Primer set determination method and device

Abstract

The invention discloses a primer set determination method and device, which can be applied to the technical field of biological medicine, and comprises the following steps: calculating the dimer score of each pair of primers and constructing a dimer score matrix, screening out primer pairs with scores lower than a threshold value, and directly excluding primer combinations which are easy to form dimers. The Gibbs free energy matrix is used for further screening, the larger the absolute value of the Gibbs free energy is, the more stable the dimer is, and the primer pairs with overlarge absolute values of the free energy are eliminated through comparison with a threshold value. Finally, the Gibbs free energy is corrected through a loss function, a corrected free energy matrix is obtained, the corrected free energy is combined with the influence of non-standard base pairing, the probability of dimer formation is further accurately evaluated, and a better primer pair is screened out again. Through multiple rounds of strict screening, the formation probability of the primer dimer is reduced to the greatest extent, so that the occurrence of false positive results caused by the primer dimer is effectively reduced, and the occurrence probability of the false positive rate is further reduced.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a method and device for determining a primer set. Background Art

[0002] Multiplex PCR amplification is a combination of super-multiplex PCR amplification and high-throughput sequencing, which can detect dozens to hundreds of known pathogens and their virulence or resistance genes in the sample to be tested. This technology has advantages over pathogen metagenomic sequencing (mNGS) in terms of clear pathogen spectrum, high detection accuracy, and low sequencing cost when detecting low concentrations of pathogens, especially in terms of detecting their virulence or resistance genes. However, how to design high-quality and high-specificity primers is the key point of this technology, which will have a very important impact on both the operation of wet experiments and the analysis of conventional second-generation sequencing sequences. Good primers can ensure the efficiency of the experiment, significantly improve the success rate of the experiment, and reduce the cost of repeated trial and error.

[0003] At present, there are many methods for designing and simulating highly multiplexed PCR primers. Among them, the wet experiment method has solved the primer design problem faced by targeted high-throughput sequencing to a certain extent, but it cannot effectively remove a large number of potential primer dimers. Primer dimers or non-specific binding may lead to false positive results, resulting in a higher probability of false positives and a higher error rate in diagnosis. Summary of the invention

[0004] The present application provides a primer set determination method to reduce the occurrence of false positive results caused by primer dimers, thereby reducing the probability of false positive rate. The present application also provides a primer set determination device.

[0005] In a first aspect, the present application provides a method for determining a primer set, comprising: Obtaining a target template sequence; designing an initial primer set according to the target template sequence; Determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and compare each dimer score with a score threshold to construct a dimer score matrix, and compare each Gibbs free energy with a Gibbs free energy threshold to construct a Gibbs free energy matrix, wherein a larger dimer score indicates a higher probability of dimer formation, and a larger absolute value of the Gibbs free energy indicates a more stable dimer; Using a loss function to correct each of the Gibbs free energies to obtain a corrected free energy, and using each of the corrected free energies to compare with a corrected free energy threshold to construct a corrected free energy matrix, wherein the corrected free energy combines the influence of non-canonical base pairing, and a larger value indicates a higher probability of dimer formation; The target primer set is determined using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix.

[0006] Optionally, the step of comparing each dimer score with a score threshold to construct a dimer score matrix comprises: Comparing each of the dimer scores with a score threshold, marking an initial primer pair whose dimer score is greater than the score threshold as 1, and marking an initial primer pair whose dimer score is less than or equal to the score threshold as 0; Construct a dimer score matrix based on the markers.

[0007] Optionally, constructing a Gibbs free energy matrix by comparing each of the Gibbs free energies with a Gibbs free energy threshold value comprises: Using each of the Gibbs free energies to compare with a Gibbs free energy threshold, marking an initial primer pair whose Gibbs free energy is greater than the Gibbs free energy threshold as 1, and marking an initial primer pair whose dimer score is less than or equal to the Gibbs free energy threshold as 0; Construct the Gibbs free energy matrix based on the labeling.

[0008] Optionally, the using a loss function to correct each of the Gibbs free energies to obtain the corrected free energy comprises: Each of the Gibbs free energies is corrected using a loss function and the distance from the matching subsequence of each pair of primers to the 3' end of the primer to obtain a corrected free energy.

[0009] Optionally, comparing the loss function value corresponding to each of the corrected free energies with a corrected free energy threshold value includes: Comparing the loss function value corresponding to each correction free energy with the correction free energy threshold, marking the initial primer pair whose correction free energy is greater than the correction free energy threshold as 1, and marking the initial primer pair whose correction free energy is less than or equal to the correction free energy threshold as 0; Construct a corrected free energy matrix based on the labeling.

