Fine classification of east asian populations by aim-snp typing system and its application

Abstract

The application belongs to the technical field of forensic medicine, and discloses an East Asian group fine division AIM-SNP typing system and application. The application is based on a machine learning algorithm, systematically screens and optimizes a set of biogeographical ancestral tracing markers in the whole genome range, optimizes 58 high ancestral information amount SNP sites from 2135 candidate di-allele SNPs, provides a core feature set for accurate tracing, and further screens 2 Y chromosome deletion / insertion sites and 1 tooth enamel gene for gender identification. The feature set can accurately trace to five major continental populations, namely, Africa, Europe, East Asia, South Asia and America (excluding mixed American descent). In particular, the problem of insufficient tracing resolution in the East Asian population is solved, and the ancestral resolution capability of the East Asian population is significantly improved, making it more applicable and feasible in Chinese forensic practice.

Description

Technical Field

[0001] This invention belongs to the field of forensic medicine technology, specifically involving the AIM-SNP typing system for fine classification of East Asian populations and its application. Background Technology

[0002] Inferring the ancestry of individuals from biological samples can provide crucial clues for case investigations, helping to narrow down the range of suspects and clarify the direction of the investigation. The core of ancestry inference is to analyze a set of ancestral information markers (AIMs) to assess the population genetic structure of individuals and thus infer their ancestral origins. Currently, domestic and international research mainly focuses on ancestry inference based on single nucleotide polymorphism (SNP) genetic markers. Compared to other markers, SNPs have significant advantages: higher polymorphism and lower mutation rate (approximately 10). -8 SNP detection methods are characterized by ease of typing and gene frequency determination, shorter fragments, and suitability for analysis of highly degraded samples. Developed SNP detection methods each have their own characteristics: TaqMan technology (real-time fluorescence detection): wide applicability and good stability, but high cost and challenging probe design. SNaPshot microsequencing (based on primer extension): stable and reliable results, widely used, but requires two-step amplification reactions. High-throughput methods such as gene chips and MALDI-TOF MS: high throughput, but require large-scale specialized equipment, are costly, and time-consuming. Allele-specific PCR (AS-PCR) is simple to operate; DNA samples can be amplified in one step and then genotyped by capillary electrophoresis (CE), making it highly compatible with existing forensic laboratory platforms and enabling rapid detection. Its core principle is that the amplification reaction should be blocked when the 3' end of the primer mismatches with the template DNA. However, experiments have shown that even with a single base mismatch at the primer end, the amplification reaction sometimes continues, leading to a decrease in the expected allele resolution. This is because PCR reactions exhibit exponential amplification; even if only a very small amount of mismatched template extends in the first cycle, the resulting product will no longer mismatch with primers in subsequent cycles, thus being amplified normally and accumulating. To address this issue, a key improvement is the introduction of an additional mismatched base at the 3' end of the specific primer. This strategy significantly improves the specificity of primer extension and effectively reduces the false positive rate in SNP analysis.

[0003] Currently, several reported ancestry inference systems based on ancestral information SNPs (AIM-SNP) (such as the 27-plex, 30-plex, 34-plex, and 56-plex SNP systems) primarily focus on intercontinental (e.g., Africa, East Asia, Europe) biogeographical tracing studies. Detailed ancestry inference studies targeting subgroups within East Asia are underway: Yuasa et al. screened 67 population-specific SNP loci in Japan. However, these research findings are mostly in the research stage, and standardized commercial testing kits have not yet been developed. In terms of ease of operation and cost-effectiveness, they still fall short of meeting the daily needs of frontline forensic identification work. The core trend for future development is to construct an ancestry inference system capable of multi-level, detailed inference from intercontinental groups to local subgroups, and to ensure its application in grassroots laboratories, meeting the urgent need for efficient and convenient technologies in public security operations.

[0004] Conventional multiplex amplification systems, limited by the number of fluorescent labeling channels, struggle to simultaneously detect multiple high-information-value ancestral SNP loci in a single reaction. This leads to two major problems: a) the resolution for identifying intercontinental populations (such as those in Africa, Europe, and East Asia) cannot be further improved; b) the tracing efficiency is significantly low, particularly for subpopulations within East Asia. Traditional genetic marker screening processes rely excessively on manual experience and limited prior knowledge, lacking advanced methods for intelligent and systematic screening at the whole-genome scale. This limits the design of tracing systems, making it difficult to effectively cover the multi-level population genetic structure from macro-intercontinental populations to local regional subpopulations, resulting in a single geographical coverage level and limited ability for precise inference. Although six-color fluorescent labeling technology has driven the development of multiplex amplification systems that integrate more SNP loci to increase information content, the increased number of loci intensifies competition among primers, significantly increasing the difficulty of optimizing primer concentration ratios and reaction conditions. Meanwhile, although machine learning algorithms have the ability to efficiently screen AIM-SNPs from whole-genome data, applying them to build a complete and fully validated AIM-SNP tracing system and form a standardized analysis process still faces significant challenges. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention integrates machine learning algorithms with capillary electrophoresis platform technology, and by screening SNP markers strongly associated with intercontinental and local regional subpopulations across the entire genome, and developing a highly compatible multiplex amplification system, it aims to achieve multi-level precise tracing from macro-intercontinental populations to internal East Asian subpopulations, providing efficient and stable technical support for forensic practice.

