A panel-in-primer compatibility screening method, system and storage medium

Abstract

The application discloses a panel inner primer compatibility screening method and system and a storage medium. The method comprises the following steps: combining a plurality of primer pools into a primer pool to be sequenced, and sequencing the primer pool according to primer length and compatibility degree; and performing primer comparison in a multi-process sliding window comparison mode. In order to speed up the comparison process, a hash algorithm is added. The complementary pairing length, the pairing region GC content, the free energy and the annealing temperature required for complementary pairing are used as the compatibility evaluation factors, the weighted scores of various factors are calculated, the primer dimer information is recorded, and the result is output. Compared with the existing primer compatibility screening method, as the scale of the primer pool increases, the time and memory required for comparison will increase exponentially, and it is too dependent on manual comparison by researchers. The application can accurately output high-risk primer pairs in a short time. The application provides another way for manufacturing a virus panel.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a panel internal primer compatibility screening method, a system and a storage medium. BACKGROUND

[0002] High-throughput sequencing technology, also known as “next-generation” sequencing technology, has the advantages of fast speed, wide detection range, high accuracy and high sensitivity. For example, metagenomic sequencing technology adopts a shotgun method to sequence all nucleic acids in a clinical specimen by breaking them into small fragments of 500 bp in length, and then assembles and annotates the results; DNA chip technology uses hybridization technology to fix the designed probes on the chip for incubation, so that the target fragments and probes are combined, and then the target sequence is identified or sequenced.

[0003] However, due to the overwhelming influence of human genome pollution and environmental microbial pollution, metagenomic sequencing or DNA chip hybridization requires a large number of clinical samples, and the data output is extremely large, and the interpretation is difficult. Targeted sequencing (Target region sequencing, tNGS) is also called target region sequencing, which is a technical means of using PCR or probe hybridization to capture and enrich the genomic region and perform high-throughput sequencing, which can detect genetic variation sites in the target genomic region and obtain variation information of the specified target region. Compared with first-generation sequencing, whole-genome sequencing and whole-exome sequencing, targeted sequencing can obtain deeper coverage and higher data accuracy, improve the detection efficiency of the target region, shorten the research period, reduce the sequencing cost, and is suitable for research on a large number of samples, which helps to find and verify disease-related candidate genes or related sites, and has great application potential in clinical diagnosis and drug development.

[0004] Panel refers to not only detecting one site, one gene, but also simultaneously detecting multiple sites and multiple genes, and these sites and genes need to be selected and combined according to a standard to form a detection panel. Panel is the application of targeted sequencing in diagnosis. The advantage of panel is its high efficiency, accuracy and rapidity. Compared with traditional detection methods such as culture and serological detection, panel can provide more accurate results in a shorter time. Panel can give accurate test results with only a small amount of sample, and the required data volume is small, which not only reduces the difficulty of data interpretation, but also has a lower requirement for the order of magnitude of data output, and the required data volume is only about 10M, which is only one percent of metagenomics.

[0005] However, the difficulty of panel production lies in the primer pool used, a good primer pool can not only reduce the number of operations, but also should have high accuracy and specificity for target sequence detection. At present, there are a considerable number of primer design software such as Primer3, but there is little research on how to construct a primer pool. Compatibility analysis of most primer pools is still based on empiricism, such as whether the primer ends match, how long the matching length is, etc., which cannot effectively guide the researchers. SUMMARY

[0006] To solve the above problems, the application provides a panel internal primer compatibility screening method, system and storage medium, which quantifies each important parameter of primer dimer through a large number of experiments to form a scoring mechanism, and can realize rapid batch dimer filtering, and the dimer filtering of 1000 pairs of primers only needs 2s.

[0007] To achieve the technical purpose of the application, on the one hand, the application provides a panel internal primer compatibility screening method, which comprises: merging a panel containing a plurality of primer pools to obtain a primer pool to be sorted; converting the primer pool to be sorted into a format based on text for representing nucleic acid sequences, and then sorting to obtain a sorted primer pool; comparing primers in a multi-process sliding window manner to evaluate primer pair compatibility and calculate a weighted score.

[0008] The factors for evaluating the compatibility of the primer pair include complementary pairing length, GC content of the pairing region, free energy and annealing temperature required for complementary pairing; according to the compatibility evaluation result, the primer dimer information is recorded, and the result is output.

[0009] The application does not make specific limitations on the source of the plurality of primer pools, and those skilled in the art can obtain them by using conventional software design, directly using conservative regions, user preferences, etc.

