, the primer parameter table, and optimization suggestions. The primer combination editing module allows users to edit and add notes to primers in recommended primer combinations through a visual display interface; The primer combination output module allows users to output the determined primer combination in multiple formats.

9. The system for automatically recommending primers according to claim 8, characterized in that, The primer selection module is used to select target fragments on the double-stranded structure by moving the mouse. Preferably, after selecting the target fragment on the double-stranded structure, A pop-up window appears on the interface, allowing users to input the preset primer search conditions.

10. A system for automatically recommending primers according to claim 8 or 9, characterized in that, The scoring module is used to score based on one or more of the following indicators: Calculate the number of mismatches for both the forward and reverse primers, and then score each mismatch individually. Calculate the melting temperature difference between the forward and reverse primers, and score the melting temperature difference individually. Calculate the probability of forming a secondary hairpin structure with both the forward and reverse primers. Then, assign a score based on the combined probabilities of both primers forming a secondary hairpin structure. Calculate the probability of the forward primer forming a primer dimer and the probability of the reverse primer forming a primer dimer, and then give a combined score based on the probabilities of both primers forming a primer dimer. Calculate the probability of primer dimer formation between the forward and reverse primers, and score the probability of primer dimer formation individually. Calculate the GC content difference between the forward and reverse primers, and score the GC content difference individually. Calculate the primer length difference between the forward and reverse primers, and score the primer length difference individually. Preferably, a comprehensive score is calculated based on at least two individual scores, and the top N optimal primer combinations with the highest comprehensive scores are selected in descending order and recommended on the interface in the form of primer combinations; and / or The visualization output includes any one or more of the following information: individual score, comprehensive score, and optimization suggestions for the recommended primer combination. Preferably, the visualization displays the primer parameter table for each of the recommended primer combinations, along with the comprehensive score and optimization suggestions. More preferably, the primer parameters displayed in the visualization refer to any one or more of the following primer parameters: Primer length; The number of bases matched at the 3' end, and whether a single mismatch is allowed at the 3' end; The number of bases matched at the 5' end; Depolymerization temperature Tm; GC count; GC content; Primer binding site; Primer secondary structure; Total free energy modifier parameter; The corresponding PCR product range and length; The Tm value corresponding to the PCR product.

11. A computer program for automatically recommending primers, characterized in that, When the computer program is executed, the method as described in any one of claims 1 to 7 is performed.

12. An automated primer recommendation device, characterized in that, include A processor coupled to a memory storing a computer program, which, when executed by the processor, causes the electronic device to perform the method as described in any one of claims 1 to 7.

13. A computer-readable storage medium, characterized in that, The computer-readable storage medium includes instructions that, when executed, cause the method as described in any one of claims 1 to 7 to be implemented.

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