System, method and device for designing specific primers of target species based on information entropy

Abstract

The invention relates to the field of biotechnology primer design, in particular to a target species specific primer design system, method and device based on information entropy. The system comprises a data acquisition module, a data screening module and a data optimization module, the data screening module is used for acquiring short sequence fragment data with identity and coverage greater than or equal to 80%, and the data optimization module is used for processing the short sequence fragment data with identity and coverage greater than or equal to 80%. According to the method, short sequence fragments covering 90% of genomes are screened out, the entropy value of each basic group in the short sequence fragments is obtained, quantitative scoring is conducted on the short sequence fragments according to the entropy values and coverage degree information, the short sequence fragments with the top scores are screened out, the basic groups are replaced with the most conservative basic groups, and the optimized short sequence fragments are obtained. And designing primers based on the short sequence fragments. According to the system, entropy analysis is introduced in the primer design process, and the design quality and application efficiency of primers are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology primer design, and in particular to a system, method and device for designing target species-specific primers based on information entropy. Background Art

[0002] Since its invention in the 1980s, polymerase chain reaction (PCR) has become an important tool in the detection of pathogenic microorganisms. The core advantages of this technology are high sensitivity and specificity, which enable researchers to detect extremely low concentrations of microbial DNA in samples. Therefore, PCR has been widely used in fields such as infectious disease diagnosis, environmental monitoring, and food safety testing. The key to the success of PCR lies in the design of primers, which need to be highly specific to ensure that they only bind to the DNA of the target pathogen. If the binding site of the primer contains a single nucleotide polymorphism (SNP), especially the SNP at the 3' end of the primer, it may cause the primer to bind to the target sequence less efficiently, affecting the amplification efficiency and resulting in false negative results. In addition, amplification product deviation may also lead to an incorrect estimate of the target DNA concentration.

[0003] Primer design usually prioritizes the conserved regions in the target sequence as binding sites to avoid the effects of SNPs as much as possible. However, existing design methods have difficulty in accurately quantifying the effects of SNPs in conserved regions, especially in evaluating the specific effects of each base position in these regions on primer performance. Summary of the invention

[0004] Based on the above technical problems, the present invention innovatively introduces entropy analysis in the primer design process, which significantly improves the design quality and application efficiency of primers and provides a more reliable solution for PCR-related fields.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a system for designing target species-specific primers based on information entropy, the system comprising the following modules: A data acquisition module is used to obtain the genome sequences of the target species and its closely related species; A data screening module is used to process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; The data optimization module is used to process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen out the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the screened short sequence fragments, and quantitatively score the short sequence fragments covering 90% of the genome according to the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; The primer design module is used to design primers based on the optimized short sequence fragment data and ensure that the 3' end of the primer avoids the high entropy value site to obtain candidate primers; The primer quality assessment module is used to perform quality assessment on candidate primers to obtain candidate primers that meet the assessment conditions.

[0006] Preferably, the data acquisition module includes the following units: A first data acquisition unit is used to process all genome sequences of the target species to obtain a reference genome sequence of the target species and other genome sequences of the target species; The second data acquisition unit is used to obtain a reference genome sequence of a closely related species of the target species.

[0007] Preferably, the data screening module includes the following units: A segmentation unit, used to segment the reference genome sequence of the target species into short sequence fragments; The first alignment unit is used to align the short sequence fragments to the reference genome sequence of the closely related species of the target species, and to screen the unaligned short sequence fragments as candidate fragments; The second alignment unit is used to align the candidate fragments to other genome sequences of the target species and select the aligned short sequence fragments as target fragments; The calculation unit is used to calculate the identity and coverage of the target fragment in other genome sequences of the target species, and to select the target fragment with identity and coverage greater than or equal to 80% as the optimized fragment.