[0010] Further, in the method provided by the application, the plurality of primer pools are merged based on as many captured target sequences as possible, and the primers in the merged primer pool are sorted according to primer length and compatibility degree in the fasta format for the primer pool to be sorted, to obtain a sorted primer pool.

[0011] Further, in the method provided by the application, the multi-process sliding window comparison first defines a process pool, the number of processes in the process pool is n, and the application does not make specific limitations on n, which can be adjusted by those skilled in the art as needed. The first n primers in the sorted primer pool are selected, and each primer is compared with itself and the primers behind it in the order. After completing a comparison, the primers are supplemented to the process pool in order until all primer comparisons are completed.

[0012] Further, in the method provided by the application, a hash algorithm is introduced in the multi-process sliding window comparison to record the k-mers that have appeared, and if the newly supplemented primer contains a recorded k-mer, the recorded result is directly extracted and the comparison is skipped.

[0013] Further, in the method provided by the application, the compatibility evaluation factors of the primer pair include the complementary pairing length, the GC content of the pairing region, the energy released by the pairing of the pairing region, and the annealing temperature required for the complementary pairing. The application determines whether the primer pair is compatible by taking the threshold value corresponding to each factor as an index, determines that the primer pair is incompatible if any factor is incompatible, records the primer pair that is incompatible and skips it, and calculates the weighted score of the primer pair that is not incompatible after weighting and scoring, and considers the primer pair that exceeds the weighted score threshold as compatible and outputs it.

[0014] Specifically, in the method provided by the application, the complementary pairing length is determined by regular matching, the weight score is the square of the complementary pairing length, and the weight is 1; the weight score of the GC content of the pairing region is the square of the GC content of the pairing region, and the weight is 1; a first threshold value is set, and the product of the complementary pairing length and the GC content of the pairing region is compared with the first threshold value, and it is considered incompatible if it exceeds the first threshold value.

[0015] Specifically, in the method provided by the application, the free energy calculation adopts a contribution model of base pairing and base stacking for double-strand formation, which is based on a large amount of experimental data and obtains a result more consistent with the actual situation. The greater the free energy, the lower the compatibility; a second threshold value is set, and the calculated free energy is compared with the second threshold value, and it is considered incompatible if it is lower than the second threshold value; if it exceeds the second threshold value, the weight is -1, and the weight score is the numerical value of the free energy calculated according to the model.

[0016] Specifically, in the method provided by the application, the calculation model of the annealing temperature required for the complementary pairing is:

[0017] ΔG=ΔH-T×ΔS×lnC

[0018] In the formula, ΔG is the Gibbs free energy change; ΔH is the enthalpy change; ΔS is the entropy change; T is the annealing temperature required for the complementary pairing; and C is the primer concentration.

[0019] The higher the annealing temperature required for the complementary pairing, the higher the incompatibility; a third threshold value is set, and the calculated annealing temperature required for the complementary pairing is compared with the third threshold value, and it is considered incompatible if it exceeds the third threshold value; if it is lower than the third threshold value, the weight is 1, and the weight score is the numerical value of the annealing temperature required for the complementary pairing obtained by the calculation model of the annealing temperature.

[0020] Further, in the method provided by the application, the calculation method of the weighted score is:

[0021] The weighted score is set as the square value of the complementary pairing length multiplied by the square value of the GC content of the pairing region / (-1x free energy x annealing temperature required for complementary pairing), a threshold value of the weighted score is set, and the incompatible primer pair is not compatible when the weighted score is lower than the threshold value, and the high-risk primer pair is output when the weighted score is higher than the threshold value.

[0022] The first threshold value, the second threshold value, the third threshold value and the threshold value of the weighted score are not specifically limited in the present application, and can be freely set by those skilled in the art according to needs.

[0023] Further, in the method provided by the present application, the finDimer software outputs all recorded incompatible primer pairs, i.e. primer dimers, so as to facilitate researchers to replace or exclude them.

[0024] Further, the method provided by the present application can quickly and accurately filter out incompatible primer pairs, and improve the efficiency and precision of primer pool design.

[0025] On the other hand, the present application claims a system for screening primer compatibility in a panel, comprising: a primer pool merging module for merging a plurality of primer pools into a primer pool to be sorted; a primer sorting module for converting the primer pool to be sorted into a format based on text for representing nucleic acid sequences, and then sorting according to primer length and compatibility degree to obtain a sorted primer pool; a primer comparison module for comparing primer sequences to be compared through multi-process sliding window comparison, adding a hash algorithm to further speed up the comparison speed, evaluating the compatibility of primer pairs according to complementary pairing length, pairing region GC content, free energy and annealing temperature required for complementary pairing, judging the compatibility of primer pairs, recording primer dimer information, i.e. incompatible primer pairs, after weighted score calculation; a result output module for outputting recorded incompatible primer pairs and outputting high-risk primer pairs.