[0006] The first objective of this invention is to provide a reagent for detecting gene panel combinations.

[0007] A second aspect of the present invention is to provide a reagent kit.

[0008] The object of a third aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention.

[0009] The fourth aspect of this invention aims to provide a method for inferring biogeographical ancestral information in forensic medicine or a method for distinguishing between the five continental groups and East Asian groups.

[0010] The fifth aspect of this invention aims to provide a detection system for inferring or distinguishing between the five continental groups and the East Asian group using forensic biogeographical ancestry information.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a reagent for detecting gene panel combinations, the gene panel combinations comprising 58 autosomal SNP sites, 2 Y chromosome deletion / insertion sites and 1 tooth enamel gene.

[0013] In some embodiments of the present invention, the 58 autosomal SNP loci include rs9285110, rs56142203, rs570435573, rs2261033, rs17207681, rs1250233, rs11104947, rs12006467, rs2920295, rs12130799, rs2851012, rs11841589, r s722869, rs9522149, rs1288367, rs28777, rs11034709, rs1063, rs10741584, rs6123723, rs53 4029120, rs4907251, rs6969817, rs2642066, rs2024566, rs6078837, rs116783706, rs2042762 , rs7596027, rs12229892, rs464165, rs4704322, rs7745461, rs7554936, rs11224765, rs79200 067, rs17822931, rs17034666, rs17451739, rs6548616, rs3809161, rs1371048, rs11652805, r s535319466, rs10497191, rs17599827, rs12498138, rs59950930, rs239031, rs7799912, rs174570, rs149768401, rs3217805, rs546642722, rs10008492, rs530357165, rs2717329 and rs434124.

[0014] In some embodiments of the present invention, the two Y chromosome deletion / insertion sites include rs759551978 and rs199815934.

[0015] In some embodiments of the present invention, the enamel gene is the Amelogenin gene.

[0016] In some embodiments of the present invention, the gene panel combination is used for forensic biogeographical ancestry information inference and differentiation of the five continental groups and the East Asian group.

[0017] In some embodiments of the present invention, the reagent includes specific primers.

[0018] In some embodiments of the present invention, the sequences of the specific primers are shown as SEQ ID NO:1-SEQ ID NO:180.