[0010] Optionally, each pair of primers includes a P1 primer and a P2 primer, and the method further includes: In the case where the P1 primer and the P2 primer are homologous primers, if the specificity of the P1 primer is verified using only a subsequence of a single read length of the product generated by the P1 primer and the verification is passed, the primer pair is marked as 0; if the subsequence does not exist or the specificity verification fails, the primer pair is marked as 1; In the case where the P1 primer and the P2 primer are different primer combinations, if both the P1 primer and the P2 primer have a single read length subsequence, the primer pair is marked as 0; if one of the P1 primer and the P2 primer does not have a single read length subsequence or fails the specificity verification, the primer pair is marked as 1; Construct a specificity matrix based on the markers.

[0011] The method of determining a target primer set by using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix comprises: The target primer set is determined using the intersection of the primer pairs in the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix, and the specificity matrix.

[0012] Optionally, determining the target primer set by using the intersection of the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix, and the primer pairs in the specificity matrix comprises: The primer pairs marked as 1 in the filter results are screened according to the dimer score matrix to obtain a candidate primer set S1 that satisfies the score matrix; The primer pairs marked as 1 in the screening and filtering results are selected according to the Gibbs free energy matrix to obtain a candidate primer set S2 of the Gibbs free energy matrix; The primer pairs marked as 1 in the filter results are screened according to the correction free energy matrix to obtain a candidate primer set S3 that satisfies the correction free energy matrix; Extract the intersection of primer pairs from S1, S2 and S3 to obtain the coexisting candidate primer set of dimers; The primer pairs marked as 0 after specificity verification were screened out according to the specificity matrix to obtain a candidate primer set S4 with high specificity; The target primer set is determined according to the intersection of the primer pairs extracted from the co-existing candidate primer set of the dimer and the high-specificity candidate primer set S4.

[0013] In a second aspect, the present application further provides a primer set determination device, the device comprising: An acquisition unit, used for acquiring a target template sequence; A design unit, used to design an initial primer set according to the target template sequence; A first determination unit is used to determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and to construct a dimer score matrix by comparing each dimer score with a score threshold, and to construct a Gibbs free energy matrix by comparing each Gibbs free energy with a Gibbs free energy threshold, wherein a larger dimer score indicates a higher probability of dimer formation, and a larger absolute value of the Gibbs free energy indicates a more stable dimer; A comparison unit, used to correct each of the Gibbs free energies using a loss function to obtain a corrected free energy, and to construct a corrected free energy matrix by comparing each of the corrected free energies with a corrected free energy threshold, wherein the corrected free energy combines the influence of non-canonical base pairing, and a larger value indicates a higher probability of dimer formation; The second determination unit is used to determine the target primer set by using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix and the corrected free energy matrix.

[0014] Optionally, the first determining unit is specifically configured to compare each of the dimer scores with a score threshold, mark an initial primer pair whose dimer score is greater than the score threshold as 1, and mark an initial primer pair whose dimer score is less than or equal to the score threshold as 0; Construct a dimer score matrix based on the markers.

[0015] Optionally, the first determination unit is specifically used to compare each of the Gibbs free energy with the Gibbs free energy threshold, mark the initial primer pair whose Gibbs free energy is greater than the Gibbs free energy threshold as 1, and mark the initial primer pair whose dimer score is less than or equal to the Gibbs free energy threshold as 0; Construct the Gibbs free energy matrix based on the labeling.

[0016] Optionally, the comparison unit is specifically configured to correct each of the Gibbs free energies using a loss function and a distance from a matching subsequence of each pair of primers to the 3' end of the primers to obtain a corrected free energy.

[0017] Optionally, the comparison unit is specifically used to compare each of the correction free energies with the correction free energy threshold, marking an initial primer pair whose correction free energy is greater than the correction free energy threshold as 1, and marking an initial primer pair whose correction free energy is less than or equal to the correction free energy threshold as 0; Construct a corrected free energy matrix based on the labeling.

[0018] Optionally, the device further comprises a marking unit, which is used for marking the primer pair as 0 if the specificity of the P1 primer is verified by using only a subsequence of a single read length of a product generated by the P1 primer and the verification is passed, and marking the primer pair as 1 if the subsequence does not exist or the specificity verification fails, when the P1 primer and the P2 primer are homologous primers; In the case where the P1 primer and the P2 primer are different primer combinations, if both the P1 primer and the P2 primer have a single read length subsequence, the primer pair is marked as 0; if one of the P1 primer and the P2 primer does not have a single read length subsequence or fails the specificity verification, the primer pair is marked as 1; Construct a specificity matrix based on the markers.

[0019] The second determination unit is specifically used to determine the target primer set by using the intersection of the primer pairs in the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix and the specificity matrix.