[0026] In addition, the present application claims the application of the method for screening primer compatibility in a panel in high-throughput sequencing technology, specifically targeted sequencing technology.

[0027] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages:

[0028] The method for detecting compatibility of primers in a disease panel provided by the application can quickly and accurately evaluate the compatibility between primer pairs, so as to filter out incompatible primer pairs, improve the efficiency and precision of the primer pool. Compared with the existing software mfeprimer, the running time of finDimer is less than 1% of the existing software, and the dimer filtering of 1000 primer pairs by the finDimer software only needs 2s, which can realize rapid batch dimer filtering; the optimized primer pool is only left with 17 primer pairs, which greatly reduces the error of primer compatibility determination, and the output primer pair risk is higher. The application simplifies the complexity of comparison through multi-process sliding window comparison, and can realize rapid comparison; at the same time, the hash algorithm is introduced in the multi-process sliding window comparison, and the k-mers that have appeared are recorded, once the new comparison object contains the recorded k-mer, the recorded result can be directly extracted and skipped, further improving the comparison speed.

[0029] The method for detecting compatibility of primers in a disease panel provided by the application has high accuracy for target sequences, and experimental verification shows that the dimer content of the primer pool in the second-generation library is about 5%, indicating that the accuracy of the results is high. The compatibility evaluation of the primer includes using the base pairing and base stacking contribution model for double-stranded formation as a free energy calculation model, compared with the adjacent base model used by Primer3, the experimental data obtained by the application is more consistent with the actual situation. The compatibility evaluation of the primer includes using the Gibbs free energy change, enthalpy change and entropy change according to the annealing temperature calculation model required by complementary pairing to obtain the annealing temperature required by complementary pairing, wherein the free energy uses a calculation model with higher accuracy, and the enthalpy change and entropy change are obtained by experiment, so the annealing temperature required by complementary pairing is more accurate.

[0030] The method for detecting compatibility of primers in a disease panel provided by the application retains more primer pairs, and the results of the existing software almost exclude all primer pairs, which is very unfriendly to the experimental end, while finDimer retains primer pairs as much as possible to facilitate researchers to replace or exclude. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description only some embodiments of the application.

[0032] Figure 1 The flowchart for making a disease panel for the finDimer software.

[0033] Figure 2Comparison chart of finDimer software and existing software mfeprimer for making disease panel. A is a comparison chart of running time of finDimer software and existing software mfeprimer for making disease panel; B is a comparison chart of the number of incompatible primers detected by finDimer software and existing software mfeprimer for making disease panel. DETAILED DESCRIPTION

[0034] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0035] Example 1

[0036] The present embodiment provides a method for screening primer compatibility in a panel, which specifically comprises the following steps:

[0037] The plurality of primer pools designed by conventional software are combined, and the combined primer pool is sorted according to the fasta format, with primer length and compatibility degree as the sorting basis. The shorter the primer length, the higher the compatibility degree, and the closer to the front in the sorting.

[0038] Multi-process sliding window comparison: define a process pool of 20 processes, select the first 20 primers in the sorted primer pool, and compare and evaluate the compatibility of each primer only with itself and the primers behind it in the order. After each comparison, the primers are supplemented into the process pool in order. In the process of primer comparison, a hash algorithm is introduced to record the k-mers that have appeared. If the new comparison contains the recorded k-mer, the recorded result can be directly extracted and skipped. The complementary pairing length, the GC content of the paired region, the free energy and the annealing temperature required for complementary pairing are used as factors to evaluate the compatibility of primers.

[0039] Wherein, the complementary pairing length is determined by the regular matching, the weight score is the square of the complementary pairing length, and the weight is 1; the pairing region GC content weight score is the square of the pairing region GC content, and the weight is 1; the first threshold value is 1000, and the product of the complementary pairing length and the pairing region GC content is compared with the first threshold value, and if it exceeds 1000, it is considered incompatible. Wherein, the calculation model of the free energy is a model of the contribution of base pairing and base stacking to the formation of double strands, and the greater the free energy, the higher the weight score; the second threshold value is set to -5, and the free energy calculation result is compared with the second threshold value, and if it is lower than -5, it is considered incompatible, and if it is higher than -5, the weight is -1, and the weight score is the free energy calculation result. Wherein, the calculation model of the annealing temperature required for complementary pairing is: ΔG = ΔH - T x ΔS x lnC, the annealing temperature required for complementary pairing is positively correlated with the primer concentration, and the higher the annealing temperature required for complementary pairing, the higher the incompatibility; the third threshold value is set to 15℃, and the annealing temperature required for complementary pairing calculated is compared with the third threshold value, and if it exceeds 15℃, it is considered incompatible, and if it is lower than 15℃, the weight is 1, and the weight score is the annealing temperature required for complementary pairing calculated.