[0008] Preferably, the data optimization module includes the following units: The first calculation unit is used to count the coverage of the optimized fragments in all genome sequences of the target species, screen short sequence fragments covering 90% of the genome, and obtain all aligned sequences; An alignment unit is used to perform a multiple sequence alignment on the target fragment sequence screened by the first calculation unit and all aligned sequences in other genomes of the target species, generate a position-aligned alignment sequence, and obtain an alignment matrix; The second calculation unit is used to calculate the entropy value of each base site in the comparison matrix to obtain the entropy value of the optimized fragment; A scoring unit is used to quantitatively score the optimized fragments according to the entropy values ​​of the optimized fragments and the coverage information of the optimized fragments, and select the optimized fragments with the highest scores; The optimization unit is used to optimize the bases in the optimized fragments with the highest scores, replace the bases with the most conservative bases in the multiple sequence alignment, and mark the base sites with high entropy values.

[0009] Preferably, the quality assessment includes one or more of primer dimerization analysis, primer conservation and genome coverage assessment, primer off-target analysis and species specificity assessment of amplification products.

[0010] Preferably, the target species is a pathogenic microorganism species, and the pathogenic microorganism species is bacteria, fungi or viruses.

[0011] More preferably, the pathogenic microbial species is bacteria.

[0012] Further preferably, the pathogenic microorganism species is Streptococcus pneumoniae.

[0013] In a second aspect, the present invention provides a method for designing target species-specific primers based on information entropy, the method comprising the following steps: Obtain the genome sequence of the target species and its closely related species; Process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; Process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the short sequence fragment covering 90% of the genome, and quantitatively score the short sequence fragments covering 90% of the genome based on the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; Primers are designed based on the optimized short sequence fragment data, and the 3' end of the primer is ensured to avoid the high entropy value site to obtain candidate primers; The quality of the candidate primers is evaluated to obtain candidate primers that meet the evaluation criteria.

[0014] Preferably, the target species is a pathogenic microorganism species, and the pathogenic microorganism species is bacteria, fungi or viruses.

[0015] More preferably, the pathogenic microbial species is bacteria.

[0016] Further preferably, the pathogenic microorganism species is Streptococcus pneumoniae.

[0017] In a third aspect, a device for designing target species-specific primers based on information entropy is provided, wherein the device comprises the system as described in the present invention.

[0018] According to a fourth aspect, a readable storage medium is provided, wherein a plurality of computer programs are stored in the readable storage medium, and when the computer programs are run on a processor, the method according to the present invention is executed.

[0019] Compared with the prior art, the beneficial effects of the present invention are: (1) The concept of information entropy introduced in the present invention provides strong support for the selection of conservative regions and primer binding sites. The entropy in the genome of the present invention is a method used to quantify the complexity of sequences or information uncertainty in the genome, which reflects the degree of sequence diversity. In the genome, regions with lower entropy values ​​are often conservative regions. These regions have stable base compositions and low mutation rates, and are suitable as primer binding sites. Regions with higher entropy values ​​are usually more variable, such as non-coding regions or highly variable regions, and are not suitable as binding sites. By analyzing the entropy distribution of genomic sequences, primer binding sites can be screened more scientifically and primer design can be optimized, thereby improving the reliability and accuracy of PCR detection.

[0020] (2) The method for designing target species-specific primers based on information entropy of the present invention pre-calculates the conserved fragments of the target species genome and the entropy value of each base site, evaluates the conservation of the fragments based on information entropy and sorts them, and avoids high entropy sites in primer design, thereby improving the specificity and amplification efficiency of the primers.

[0021] (3) Traditional primer design methods mainly focus on the conservation and specificity of the primer region, but do not effectively evaluate the number of SNPs in the region and the primer mismatches that may be caused. The present invention calculates the entropy value to accurately screen out high SNP regions and avoid them, especially to avoid the 3' end of the primer being located at a high entropy value site.

[0022] (4) If the primer is located at the SNP site and the amplified target sequence does not match the base of the primer at that site, the amplification efficiency will be significantly affected. The present invention effectively reduces the risk of primer mismatch through entropy screening and fragment optimization, ensuring that the designed primers achieve a higher level of conservatism and amplification efficiency.

[0023] (5) The present invention provides a complete and scientific optimization process from genome screening to primer design, evaluation and verification, which can quickly design specific primers with strong conservation and high amplification efficiency.