[0020] Optionally, the second determining unit is specifically configured to screen the primer pairs whose filtering results are marked as 1 according to the dimer score matrix to obtain a candidate primer set S1 that satisfies the score matrix; The primer pairs marked as 1 in the screening and filtering results are selected according to the Gibbs free energy matrix to obtain a candidate primer set S2 of the Gibbs free energy matrix; The primer pairs marked as 1 in the filter results are screened according to the correction free energy matrix to obtain a candidate primer set S3 that satisfies the correction free energy matrix; Extract the intersection of primer pairs from S1, S2 and S3 to obtain the coexisting candidate primer set of dimers; The primer pairs marked as 0 after specificity verification were screened out according to the specificity matrix to obtain a candidate primer set S4 with high specificity; The target primer set is determined according to the intersection of the primer pairs extracted from the co-existing candidate primer set of the dimer and the high-specificity candidate primer set S4.

[0021] In a third aspect, an embodiment of the present application provides a device, comprising a memory and a processor, wherein the memory is used to store instructions or codes, and the processor is used to execute the instructions or codes so that the device executes the method described in the first aspect above.

[0022] In a fourth aspect, an embodiment of the present application provides a computer storage medium, in which a code is stored. When the code is executed, a device executing the code implements the method described in the first aspect above. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in this embodiment or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 A flowchart of a primer set determination method provided in an embodiment of the present application; Figure 2 A structural block diagram of a primer set determination method provided in an embodiment of the present application; Figure 3 A schematic structural diagram of a specific implementation of the primer set determination device provided in the present application. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] It should be noted that the primer set determination method and device provided in the present application are used in the field of biomedical technology. The above is only an example and does not limit the application field of the method and device provided in the present application.

[0027] Multiplex PCR amplification is a combination of super-multiplex PCR amplification and high-throughput sequencing, which can detect dozens to hundreds of known pathogens and their virulence or resistance genes in the sample to be tested. This technology has advantages over pathogen metagenomic sequencing (mNGS) in terms of clear pathogen spectrum, high detection accuracy, and low sequencing cost when detecting low concentrations of pathogens, especially in terms of detecting their virulence or resistance genes. However, how to design high-quality and high-specificity primers is the key point of this technology, which will have a very important impact on both the operation of wet experiments and the analysis of conventional second-generation sequencing sequences. Good primers can ensure the efficiency of the experiment, significantly improve the success rate of the experiment, and reduce the cost of repeated trial and error.

[0028] At present, there are many methods for designing and simulating highly multiplexed PCR primers. Among them, the wet experiment method has solved the primer design problem faced by targeted high-throughput sequencing to a certain extent, but it cannot effectively remove a large number of potential primer dimers. Primer dimers or non-specific binding may lead to false positive results, resulting in a higher probability of false positives and a higher error rate in diagnosis.

[0029] In view of this, a method for determining a primer set is proposed in an embodiment of the present application, including: obtaining a target template sequence, designing an initial primer set according to the target template sequence, determining the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and using each dimer score to compare with the score threshold to construct a dimer score matrix, using each Gibbs free energy to compare with the Gibbs free energy threshold to construct a Gibbs free energy matrix. Wherein, the greater the dimer score, the higher the probability of dimer formation, the greater the absolute value of the Gibbs free energy, the more stable the dimer, and the loss function is used to correct each Gibbs free energy, and the correction free energy is obtained. Each correction free energy is compared with the correction free energy threshold to construct a correction free energy matrix, and the correction free energy combines the influence of non-canonical base pairing, and the greater the value, the higher the probability of dimer formation, and the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the correction free energy matrix is ​​used to determine the target primer set. The present application effectively reduces the formation of primer dimers through multi-dimensional evaluation and screening, thereby reducing the false positive rate. First, by calculating the dimer score of each pair of primers and constructing a dimer score matrix, primer pairs with scores below the threshold were screened out, and primer combinations that were prone to dimer formation were directly excluded. Secondly, the Gibbs free energy matrix was used for further screening. The larger the absolute value of the Gibbs free energy, the more stable the dimer. By comparing with the threshold, primer pairs with excessively large absolute values ​​of free energy were excluded. Finally, the Gibbs free energy was corrected by the loss function to obtain the corrected free energy matrix. The corrected free energy combined the influence of non-canonical base pairing, further accurately evaluated the probability of dimer formation, and screened out better primer pairs again. The target primer set was finally determined by the intersection of the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix. After multiple rounds of rigorous screening, the probability of primer dimer formation was minimized, thereby effectively reducing the occurrence of false positive results caused by primer dimers, thereby reducing the probability of false positive rate.

[0030] The method provided in the embodiment of the present application may be executed by software on a terminal device, which may be, for example, a computer or other device.

[0031] In order to enable people in the technical field to better understand the present application scheme, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. The following is an example of the method provided in the embodiment of the present application being executed by a computing device. This embodiment can be referred to as embodiment 1. Figure 1 The flowchart provided in Example 1. Figure 1 As shown, the method includes: S101, obtaining a target template sequence.

[0032] The computing device can obtain a target template sequence, which may also be called a target nucleic acid sequence, that is, a DNA or cDNA sequence that needs to be amplified or detected.

[0033] Specifically, the computing device can collect specific gene sequences from literature or use "species-specific" genomic regions as templates.

