SNP combination and paternity identification method for paternity identification of cynomolgus monkey

Abstract

This invention relates to the field of molecular marker screening technology, specifically to a SNP combination and method for paternity testing in cynomolgus monkeys. The method provided by this invention utilizes 100 selected specific SNP combinations (SNP1~SNP100) combined with low-depth sequencing for paternity testing in cynomolgus monkeys. The cost per sample is only 1 / 5 to 1 / 10 of existing high-depth sequencing and 1 / 2 of SNP chips. For large-scale experimental monkey populations (such as populations of thousands), this can save hundreds of thousands of yuan in testing costs annually, significantly reducing the economic burden of breeding and management of experimental monkeys, demonstrating extremely high economic efficiency. The method provided by this invention is applicable to cynomolgus monkey population pedigree management, kinship tracing, breeding pair optimization, and genetic quality control, and can be widely applied in experimental monkey breeding farms, primate research centers, and biomedical research institutions.

Description

Technical Field

[0001] This invention relates to the field of molecular marker screening technology, specifically to an SNP combination and a parentage testing method for cynomolgus monkeys. Background Technology

[0002] As a core model animal in fields such as biomedical research, preclinical drug evaluation, and gene editing research, the accuracy of the genetic background and the integrity of the pedigree of cynomolgus monkeys directly determine the reliability, repeatability, and scientific validity of experimental data. Parentage testing is a key technical means to ensure the genetic quality of experimental monkey populations, correct pedigree errors, avoid inbreeding depression, and optimize breeding pairings. It plays an irreplaceable role in the large-scale breeding and standardized management of experimental monkeys.

[0003] Currently, existing technologies in monkey paternity testing mainly fall into two categories: one is typing technology based on short tandem repeats (STRs), and the other is typing technology based on single nucleotide polymorphisms (SNPs). Both technologies have significant drawbacks and limitations, making it difficult to meet the needs of large-scale, low-cost, and high-precision paternity testing in experimental monkeys. Specifically: Existing Technology 1: STR Marker Paternity Testing Technology. STR markers were the mainstream technology for early monkey paternity testing. Its core principle is to detect differences in the number of repetitions of short tandem repeats in the genome to achieve individual typing and determine kinship. The main drawbacks of this technology include: High mutation rate: The mutation rate of STR markers is as high as 10%. - 3~10 - 4. Much higher than SNP markers (10 -8) In multi-generational pedigree identification, typing discrepancies are prone to occur, leading to inaccurate identification results and making it difficult to use for long-term population pedigree construction; Limited number of loci: The number of highly polymorphic STR loci available for monkey parentage identification is relatively small (usually no more than 20), with insufficient polymorphism and a low cumulative exclusion probability, making it difficult to meet the needs of high-precision identification, especially prone to misjudgment in the identification of closely related individuals; Low throughput and low efficiency: Traditional STR typing uses multiplex PCR combined with capillary electrophoresis detection, which can only process 24-48 samples per test, with a long detection cycle (usually requiring 3-5 days), making it difficult to meet the batch identification needs of large-scale experimental monkey populations (hundreds or even thousands); Poor stability: STR typing is prone to interference signals such as shadow peaks and spurious peaks, requiring high proficiency in experimental operation, and the consistency of test results between different laboratories and different operators is poor, with insufficient repeatability. Existing technology two: SNP marker parentage identification technology. With the development of sequencing technology, SNP markers, due to their advantages such as large number, uniform distribution, low mutation rate, stable genotyping, and suitability for high-throughput detection, are gradually replacing STR markers as the development direction for animal paternity testing. However, existing monkey SNP paternity testing technologies still have the following core pain points, preventing large-scale application: Lack of monkey species-specific and family-verified SNP combinations: The SNP loci used in existing technologies are mostly universal loci (not optimized for monkey species) or have only been validated with small samples, without verifying their genetic stability and identification effectiveness in multiple families. This results in problems such as unstable genotyping, high Mendelian genetic contradiction rate, and insufficient identification accuracy, failing to meet the precise identification needs of experimental monkey populations; High testing costs: Existing SNP paternity testing mostly uses high-depth whole-genome sequencing. Genome sequencing (sequencing depth ≥10×) or SNP chip detection costs hundreds of yuan per sample, which is too expensive for large-scale experimental monkey populations (such as routine identification in breeding farms) and difficult to popularize. High technical threshold and complex operation: High-depth sequencing and chip detection require professional experimental equipment, complex library construction processes and high-level bioinformatics analysis capabilities, which are difficult for ordinary breeding farms and small research institutions to meet, thus limiting their practicality. Technical bias limits application: Although low-depth sequencing (sequencing depth 0.1×~1×) can significantly reduce costs, it is traditionally believed that its genome coverage is low and its genotype interpretation error is high, making it unsuitable for accurate paternity testing. As a result, current technologies all rely on high-depth sequencing or chips, failing to achieve a balance between cost and accuracy.