[0024] In summary, the present invention innovatively introduces entropy analysis in the primer design process, significantly improves the design quality and application efficiency of primers, and provides a more reliable solution for PCR-related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the workflow of a system for designing target species-specific primers based on information entropy provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0027] Example 1 This embodiment provides a system for designing target species-specific primers based on information entropy, and the system includes the following modules: A data acquisition module is used to obtain the genome sequences of the target species and its closely related species; A data screening module is used to process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; The data optimization module is used to process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen out the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the short sequence fragment covering 90% of the genome, and quantitatively score the short sequence fragments covering 90% of the genome according to the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; The primer design module is used to design primers based on the optimized short sequence fragment data and ensure that the 3' end of the primer avoids the high entropy value site to obtain candidate primers; The primer quality assessment module is used to perform quality assessment on candidate primers to obtain candidate primers that meet the assessment conditions.

[0028] In this embodiment, the data acquisition module includes the following units: A first data acquisition unit is used to process all genome sequences of the target species to obtain a reference genome sequence of the target species and other genome sequences of the target species; The second data acquisition unit is used to obtain a reference genome sequence of a closely related species of the target species.

[0029] In this embodiment, the data screening module includes the following units: A segmentation unit, used to segment the reference genome sequence of the target species into short sequence fragments; The first alignment unit is used to align the short sequence fragments to the reference genome sequence of the closely related species of the target species, and to screen the unaligned short sequence fragments as candidate fragments; The second alignment unit is used to align the candidate fragments to other genome sequences of the target species and select the aligned short sequence fragments as target fragments; The calculation unit is used to calculate the identity and coverage of the target fragment in other genome sequences of the target species, and to select the target fragment with identity and coverage greater than or equal to 80% as the optimized fragment.

[0030] In this embodiment, the data optimization module includes the following units: The first calculation unit is used to count the coverage of the optimized fragment in all genome sequences of the target species, screen short sequence fragments with a coverage greater than 90%, and obtain all aligned sequences; An alignment unit is used to perform a multiple sequence alignment on the target fragment sequence screened by the first calculation unit and all aligned sequences in other genomes of the target species, generate a position-aligned alignment sequence, and obtain an alignment matrix; The second calculation unit is used to calculate the entropy value of each base site in the comparison matrix to obtain the entropy value of the optimized fragment; A scoring unit is used to quantitatively score the optimized fragments according to the entropy values ​​of the optimized fragments and the coverage information of the optimized fragments, and select the optimized fragments with the highest scores; The optimization unit is used to optimize the bases in the optimized fragments with the highest scores, replace the bases with the most conservative bases in the multiple sequence alignment, and mark the base sites with high entropy values.

[0031] In this embodiment, the quality assessment includes one or more of primer dimerization analysis, primer conservation and genome coverage assessment, primer off-target analysis, and species specificity assessment of amplified products.

[0032] In this embodiment, the target species is Streptococcus pneumoniae.

[0033] Example 2 This embodiment provides a method for designing target species-specific primers based on information entropy, the method comprising the following steps: Obtain the genome sequence of the target species and its closely related species; Process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; Process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the short sequence fragment covering 90% of the genome, and quantitatively score the short sequence fragments covering 90% of the genome based on the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; Primers are designed based on the optimized short sequence fragment data, and the 3' end of the primer is ensured to avoid the high entropy value site to obtain candidate primers; The quality of the candidate primers is evaluated to obtain candidate primers that meet the evaluation criteria.

[0034] In this example, the target species is Streptococcus pneumoniae.

[0035] Example 3 This embodiment provides a method for designing target species-specific primers based on information entropy, the method comprising the following steps: Obtain genome sequences of the target species and its closely related species.

[0036] On the one hand, all genome sequences of the target species (Streptococcus pneumoniae) were obtained from NCBI's GenBank or RefSeq public genome database, and the reference genome number of Streptococcus pneumoniae was obtained from NCBI's Genome database; if all genome sequences of Streptococcus pneumoniae were more than 1000, 5000 or 10,000, redundancy removal was required, and dRep software was used to remove highly similar genomes with an ANI value greater than 0.995; on the other hand, reference genome sequences of 5 to 10 closely related species of Streptococcus pneumoniae were obtained from GenBank or RefSeq public genome databases.