[0034] For example, in the embodiment of the present application, 32 target template sequences can be collected as an example, and the relevant information of the target template sequences can be collected at the same time, including: the corresponding sequence name (Template_Sequences_ID), species name (Species_Name), custom species abbreviation name (Abbreviation), species classification identifier (Taxid), gene name (Gene) corresponding to the template sequence, and these target template sequences and related information are stored as an Excel table. For example, the relevant information and format of the first 10 template sequences among the 32 target template sequences can be as shown in Tables 1-1 to 1-2: Table 1-1

[0035] Table 1-2

[0036] S102, designing an initial primer set according to the target template sequence.

[0037] The computing device can design an initial set of primers based on the target template sequence.

[0038] Specifically, the computing device can take 32 target template sequences as input and design an initial primer set for each template. The parameters in the primer design step can be: the temperature can be 55-65°C, the primer size can be 18-30bp, the GC content (the ratio of guanine (G) and cytosine (C) base pairs in the nucleic acid sequence) can be 45-55%, and the amplification product size range can be 130bp to 230bp.

[0039] In addition, thermodynamic parameters are preset, for example, the maximum self-complementarity can be set to 8.0 kcal / mol and the maximum 3' end self-complementarity can be set to 3.0 kcal / mol.

[0040] For each target template sequence, a primer set of 10 to 12 candidates can be designed and output. Use the information in Table 1 to define the primer name for each primer and supplement related information. The related information may include TaxID (species classification identifier), Sciname (species scientific name), PrimerIndex (primer name), ForwardID (Forward primer ID), ForwardSeq (Forward primer sequence), ReverseID (Reverse primer ID) and ReverseSeq (Reverse primer sequence), etc. Finally, 280 primer pairs for 17 species can be obtained (here, 32 target template sequences are taken as an example). The first 20 primer pairs output can be shown in Tables 2-1 to 2-3, where each row can represent a pair of primers. (Only the results of the first two genes are selected for display): Table 2-1

[0041] Table 2-2

[0042] Table 2-3

[0043] S103, determining the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and constructing a dimer score matrix by comparing each dimer score with a score threshold, and constructing a Gibbs free energy matrix by comparing each Gibbs free energy with a Gibbs free energy threshold.

[0044] After the initial primer set is designed in step S102, Figure 2 As shown, the computing device can determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and construct a dimer score matrix by comparing each dimer score with a score threshold, and construct a Gibbs free energy matrix by comparing each of the Gibbs free energy with a Gibbs free energy threshold.

[0045] Specifically, the higher the dimer score of the initial primer, the more likely it is to form a dimer. The obtained dimer score is compared with the score threshold set in advance through the experiment, and then the initial primer pairs greater than the score threshold can be marked as 1, and the initial primer pairs less than or equal to the score threshold can be marked as 0. The relationship between the dimer score and the score threshold of each initial primer pair in the initial primer set is compared in turn, so as to construct a dimer score matrix according to the label, and then the initial primer pairs marked as 1 in the filtering results can be screened based on the dimer score matrix to obtain a candidate primer set S1 that meets the dimer score matrix.

[0046] The more negative the Gibbs free energy value (the higher the absolute value), the more stable the dimer is, and the more likely the dimer is to be formed. The same principle can be used to compare the Gibbs free energy obtained for each initial primer pair with the Gibbs free energy threshold value pre-set in the experiment. The initial primer pairs with a value greater than the Gibbs free energy threshold value are marked as 1, and the initial primer pairs with a value less than or equal to the Gibbs free energy threshold value are marked as 0. The relationship between the Gibbs free energy and the Gibbs free energy threshold value of all initial primers in the initial primer set is compared in turn, and the Gibbs free energy matrix is ​​obtained based on the markings. Then, the initial primer pairs marked as 1 can be filtered out based on the Gibbs free energy matrix to obtain the candidate primer set S2 of the Gibbs free energy matrix.

[0047] S104, using a loss function to correct each of the Gibbs free energies to obtain a corrected free energy, and comparing each of the corrected free energies with a corrected free energy threshold to construct a corrected free energy matrix.

[0048] The computing device can use the loss function to correct each Gibbs free energy to obtain the corrected free energy, and construct a corrected free energy matrix by comparing each corrected free energy with a corrected free energy threshold.

[0049] The purpose of using the loss function to correct the Gibbs free energy is to more accurately evaluate the tendency and stability of primer dimer formation. Although Gibbs free energy is an important indicator for measuring the stability of primer dimers, its original value alone may not fully reflect the actual dimer formation, because the actual primer dimer formation is also affected by factors such as non-canonical base pairing and local sequence structure. By introducing the loss function, the influence of these complex factors can be taken into consideration, and the Gibbs free energy can be adjusted and corrected to obtain a corrected free energy that is closer to the actual situation. The corrected free energy can more accurately reflect the probability and stability of primer dimer formation, and then by comparing with the corrected free energy threshold, more accurately screen out those primer pairs that are not easy to form dimers, thereby effectively reducing the probability of nonspecific amplification and false positive results caused by primer dimers.