[0004] Furthermore, existing technologies have not addressed the core requirements of "low cost, high throughput, high precision, and monkey-specificity." They are either costly and have low throughput, or they lack precision and stability, failing to meet the practical needs of daily pedigree management and genetic quality control in experimental monkey breeding farms. Therefore, developing a family-verified, species-specific, low-cost, high-throughput, and high-precision SNP parentage testing technology has become an urgent need in this field. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one objective of this invention is to provide the use of cynomolgus monkey SNP locus combinations in cynomolgus monkey paternity testing, and a paternity testing method for cynomolgus monkeys designed based on said SNP locus combinations. The identification method provided by this invention utilizes 100 selected specific SNP combinations combined with low-depth sequencing for cynomolgus monkey paternity testing. The cost per sample is only 1 / 5 to 1 / 10 of existing high-depth sequencing and 1 / 2 of SNP chip costs. For large-scale experimental monkey populations (such as populations of thousands), this can save hundreds of thousands of yuan in testing costs annually, significantly reducing the economic burden of experimental monkey breeding and management, and possessing extremely high economic efficiency. It is applicable to cynomolgus monkey population pedigree management, kinship tracing, breeding pair optimization, and genetic quality control, and can be widely applied in experimental monkey breeding farms, primate research centers, biomedical research institutions, and other scenarios.

[0006] Therefore, the first aspect of this invention provides the use of cynomolgus monkey SNP locus combinations in cynomolgus monkey paternity testing. According to an embodiment of the invention, the cynomolgus monkey SNP locus combinations include SNP1 to SNP100, with location information according to Macaca_fascicularis_6.0 (cynomolgus monkey genome annotation version 6.0). The location information of SNP1 to SNP100 is shown in Table 1 below.

[0007] Table 1

[0008] The inventors of this application have selected a set of SNP combinations (SNP1~SNP100) specifically for cynomolgus monkeys. These combinations are all derived from the autosomes of the cynomolgus monkey genome, exhibit high polymorphism (observed heterozygosity >0.3, polymorphism information content PIC>0.35), are evenly distributed across all autosomes, and have no linkage disequilibrium. Validated by more than 20 monkey families, the genotypes are stable and can be effectively used for cynomolgus monkey parentage testing, addressing the shortcomings of existing SNP loci, such as poor specificity and lack of validation.

[0009] A second aspect of this invention provides a method for paternity testing in cynomolgus monkeys based on SNP locus combinations. According to an embodiment of the invention, the method includes: S1: Extract genomic DNA from biological samples of cynomolgus monkeys to be identified; S2: Perform SNP site combination genotyping on the genomic DNA to obtain genotyping results, wherein the SNP site combination is SNP1~SNP100 as described in Table 1 of the first aspect; S3: Based on the classification results, calculate the parentage index or exclusion probability to determine the parent-child relationship.