[0040] When any of the above compatibility evaluation factors is incompatible, the primer pair information is recorded and skipped, and the non-incompatible primer pair is calculated for the weighted score, the weighted score = (the square value of the complementary pairing length) x (the square value of the pairing region GC content) / (-1 x the free energy x the annealing temperature required for complementary pairing), the threshold value of the weighted score is 3, and if it is lower than the threshold value, it is incompatible, and if it exceeds the threshold value, it is output as a high-risk primer pair.

[0041] Output all the recorded primer dimer information, i.e. incompatible primer pairs, and output the high-risk primer pairs.

[0042] The flowchart of the panel-in primer compatibility screening method provided in this embodiment is shown in Figure 1 .

[0043] Embodiment 2

[0044] The software for panel-in primer compatibility screening provided in this embodiment is compared with the existing software mfeprimer and primer3 for making disease panels.

[0045] The compatibility of a total of 30 viral primer pools, including influenza A, enterovirus, HIV, parainfluenza virus, norovirus, etc., is screened, and the compatibility is specifically screened by finDimer software and mfeprimer software. In this embodiment, the compatibility screening results of 1000 primer pairs are obtained.

[0046] The comparison results of finDimer software and the existing software mfeprimer are as follows Figure 2The high-risk primer pairs output by the finDimer software are shown in Tables 1 and 2.

[0047] The primer dimer contents obtained by the finDimer software and the existing software mfeprimer in 10 batches of experiments are shown in Table 3.

[0048] The annealing temperatures required for 1000 pairs of primer complementary pairing obtained by the finDimer software and the existing software primer3 are shown in Table 4, wherein the annealing temperatures required for twenty pairs of primer complementary pairing are shown in Table 4.

[0049] Table 1: High-risk primer pairs output by the finDimer

[0050]

[0051]

[0052] Table 2: High-risk primer pairs output by the finDimer

[0053]

[0054]

[0055] Table 3: Dimer content results obtained in 10 batches of experiments

[0056]

[0057] Note: JK is the experimental batch.

[0058] Table 4: Annealing temperatures required for complementary pairing of twenty primers obtained by the finDimer software and the existing software primer3

[0059]

[0060]

[0061] As shown in Tables 1 and 2, the weighted scores of the primer pairs output by the finDimer software are all higher than 3, and the risk of the output primer pairs is relatively high.

[0062] As shown in Table 3, the primer pool dimer content in the second-generation library obtained by the finDimer is about 5%, indicating that the results of the finDimer are more accurate.

[0063] From table 4, the difference between the annealing temperature required for complementary pairing calculated by finDimer and the annealing temperature required for complementary pairing calculated by primer3 is 3-5 DEG C, the annealing temperature required for complementary pairing is calculated by finDimer according to the annealing temperature required for complementary pairing calculation model, the free energy in the calculation model is higher in accuracy, and the enthalpy change and entropy change are obtained by experiment, so the annealing temperature required for complementary pairing obtained by finDimer is more accurate.

[0064] From Figure 2 It can be seen that the software finDimer provided by the application takes less time to make a disease panel, and the number of incompatible primers detected is less. The time taken by finDimer is less than 1% of the time taken by the existing software, and the dimer filtering of 1000 primer pairs only takes 2s; the number of potential incompatible primer pairs screened by the existing software is more than 80 times the number of potential incompatible primer pairs screened by finDimer, greatly increasing the error of primer compatibility determination. After optimization by the existing software mfeprimer, there are still 815 pairs of risk primers left, which are not realistic to replace completely and may still contain actual effective primer pairs. However, after optimization by finDimer, there are only 17 pairs of risk primers left, which facilitates researchers to replace or exclude.

[0065] From the above test results, it can be seen that the software finDimer provided by the application takes less time to make a disease panel than the existing software mfeprimer, and only 17 pairs of risk primers are left after optimization, which greatly reduces the error of primer compatibility determination compared with the 815 pairs of risk primers left after optimization by the existing software mfeprimer, and the risk of the output primer pairs is higher, and it is found that the dimer content in the primer pool obtained by finDimer is only about 5%, which is higher in accuracy.