[0050] Specifically, taking a pair of initial primers including a P1 primer and a P2 primer as an example, the loss function is a function for calculating the primer-dimer formation, based on the Gibbs free energy between the primers and the distance between the subsequence matched by each primer and the 3' end of the primer. The calculation formula is as follows:

[0051] Where d1 and d2 represent the distance from the matching subsequence to the 3' end of the p1 primer and the p2 primer, respectively, in base numbers. The absolute value of the Gibbs free energy (i.e., the numerator of the formula) is used for calculation in the formula. Since different matching subsequences will lead to different combinations of d1 and d2, different loss functions will be calculated. Finally, the minimum value of these loss functions is taken as the correction free energy, i.e., correction free energy=min(lossfunction), where "min" is a function used to find the minimum value from a set of values.

[0052] The level of correction free energy is closely related to the possibility of primer dimer formation. The higher the correction free energy, the greater the probability of primer dimer formation; conversely, the lower the correction free energy, the smaller the probability of primer dimer formation. Therefore, when the loss function reaches the minimum value, it can be used to determine whether the P1 primer and the P2 primer constitute a true primer pair.

[0053] It should be noted that for each pair of primers containing a matching subsequence, the larger the loss function, the more likely it is that a dimer will be formed. The correction free energy is compared with the loss function threshold value pre-set for the experiment, and the initial primer pairs greater than the correction free threshold value are marked as 1, and the initial primer pairs less than or equal to the correction free threshold value are marked as 0. The relationship between the correction free energy and the correction free threshold value of all initial primer pairs in the initial primer set is compared in turn to obtain a correction free energy matrix. Finally, the primer combination marked as 1 in the filter result can be screened based on the correction free energy matrix to obtain a candidate primer set S3 that satisfies the correction free energy matrix.

[0054] S105, determining a target primer set using the intersection of the dimer score matrix, the Gibbs free energy matrix, and the initial primer pairs in the corrected free energy matrix.

[0055] The computing device may determine a target primer set using the intersection of the dimer score matrix, the Gibbs free energy matrix, and the initial primer pairs in the corrected free energy matrix.

[0056] Specifically, the computing device can screen compatible primer combinations based on the three matrices constructed in the above steps, and each matrix can be an n×n 01 symmetric matrix, where n is the total number of initial primer pairs generated in the primer design step, and the primer pair with a value of 1 indicates that the more stable the dimer binding is, the higher the probability of primers binding to each other in PCR, resulting in a higher risk of amplification efficiency reduction or failure, that is, the two primer sets are incompatible, and the primer pair with a value of 0 indicates that the more unstable the dimer binding is, the lower the probability of primers binding to each other in PCR, the higher the amplification efficiency or the higher the probability of success, that is, this primer pair can coexist. Therefore, based on the score matrix, the primer combination marked as 1 in the filtering result is screened to obtain a candidate primer set S1 that satisfies the dimer scoring matrix; based on the Gibbs free energy matrix, the primer combination marked as 1 in the filtering result is screened to obtain a candidate primer set S2 that satisfies the Gibbs free energy matrix; based on the corrected free energy matrix, the primer combination marked as 1 in the filtering result is screened to obtain a candidate primer set S3 that satisfies the corrected free energy matrix. The intersection of three matrices can be extracted from the data sets `S1`, `S2` and `S3`, that is, the above matrices determine the compatibility between primer pairs in turn. When there is a primer pair that is compatible between all matrices, it is considered that they can coexist, and finally a primer set with lower dimer content is obtained, which is determined as the target primer set.

[0057] The embodiment of the present application can obtain the target template sequence, design the initial primer set according to the target template sequence, determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and use each dimer score to compare with the score threshold to construct a dimer score matrix, and use each Gibbs free energy to compare with the Gibbs free energy threshold to construct a Gibbs free energy matrix. Wherein, the greater the dimer score, the higher the probability of dimer formation, the greater the absolute value of the Gibbs free energy, the more stable the dimer, and the loss function is used to correct each of the Gibbs free energy, and the correction free energy is obtained. Each correction free energy is compared with the correction free energy threshold to construct a correction free energy matrix, and the correction free energy combines the influence of non-canonical base pairing, and the greater the value, the higher the probability of dimer formation, and the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the correction free energy matrix is ​​used to determine the target primer set. The present application effectively reduces the formation of primer dimers through multi-dimensional evaluation and screening, thereby reducing the false positive rate. First, by calculating the dimer score of each pair of primers and constructing a dimer score matrix, primer pairs with scores below the threshold were screened out, and primer combinations that were prone to dimer formation were directly excluded. Secondly, the Gibbs free energy matrix was used for further screening. The larger the absolute value of the Gibbs free energy, the more stable the dimer. By comparing with the threshold, primer pairs with excessively large absolute values ​​of free energy were excluded. Finally, the Gibbs free energy was corrected by the loss function to obtain the corrected free energy matrix. The corrected free energy combined the influence of non-canonical base pairing, further accurately evaluated the probability of dimer formation, and screened out better primer pairs again. The target primer set was finally determined by the intersection of the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix. After multiple rounds of rigorous screening, the probability of primer dimer formation was minimized, thereby effectively reducing the occurrence of false positive results caused by primer dimers, thereby reducing the probability of false positive rate.