[0037] Segment and compare closely related species.

[0038] A reference genome sequence of Streptococcus pneumoniae is selected and divided into segments of length n, for example, into short sequence segments of length 50, 100, 200 or 500 bp; these short sequence segments are aligned to the reference genome sequences of their closely related species using an alignment tool (such as bowtie2), and only the short sequence segments that are not aligned to their closely related species are retained.

[0039] Screening for specific fragments of Streptococcus pneumoniae.

[0040] Use blastn and other alignment tools to align the above-retained short sequence fragments to all the redundant genome sequences (other genome sequences) of Streptococcus pneumoniae, and screen out the fragments that meet the following criteria: Identity ≥ 80%, 90% or 95%; Coverage ≥ 80%, 90% or 95%.

[0041] Coverage filtering and multiple sequence alignment were performed.

[0042] According to the screening results in the previous step, the coverage of the fragments in all genome sequences of Streptococcus pneumoniae was counted, and the fragments with genome coverage greater than or equal to 90% were screened out; the sequences of all aligned fragments in the Streptococcus pneumoniae genome were collected, and tools such as Muscle were used to perform multiple sequence alignment on the above-screened target fragment sequences and all aligned sequences in other genomes of the target species to generate position-aligned alignment sequences and obtain an alignment matrix.

[0043] Calculate the entropy values ​​of sites and fragments.

[0044] According to the results of multiple sequence alignment, the entropy value of each base site in the alignment matrix is ​​calculated to measure the uncertainty of the base at that position; a high entropy value indicates that the base variability at that site is large (SNP site), and the risk of primer mismatch at this site is high. The entropy of a single site is defined as follows:

[0045] in, is the frequency of occurrence of bases A, T, C, G and deletion signals, H is the entropy of a single site, i is a natural number from 1 to 5.

[0046] Fragment quality scoring.

[0047] The entropy value of a fragment is defined as the sum of the entropy values ​​of all its sites, and the coverage of the fragment is subtracted from the entropy value of the fragment to obtain the quality score of the fragment; the fragments are sorted based on the quality score, and the optimized fragments with the highest scores are retained as candidate fragments for designing primers.

[0048] Optimize the base sequences of candidate fragments for designing primers.

[0049] Since some sites in the reference genome may not represent the sequences of most of the genome of S. pneumoniae, the bases of the candidate fragments need to be optimized: according to the results of multiple sequence alignment, the bases at each site are replaced with the bases with the highest probability of occurrence (the most conservative bases); sites with entropy values ​​higher than 0.5 are marked so that these high entropy sites can be avoided when designing primers.

[0050] Primer design.

[0051] Primer3 software was used to design primers based on the optimized candidate fragment sequences. The parameter PRIMER_LOWERCASE_MASKING=1 was set to ensure that the primers avoided high entropy sites. Furthermore, the 3' end of the primers avoided high entropy sites. 100-10000 pairs of primers were designed for each optimized candidate fragment to obtain candidate primers.

[0052] Primer quality assessment.

[0053] A comprehensive quality assessment was performed on the designed candidate primers, including the following aspects: primer dimerization analysis, primer conservation and genome coverage assessment, primer off-target analysis, and species specificity assessment of the amplification product. Only candidate primers that met the assessment criteria were retained.

[0054] Experimental verification.

[0055] Qualified candidate primers are evaluated for experimental verification, and finally reliable target species-specific primers are obtained.

[0056] In addition, an embodiment of the present invention also provides a device for designing target species-specific primers based on information entropy, and the device includes the system as described in Example 1 of the present invention.

[0057] Furthermore, an embodiment of the present invention further provides a readable storage medium, wherein the readable storage medium stores a plurality of computer programs, and when the computer programs are run on a processor, the method described in Embodiment 2 of the present invention is executed.