[0010] A third aspect of this invention provides a system for paternity testing in cynomolgus monkeys based on SNP locus combinations. According to an embodiment of the invention, the system includes: The DNA extraction module is configured to extract genomic DNA from biological samples of cynomolgus monkeys to be identified. The SNP genotyping detection module is configured to perform SNP site combination genotyping detection on the genomic DNA to obtain genotyping results; The data analysis module is configured to calculate the parentage index or exclusion probability based on the typing results, and output the judgment result. The SNP site combinations are SNP1 to SNP100 as described in Table 1 of the first aspect.

[0011] The paternity testing system provided by this invention can accurately genotype 100 target SNPs using low-depth whole-genome sequencing, without the need for high-depth sequencing or SNP chips, significantly reducing testing costs and technical barriers, and solving the pain points of high cost and complex operation of existing technologies. Based on Mendelian inheritance laws, it establishes clear criteria for determining parentage, enabling rapid and accurate paternity exclusion and confirmation, and can realize automated analysis of batch samples, improving testing efficiency and solving the pain points of low efficiency and poor consistency of results of existing technologies.

[0012] In response to the aforementioned pain points of existing monkey paternity testing technologies, this invention provides a set of family-verified SNP marker combinations specifically for cynomolgus monkeys, and a low-cost, high-throughput, and high-precision paternity testing method based on these combinations. The specific objectives are as follows: (1) To provide a set of SNP marker combinations applicable to monkeys (cynomolgus monkeys), with stable typing, high polymorphism, and verified by multiple pedigrees, solving the problems of poor specificity, insufficient stability, and lack of verification of existing SNP sites; (2) To provide a cynomolgus monkey paternity testing method based on low-depth sequencing, breaking the technical prejudice that "low-depth sequencing cannot be used for accurate paternity testing," significantly reducing detection costs, and solving the problems of high cost, complex operation, and high threshold of existing technologies; (3) To provide a set of standardized, repeatable, and scalable rules for determining parentage relationships, ensuring the accuracy and consistency of the identification results, and meeting the daily and large-scale paternity testing and pedigree management needs of experimental monkey populations; (4) To achieve precise control of the genetic quality of experimental monkey populations, avoid inbreeding depression, optimize breeding pairing, improve the overall quality of experimental monkey populations, and provide experimental animals with clear genetic backgrounds for biomedical research.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0014] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0015] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0019] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0020] According to a specific embodiment of the present invention, the present invention provides the use of cynomolgus monkey SNP locus combinations in cynomolgus monkey parentage identification, wherein the cynomolgus monkey SNP locus combinations include SNP1 to SNP100, and the location information is according to Macaca_fascicularis_6.0 version (cynomolgus monkey genome annotation version 6.0), and the location information of SNP1 to SNP100 is as described in Table 1 above.

[0021] According to a specific embodiment of the present invention, the present invention provides a method for paternity testing of cynomolgus monkeys based on SNP locus combinations, comprising: S1: Extract genomic DNA from biological samples of cynomolgus monkeys to be identified; S2: Perform SNP site combination genotyping detection on the genomic DNA to obtain genotyping results, wherein the SNP site combination is SNP1~SNP100 as described above; S3: Based on the classification results, calculate the parentage index or exclusion probability to determine the parent-child relationship.

[0022] It should be noted that, based on the typing results, the methods for calculating the parentage index or exclusion probability can be the classic PI / CPI method, PE / CPE method, or likelihood ratio / Bayesian method, etc., and some calculation tools / software (such as Familias, Cervus, COLONY, etc.) can also be used as auxiliary tools.

[0023] It should be noted that there are no particular restrictions on the methods for extracting genomic DNA. For example, phenol-chloroform extraction, magnetic bead extraction, column extraction, etc., can be used, or genomic DNA can be extracted directly using a special kit.

[0024] According to a specific embodiment of the present invention, step S2 further includes: By performing low-depth whole-genome sequencing, SNP genotyping was performed on the genomic DNA based on the sequencing results to obtain the genotyping results.

[0025] According to a specific embodiment of the present invention, the biological sample includes, but is not limited to, blood, hair, tissue, oropharyngeal swabs, etc.