[0058] In addition, the present application also provides an embodiment, which may be referred to as Embodiment 2. In Embodiment 2, the initial primer set may also be specifically evaluated and a specificity matrix may be established to ensure that the primers can accurately and specifically bind to the target sequence in the PCR reaction, avoiding nonspecific amplification, thereby improving the accuracy and reliability of the experiment.

[0059] Specifically, in this embodiment, the primer pairs in the initial primer set can be compared with the complete nucleic acid database (NT) to evaluate the specificity of the primer pairs and construct a specificity matrix. The specificity evaluation is based on the following two aspects. One is the "single read length" specificity of base extension, and the other is the "species level classification consistency" specificity of the PCR product. For each product produced by the primer combination of p1 and p2 (taking an initial primer pair including P1 primer and P2 primer as an example), there can be two subsequences with a single end read length (for example, 50bp is taken as an example here) that will be used for specificity evaluation. If these subsequences can be identified as a single species, it indicates that the specificity evaluation has been passed. In other words, a pair of effective specific primers can produce one or more different 50bp fragments at the end of the PCR amplification product, and at least one set of 50bp sequences can be identified as a unique microbial species (species level). When p1 and p2 are the same, the initial primer pair can be marked as 0 to indicate that only the subsequence of the single read length (default is 50bp) of the product generated by primer set p1 is used to verify the specificity of p1 and the verification is passed, and marked as 1 to indicate that such a valid subsequence is missing in P1 and P2. In the case that p1 and p2 are different, if the primer pairs p1 and p2 can both maintain a valid single read length subsequence, the primer pair is marked as 0 to indicate that the subsequence of the single read length (default is 50bp) of the product generated by p1 and p2 is used to verify the specificity of p1 and p2 and the verification is passed, and marked as 1 to indicate that the primer pairs p1 and p2 lack a valid single read length subsequence or the specificity verification fails. At this point, the construction of the specificity matrix can be realized by using the markers.

[0060] Subsequently, candidate primer pairs with high specificity are screened, that is, primer combinations marked as 0 through specificity verification are screened based on the specificity matrix to obtain candidate primer set s4 with high specificity.

[0061] Finally, the intersection of the data of the primer set with lower dimer content (the primer set determined according to S1, S2 and S3 in Example 1) and the high-specificity candidate primer set s4 can be extracted to finally obtain a high-specificity co-existing primer data set, which can then be determined as the target primer set.

[0062] The above are some specific implementations of the primer set determination method provided in the embodiments of the present application. Based on this, the present application also provides a corresponding device. The device provided in the embodiments of the present application will be introduced from the perspective of functional modularization. The device and the primer set determination method described above can be referenced to each other.

[0063] Figure 3 The structural block diagram of the primer set determination device provided in the embodiment of the present invention is referred to as specific implementation mode three, and reference is made to Figure 3 The device may include: An acquisition unit 300 is used to acquire a target template sequence; A design unit 310 is used to design an initial primer set according to the target template sequence; A first determining unit 320 is used to determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and to construct a dimer score matrix by comparing each dimer score with a score threshold, and to construct a Gibbs free energy matrix by comparing each Gibbs free energy with a Gibbs free energy threshold, wherein a larger dimer score indicates a higher probability of dimer formation, and a larger absolute value of the Gibbs free energy indicates a more stable dimer; A comparison unit 330 is used to correct each of the Gibbs free energies using a loss function to obtain a corrected free energy, and to construct a corrected free energy matrix by comparing each of the corrected free energies with a corrected free energy threshold, wherein the corrected free energy combines the influence of non-canonical base pairing, and a larger value indicates a higher probability of dimer formation; The second determining unit 340 is used to determine a target primer set by using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix.

[0064] Optionally, the first determining unit is specifically configured to compare each of the dimer scores with a score threshold, mark an initial primer pair whose dimer score is greater than the score threshold as 1, and mark an initial primer pair whose dimer score is less than or equal to the score threshold as 0; Construct a dimer score matrix based on the markers.

[0065] Optionally, the first determination unit is specifically used to compare each of the Gibbs free energy with the Gibbs free energy threshold, mark the initial primer pair whose Gibbs free energy is greater than the Gibbs free energy threshold as 1, and mark the initial primer pair whose dimer score is less than or equal to the Gibbs free energy threshold as 0; Construct the Gibbs free energy matrix based on the labeling.