[0066] As described above, the basic principles, main features and advantages of the application are better described. The above examples and descriptions only describe the preferred embodiments of the application, and the application is not limited by the above examples. Various changes and improvements to the technical solutions of the application made by those skilled in the art without departing from the spirit and scope of the application shall fall within the scope of protection of the application.

Claims

1. A method for screening primer compatibility within a panel, wherein the panel contains multiple primer pools, characterized in that, The primer compatibility screening method includes: The multiple primer pools are merged to obtain primer pools to be sorted; The primers in the primer pool to be sorted are converted into a text-based format for representing nucleic acid sequences, and then sorted according to primer length and degeneracy to obtain a sorted primer pool. The sorted primer pools were compared using a multi-process sliding window comparison method to evaluate primer pair compatibility and calculate weighted scores. The multi-process sliding window comparison first defines a process pool with n processes. The first n primers in the sorted primer pool are selected, and each primer is compared with itself and the primers sorted after it. After a comparison is completed, primers are added to the process pool in order until all primer comparisons are completed. Factors for evaluating primer pair compatibility include complementary pair length, GC content in the pairing region, free energy, and annealing temperature required for complementary pairing. The weighted score is calculated as follows: the square of the complementary pairing length × the square of the GC content in the pairing region / ((-1 × free energy) × annealing temperature required for complementary pairing). Based on the compatibility assessment results, record the primer dimer information and output the results.

2. The panel primer compatibility screening method according to claim 1, characterized in that, A hash algorithm is added to the multi-process sliding window comparison. The hash algorithm is used to record each primer comparison and output the k-mers record. The primer pairs compared in the multi-process sliding window comparison process include those that have been recorded k-mers. The recorded results are directly extracted and the comparison is skipped.

3. The panel primer compatibility screening method according to claim 1, characterized in that, The complementary pairing length is determined by regular matching, the weight score of the complementary pairing length is the square of the complementary pairing length, and the weight of the complementary pairing length is 1; The weight score of the GC content in the paired region is the square of the GC content in the paired region, and the weight of the GC content in the paired region is 1.

4. The panel primer compatibility screening method according to claim 1, characterized in that, The free energy calculation model is a contribution model of base pairing and base stacking to the formation of double strands; The weighted score of the free energy is the free energy value obtained through the calculation model of the free energy; The weight of the free energy is -1.

5. The method according to claim 1, characterized in that, The calculation model for the annealing temperature required for complementary pairing is: ΔG = ΔH - T × ΔS × lnC; where ΔG is the Gibbs free energy change, ΔH is the enthalpy change, ΔS is the entropy change, T is the annealing temperature required for complementary pairing, and C is the primer concentration. The weighted score of the annealing temperature required for complementary pairing is the value of the annealing temperature required for complementary pairing obtained by the calculation model of the annealing temperature required for complementary pairing. The weight of the annealing temperature required for the complementary pairing is 1.

6. The method according to claim 1, characterized in that, The output results include: primer dimer information recorded in the output and high-risk primer pairs.

7. A system for screening primer compatibility within a panel, characterized in that, include: The primer pool merging module is used to merge multiple primer pools into a single primer pool to be sorted. The primer sorting module is used to convert the primer pools to be sorted into a text-based format for representing nucleic acid sequences, and then sort them according to primer length and degeneracy to obtain sorted primer pools. The primer comparison module is used to compare primer sequences to be compared through a multi-process sliding window method, adding a hash algorithm to further accelerate the comparison speed, and combining primer pair compatibility evaluation factors, including: complementary pair length, GC content of the pairing region, free energy and annealing temperature required for complementary pairing, to determine primer pair compatibility, calculate weighted scores, and record primer dimer information. The multi-process sliding window comparison first defines a process pool with n processes. The first n primers in the sorted primer pool are selected, and each primer is compared with itself and the primers sorted after it. After a comparison is completed, primers are added to the process pool in order until all primer comparisons are completed. The weighted score is calculated as follows: the square of the complementary pairing length × the square of the GC content in the pairing region / ((-1 × free energy) × annealing temperature required for complementary pairing). The results output module is used to output the recorded incompatible primer pairs and high-risk primer pairs.

8. A computer storage medium, characterized in that, The computer storage medium stores a plurality of computer instructions, which are used to cause the computer to perform the method described in any one of claims 1 to 6.

9. The application of the method according to any one of claims 1 to 6 in the production of a virus panel.

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