[0026] According to a specific embodiment of the present invention, the present invention provides a system for paternity testing of cynomolgus monkeys based on SNP locus combinations, comprising: The DNA extraction module is configured to extract genomic DNA from biological samples of cynomolgus monkeys to be identified. The SNP genotyping detection module is configured to perform SNP site combination genotyping detection on the genomic DNA to obtain genotyping results; The data analysis module is configured to calculate the parentage index or exclusion probability based on the typing results, and output the judgment result. The SNP site combination is SNP1~SNP100 as described above.

[0027] According to a specific embodiment of the present invention, low-depth whole-genome sequencing is used to perform SNP genotyping detection on the genomic DNA based on the sequencing results to obtain genotyping results.

[0028] The present invention, through the above-described technical solution, solves many pain points of the prior art, and produces significant technical, economic, and application effects, as detailed below: Technical advantages: 1) Stable and reliable genotyping: 100 SNP loci have been verified by more than 20 families, following Mendelian laws of inheritance; 2) Low technical threshold: standardized experimental procedures, simple DNA extraction, sequencing, and genotyping operations, no need for professional high-end equipment, and can be widely promoted; 3) Strong adaptability: can handle various sample types such as peripheral blood, ear tissue, hair with follicles, and oral swabs, and sample collection is convenient without damaging experimental monkeys. Economic benefits: The cost of single-sample testing is only 1 / 5 to 1 / 10 of that of existing high-depth sequencing and 1 / 2 of that of SNP chips. For large-scale experimental monkey populations (such as populations of thousands), it can save hundreds of thousands of yuan in testing costs per year, greatly reducing the economic burden of breeding and managing experimental monkeys, and has extremely high economic efficiency. Application effects: 1) It can be directly applied to the daily pedigree management, parentage identification, and breeding pair optimization of experimental monkey breeding farms, effectively avoiding inbreeding depression and improving the genetic quality of experimental monkey populations; 2) It can be used for kinship tracing and population genetic diversity monitoring in primate research centers, providing technical support for conservation and introduction of new species; 3) It can be used for tracing the source of experimental animals in biomedical research institutions, ensuring a clear genetic background of experimental animals and improving the reliability and reproducibility of experimental data; 4) It can lead to the development of products such as detection kits and primer combinations, further expanding application scenarios and possessing broad market application prospects. Industry Value: This invention breaks the technical bias of low-depth sequencing in monkey paternity testing, establishes a "low-cost, high-throughput, high-precision, and monkey-specific" paternity testing technology system, fills the gap in existing technologies, promotes the standardization and large-scale development of monkey paternity testing technology, and plays an important role in promoting the standardization and high-quality development of the experimental monkey industry.

[0029] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0030] Example 1: Screening and Validation of SNP Sites The methods for SNP locus screening and family verification are as follows: 1. SNP site screening: One hundred cynomolgus monkeys underwent low-depth genome sequencing, and 30 cynomolgus monkeys underwent 30× genome resequencing. Genotyping of the low-depth sequenced individuals was performed using GLIMPSE2 software. The 100 SNP loci with the highest information content were selected, and detailed SNP information is shown in Table 1 below.

[0031] Table 1 Detailed location information of SNPs

[0032] 2. Family lineage verification: In an experimental population of 130 cynomolgus monkeys, 28 known cynomolgus monkey families were selected for paternity testing. The pedConstruct tool was used to determine parentage among candidate paternal, maternal, and offspring genotype data. The program calculated the Mendelian error rate per sample locus: when both parents and offspring were provided, all parental combinations were listed and the three-way Mendelian consistency was calculated; the parental combination with the lowest error rate was selected as the candidate kinship. When only one parent was available, the Mendelian error rate between that parent and offspring was calculated and judged according to a threshold.

[0033] Mendel's error rate was calculated using the following formula: Let N be the number of comparable sites and E be the number of erroneous sites, then

[0034] Pairwise samples

[0035] Father-mother-child three parties For each locus K, if a set can be generated by Mendel that does not belong to the parent genotype, it is counted as an error.