[0066] Optionally, the comparison unit is specifically configured to correct each of the Gibbs free energies using a loss function and a distance from a matching subsequence of each pair of primers to the 3' end of the primers to obtain a corrected free energy.

[0067] Optionally, the comparison unit is specifically used to compare the loss function value corresponding to each of the correction free energies with the correction free energy threshold, marking the initial primer pair whose correction free energy is greater than the correction free energy threshold as 1, and marking the initial primer pair whose correction free energy is less than or equal to the correction free energy threshold as 0; Construct a corrected free energy matrix based on the labeling.

[0068] Optionally, the device further comprises a marking unit, which is used for marking the primer pair as 0 if the specificity of the P1 primer is verified by using only a subsequence of a single read length of a product generated by the P1 primer and the verification is passed, and marking the primer pair as 1 if the subsequence does not exist or the specificity verification fails, when the P1 primer and the P2 primer are homologous primers; In the case where the P1 primer and the P2 primer are different primer combinations, if both the P1 primer and the P2 primer have a single read length subsequence, the primer pair is marked as 0; if one of the P1 primer and the P2 primer does not have a single read length subsequence or fails the specificity verification, the primer pair is marked as 1; Construct a specificity matrix based on the markers.

[0069] The second determination unit is specifically used to determine the target primer set by using the intersection of the primer pairs in the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix and the specificity matrix.

[0070] Optionally, the second determining unit is specifically configured to screen the primer pairs whose filtering results are marked as 1 according to the dimer score matrix to obtain a candidate primer set S1 that satisfies the score matrix; The primer pairs marked as 1 in the screening and filtering results are selected according to the Gibbs free energy matrix to obtain a candidate primer set S2 of the Gibbs free energy matrix; The primer pairs marked as 1 in the filter results are screened according to the correction free energy matrix to obtain a candidate primer set S3 that satisfies the correction free energy matrix; Extract the intersection of primer pairs from S1, S2 and S3 to obtain the coexisting candidate primer set of dimers; The primer pairs marked as 0 after specificity verification were screened out according to the specificity matrix to obtain a candidate primer set S4 with high specificity; The target primer set is determined according to the intersection of the primer pairs extracted from the co-existing candidate primer set of the dimer and the high-specificity candidate primer set S4.

[0071] The embodiments of the present application also provide corresponding devices and computer storage media for implementing the solutions provided by the embodiments of the present application.

[0072] The device includes a memory and a processor, the memory is used to store instructions or codes, and the processor is used to execute the instructions or codes so that the device executes the method described in any embodiment of the present application.

[0073] The computer storage medium stores codes, and when the codes are executed, a device executing the codes implements the method described in any embodiment of the present application.

[0074] The "first" and "second" in the names such as "first" and "second" (if any) mentioned in the embodiments of the present application are only used as name identifiers and do not represent the first or second in order.

[0075] Through the description of the above implementation methods, it can be known that those skilled in the art can clearly understand that all or part of the steps in the above-mentioned embodiment method can be implemented by means of software plus a general hardware platform. Based on such an understanding, the technical solution of the present application can be embodied in the form of a software product, and the computer software product can be stored in a storage medium, such as a read-only memory (ROM) / RAM, a disk, an optical disk, etc., including a number of instructions for a computer device (which can be a personal computer, a server, or a network communication device such as a router) to execute the methods described in each embodiment of the present application or some parts of the embodiments.

[0076] Each embodiment in this specification is described in a progressive manner, and the same and similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the device embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment. Some or all of the modules can be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Ordinary technicians in this field can understand and implement it without paying creative work.

[0077] The above description is merely an exemplary embodiment of the present application and is not intended to limit the protection scope of the present application.

Claims

1. A method for determining a primer set, characterized in that: include: Obtaining a target template sequence; designing an initial primer set according to the target template sequence; Determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and compare each dimer score with a score threshold to construct a dimer score matrix, and compare each Gibbs free energy with a Gibbs free energy threshold to construct a Gibbs free energy matrix, wherein a larger dimer score indicates a higher probability of dimer formation, and a larger absolute value of the Gibbs free energy indicates a more stable dimer; Using a loss function to correct each of the Gibbs free energies to obtain a corrected free energy, and using each of the corrected free energies to compare with a corrected free energy threshold to construct a corrected free energy matrix, wherein the corrected free energy combines the influence of non-canonical base pairing, and a larger value indicates a higher probability of dimer formation; The target primer set is determined using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix.

2. The method according to claim 1, characterized in that The step of comparing each dimer score with a score threshold to construct a dimer score matrix comprises: Comparing each of the dimer scores with a score threshold, marking an initial primer pair whose dimer score is greater than the score threshold as 1, and marking an initial primer pair whose dimer score is less than or equal to the score threshold as 0; Construct a dimer score matrix based on the markers.