[0058] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0059] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A system for designing target species-specific primers based on information entropy, characterized in that: The system includes the following modules: A data acquisition module is used to obtain the genome sequences of the target species and its closely related species; A data screening module is used to process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; The data optimization module is used to process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen out the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the screened short sequence fragments, and quantitatively score the short sequence fragments covering 90% of the genome according to the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; The primer design module is used to design primers based on the optimized short sequence fragment data and ensure that the 3' end of the primer avoids the high entropy value site to obtain candidate primers; The primer quality assessment module is used to perform quality assessment on candidate primers to obtain candidate primers that meet the assessment conditions.

2. The system according to claim 1, characterized in that The data acquisition module includes the following units: A first data acquisition unit is used to process all genome sequences of the target species to obtain a reference genome sequence of the target species and other genome sequences of the target species; The second data acquisition unit is used to obtain a reference genome sequence of a closely related species of the target species.

3. The system according to claim 1, characterized in that The data screening module includes the following units: A segmentation unit, used to segment the reference genome sequence of the target species into short sequence fragments; The first alignment unit is used to align the short sequence fragments to the reference genome sequence of the closely related species of the target species, and to screen the unaligned short sequence fragments as candidate fragments; The second alignment unit is used to align the candidate fragments to other genome sequences of the target species and select the aligned short sequence fragments as target fragments; The calculation unit is used to calculate the identity and coverage of the target fragment in other genome sequences of the target species, and to select the target fragment with identity and coverage greater than or equal to 80% as the optimized fragment.

4. The system according to claim 1, characterized in that The data optimization module includes the following units: The first calculation unit is used to count the coverage of the optimized fragments in all genome sequences of the target species, screen short sequence fragments covering 90% of the genome, and obtain all aligned sequences; An alignment unit is used to perform a multiple sequence alignment on the target fragment sequence screened by the first calculation unit and all aligned sequences in other genomes of the target species, generate a position-aligned alignment sequence, and obtain an alignment matrix; The second calculation unit is used to calculate the entropy value of each base site in the comparison matrix to obtain the entropy value of the optimized fragment; A scoring unit is used to quantitatively score the optimized fragments according to the entropy values ​​of the optimized fragments and the coverage information of the optimized fragments, and select the optimized fragments with the highest scores; The optimization unit is used to optimize the bases in the optimized fragments with the highest scores, replace the bases with the most conservative bases in the multiple sequence alignment, and mark the base sites with high entropy values.

5. The system according to claim 1, characterized in that The quality assessment includes one or more of primer dimerization analysis, primer conservation and genome coverage assessment, primer off-target analysis and amplification product species specificity assessment.

6. The system according to claim 1, characterized in that The target species is a pathogenic microorganism species, and the pathogenic microorganism species is bacteria, fungi or viruses.

7. A method for designing target species-specific primers based on information entropy, characterized in that: The method comprises the following steps: Obtain the genome sequence of the target species and its closely related species; Process the reference genome sequence of the target species to obtain short sequence fragment data with identity and coverage greater than or equal to 80%; Process the short sequence fragment data with identity and coverage greater than or equal to 80%, screen the short sequence fragment data covering 90% of the genome, obtain the entropy value of each base in the short sequence fragment covering 90% of the genome, and quantitatively score the short sequence fragments covering 90% of the genome based on the entropy value and coverage information, screen the short sequence fragments covering 90% of the genome with the top scores, and replace the bases with the most conservative bases to obtain the optimized short sequence fragment data; Primers are designed based on the optimized short sequence fragment data, and the 3' end of the primer is ensured to avoid the high entropy value site to obtain candidate primers; The quality of the candidate primers is evaluated to obtain candidate primers that meet the evaluation criteria.

8. The method according to claim 7, characterized in that The target species is a pathogenic microorganism species, and the pathogenic microorganism species is bacteria, fungi or viruses.

9. A device for designing target species-specific primers based on information entropy, characterized in that: The device comprises the system according to any one of claims 1-6.

10. A readable storage medium, characterized in that: The readable storage medium stores a plurality of computer programs, and when the computer programs are executed on a processor, the method according to claim 7 is executed.

Images