[0036] )] Twenty-eating macaque families (each family includes the maternal parent, offspring, and a known paternal parent) were selected to verify the genotyping stability of the loci. The results are shown in Table 2.

[0037] Table 2. Genotype matching results between crab-eating monkeys and candidate parents.

[0038] The results in Table 2 above show that the 100 SNP loci screened in this invention, after verification by cynomolgus monkey families, follow Mendelian inheritance laws, have stable typing, and can be effectively used for cynomolgus monkey parentage testing.

[0039] Example 2: Paternity Testing Process Samples were collected, DNA was extracted, and low-depth sequencing was performed on the monkeys to be tested (offspring, suspected paternal parents, and suspected maternal parents). Genotypes of 100 target SNP loci were extracted, and the pedConstruct tool was used to determine parentage between the genotype data of the candidate paternal parents, maternal parents, and offspring. The specific parentage testing process is as follows: 1. Sample collection: Peripheral blood samples were collected from the crab-eating monkey offspring to be tested, the suspected paternal parent, and the suspected maternal parent. EDTA was added as an anticoagulant to avoid sample contamination and DNA degradation. 2. DNA extraction: Genomic DNA was extracted from the sample. Quality control standards: DNA concentration 50~100 ng / μL, OD260 / 280=1.8~2.0, no obvious degradation; 3. Low-depth sequencing: Construct a second-generation sequencing library and perform low-depth whole-genome sequencing using an Illumina NovaSeq sequencer. The sequencing depth is 0.1×~1× (which can be adjusted according to sample quality), with a paired-end read length of 150bp. Sequencing data are obtained after data quality control. 4. SNP genotyping: The quality-controlled sequencing data was aligned to the monkey reference genome, and the genotypes of 100 target SNP loci in Table 1 were extracted to obtain the genotyping results (AA / AB / BB) for each individual. Low-quality data were removed after quality control. 5. Parentage determination: Based on Mendelian inheritance laws, the following criteria are used for determination: The pedConstruct tool is used to determine parentage among candidate fathers, mothers, and offspring genotype data.

[0040] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0041] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of cynomolgus monkey SNP locus combinations in cynomolgus monkey paternity testing, characterized in that, The cynomolgus monkey SNP locus combination includes SNP1 to SNP100, and the location information is based on Macaca_fascicularis_6.0 version. The location information of SNP1 to SNP100 is as follows: 。 2. A method for paternity testing in cynomolgus monkeys based on SNP locus combinations, characterized in that, include: S1: Extract genomic DNA from biological samples of cynomolgus monkeys to be identified; S2: Perform SNP site combination genotyping on the genomic DNA to obtain genotyping results, wherein the SNP site combination is SNP1~SNP100 as described in claim 1; S3: Based on the classification results, calculate the parentage index or exclusion probability to determine the parent-child relationship.

3. The method according to claim 2, characterized in that, Step S2 further includes: Low-depth whole-genome sequencing was used to perform SNP site combination genotyping detection on the genomic DNA based on the sequencing results, and genotyping results were obtained.

4. The method according to claim 2, characterized in that, The biological samples include blood, hair, tissue, and oropharyngeal swabs.

5. A system for paternity testing in cynomolgus monkeys based on SNP locus combinations, characterized in that, include: The DNA extraction module is configured to extract genomic DNA from biological samples of cynomolgus monkeys to be identified. The SNP genotyping detection module is configured to perform SNP site combination genotyping detection on the genomic DNA to obtain genotyping results; The data analysis module is configured to calculate the parentage index or exclusion probability based on the typing results, and output the judgment result. Wherein, the SNP site combination is SNP1~SNP100 as described in claim 1.

6. The system according to claim 5, characterized in that, By performing low-depth whole-genome sequencing, SNP genotyping was performed on the genomic DNA based on the sequencing results to obtain the genotyping results.

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