3. The method according to claim 1, characterized in that: The method of constructing a Gibbs free energy matrix by comparing each of the Gibbs free energies with a Gibbs free energy threshold value comprises: Using each of the Gibbs free energies to compare with a Gibbs free energy threshold, marking an initial primer pair whose Gibbs free energy is greater than the Gibbs free energy threshold as 1, and marking an initial primer pair whose dimer score is less than or equal to the Gibbs free energy threshold as 0; Construct the Gibbs free energy matrix based on the labeling.

4. The method according to claim 1, characterized in that: The method of correcting each of the Gibbs free energies using a loss function to obtain a corrected free energy comprises: Each of the Gibbs free energies is corrected using a loss function and the distance from the matching subsequence of each pair of primers to the 3' end of the primer to obtain a corrected free energy.

5. The method according to claim 4, characterized in that The method of comparing each of the corrected free energies with a corrected free energy threshold to correct the free energy matrix comprises: Using each of the correction free energies to compare with the correction free energy threshold, marking the initial primer pairs whose correction free energy is greater than the correction free energy threshold as 1, and marking the initial primer pairs whose correction free energy is less than or equal to the correction free energy threshold as 0; Construct a corrected free energy matrix based on the labeling.

6. The method according to claim 1, characterized in that Each pair of primers includes a P1 primer and a P2 primer, and the method further includes: In the case where the P1 primer and the P2 primer are homologous primers, if the specificity of the P1 primer is verified using only a subsequence of a single read length of the product generated by the P1 primer and the verification is passed, the primer pair is marked as 0; if the subsequence does not exist or the specificity verification fails, the primer pair is marked as 1; In the case where the P1 primer and the P2 primer are different primer combinations, if both the P1 primer and the P2 primer have a single read length subsequence, the primer pair is marked as 0; if one of the P1 primer and the P2 primer does not have a single read length subsequence or fails the specificity verification, the primer pair is marked as 1; Construct a specificity matrix based on the markers; The method of determining a target primer set by using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix, and the corrected free energy matrix comprises: The target primer set is determined using the intersection of the primer pairs in the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix, and the specificity matrix.

7. The method according to claim 6, characterized in that The method of determining a target primer set by using the intersection of the primer pairs in the dimer score matrix, the Gibbs free energy matrix, the corrected free energy matrix, and the specificity matrix comprises: The primer pairs marked as 1 in the filter results are screened according to the dimer score matrix to obtain a candidate primer set S1 that satisfies the score matrix; The primer pairs marked as 1 in the screening and filtering results are selected according to the Gibbs free energy matrix to obtain a candidate primer set S2 of the Gibbs free energy matrix; The primer pairs marked as 1 in the filter results are screened according to the correction free energy matrix to obtain a candidate primer set S3 that satisfies the correction free energy matrix; Extract the intersection of primer pairs from S1, S2 and S3 to obtain the coexisting candidate primer set of dimers; The primer pairs marked as 0 after specificity verification were screened out according to the specificity matrix to obtain a candidate primer set S4 with high specificity; The target primer set is determined according to the intersection of the primer pairs extracted from the co-existing candidate primer set of the dimer and the high-specificity candidate primer set S4.

8. A primer set determination device, characterized in that: The device comprises: An acquisition unit, used for acquiring a target template sequence; A design unit, used to design an initial primer set according to the target template sequence; A first determination unit is used to determine the dimer score and Gibbs free energy of each pair of initial primers in the initial primer set, and to construct a dimer score matrix by comparing each dimer score with a score threshold, and to construct a Gibbs free energy matrix by comparing each Gibbs free energy with a Gibbs free energy threshold, wherein a larger dimer score indicates a higher probability of dimer formation, and a larger absolute value of the Gibbs free energy indicates a more stable dimer; A comparison unit, used to correct each of the Gibbs free energies using a loss function to obtain a corrected free energy, and to construct a corrected free energy matrix by comparing each of the corrected free energies with a corrected free energy threshold, wherein the corrected free energy combines the influence of non-canonical base pairing, and a larger value indicates a higher probability of dimer formation; The second determination unit is used to determine the target primer set by using the intersection of the initial primer pairs in the dimer score matrix, the Gibbs free energy matrix and the corrected free energy matrix.

9. The device according to claim 8, characterized in that The first determining unit is specifically configured to compare each of the dimer scores with a score threshold, mark an initial primer pair whose dimer score is greater than the score threshold as 1, and mark an initial primer pair whose dimer score is less than or equal to the score threshold as 0; Construct a dimer score matrix based on the markers.

10. The device according to claim 8, characterized in that The first determination unit is specifically used to compare each of the Gibbs free energies with a Gibbs free energy threshold, mark an initial primer pair whose Gibbs free energy is greater than the Gibbs free energy threshold as 1, and mark an initial primer pair whose dimer score is less than or equal to the Gibbs free energy threshold as 0; Construct the Gibbs free energy matrix based on the labeling.

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