[0019] In some embodiments of the present invention, the sequences of the specific primers for rs9285110 are shown in SEQ ID NO:1-3; the sequences of the specific primers for rs56142203 are shown in SEQ ID NO:4-6; the sequences of the specific primers for rs570435573 are shown in SEQ ID NO:7-9; the sequences of the specific primers for rs2261033 are shown in SEQ ID NO:10-12; the sequences of the specific primers for rs17207681 are shown in SEQ ID NO:13-15; the sequences of the specific primers for rs1250233 are shown in SEQ ID NO:16-18; the sequences of the specific primers for rs11104947 are shown in SEQ ID NO:19-21; the sequences of the specific primers for rs12006467 are shown in SEQ ID NO:22-24; and the sequences of the specific primers for rs2920295 are shown in SEQ ID NO:1-24. The sequences of the rs12130799 specific primers are shown in SEQ ID NO: 25-27; the sequences of the rs2851012 specific primers are shown in SEQ ID NO: 31-33; the sequences of the rs11841589 specific primers are shown in SEQ ID NO: 34-36; the sequences of the rs759551978 specific primers are shown in SEQ ID NO: 37-38; the sequences of the rs722869 specific primers are shown in SEQ ID NO: 39-41; the sequences of the rs9522149 specific primers are shown in SEQ ID NO: 42-44; the sequences of the rs1288367 specific primers are shown in SEQ ID NO: 45-47; the sequences of the rs28777 specific primers are shown in SEQ ID NO: 48-50; and the sequences of the rs11034709 specific primers are shown in SEQ ID NO: 28-30. The sequences of the rs1063 specific primers are shown in SEQ ID NO:51-53; the sequences of the rs10741584 specific primers are shown in SEQ ID NO:57-59; the sequences of the rs6123723 specific primers are shown in SEQ ID NO:60-62; the sequences of the rs534029120 specific primers are shown in SEQ ID NO:63-65; the sequences of the rs4907251 specific primers are shown in SEQ ID NO:66-68; the sequences of the rs6969817 specific primers are shown in SEQ ID NO:69-71; and the sequences of the rs199815934 specific primers are shown in SEQ ID NO:72-73.The sequences of the rs2642066 specific primers are shown in SEQ ID NO:74-76; the sequences of the rs2024566 specific primers are shown in SEQ ID NO:77-79; the sequences of the rs6078837 specific primers are shown in SEQ ID NO:80-82; the sequences of the rs116783706 specific primers are shown in SEQ ID NO:83-85; the sequences of the rs2042762 specific primers are shown in SEQ ID NO:86-88; the sequences of the rs7596027 specific primers are shown in SEQ ID NO:89-91; the sequences of the rs12229892 specific primers are shown in SEQ ID NO:92-94; the sequences of the rs464165 specific primers are shown in SEQ ID NO:95-97; the sequences of the rs4704322 specific primers are shown in SEQ ID NO:98-100; and the sequences of the rs7745461 specific primers are shown in SEQ ID NO:74-76. The sequences of the rs7554936 specific primers are shown in SEQ ID NO: 101-103; the sequences of the rs11224765 specific primers are shown in SEQ ID NO: 107-109; the sequences of the rs79200067 specific primers are shown in SEQ ID NO: 110-112; the sequences of the rs17822931 specific primers are shown in SEQ ID NO: 113-115; the sequences of the Amel specific primers are shown in SEQ ID NO: 116-117; the sequences of the rs17034666 specific primers are shown in SEQ ID NO: 118-120; the sequences of the rs17451739 specific primers are shown in SEQ ID NO: 121-123; the sequences of the rs6548616 specific primers are shown in SEQ ID NO: 124-126; and the sequences of the rs3809161 specific primers are shown in SEQ ID NO: 104-106. The sequences of the rs1371048 specific primers are shown in SEQ ID NO: 130-132; the sequences of the rs11652805 specific primers are shown in SEQ ID NO: 133-135; the sequences of the rs535319466 specific primers are shown in SEQ ID NO: 136-138; the sequences of the rs10497191 specific primers are shown in SEQ ID NO: 139-141; the sequences of the rs17599827 specific primers are shown in SEQ ID NO: 142-144; and the sequences of the rs12498138 specific primers are shown in SEQ ID NO: 145-147.The sequences of the specific primers for rs59950930 are shown in SEQ ID NO:148-150; the sequences of the specific primers for rs239031 are shown in SEQ ID NO:151-153; the sequences of the specific primers for rs7799912 are shown in SEQ ID NO:154-156; the sequences of the specific primers for rs174570 are shown in SEQ ID NO:157-159; the sequences of the specific primers for rs149768401 are shown in SEQ ID NO:160-162; the sequences of the specific primers for rs3217805 are shown in SEQ ID NO:163-165; the sequences of the specific primers for rs546642722 are shown in SEQ ID NO:166-168; the sequences of the specific primers for rs10008492 are shown in SEQ ID NO:169-171; and the sequences of the specific primers for rs530357165 are shown in SEQ ID NO:148-150. The sequences of the rs2717329 specific primers are shown in SEQ ID NO:175-177; the sequences of the rs434124 specific primers are shown in SEQ ID NO:178-180.

[0020] The primer design principle of this invention is to design three primers for each SNP site. Two specific upstream (downstream) primers have their 3' ends matched with mutant and wild-type bases, respectively. One primer has a 2-4 base mismatched nucleotide appended to its 5' end, resulting in a length difference in the amplified fragment, converting sequence polymorphism into length polymorphism for easier electrophoresis detection. A common downstream (upstream) primer divides the 58 SNP sites into five groups based on their fluorescent labeling groups. To effectively prevent non-specific extension, a mismatched base is artificially introduced at the penultimate or third-to-last position of the 3' end of the specific primers. The Tm value of all primers is set to (58 + 0.5) °C, and the amplified product fragment size is less than 200 bp. For the confirmatory sequencing experiments, 58 sequencing primer pairs were designed with a sequencing length of 300-400 bp as the standard. After primer design, primer specificity was compared using Primer-Blast and the UCSC In-Silico PCR Tool to select primer pairs with human specificity. In addition, published reference sequences for the Y-InDel site were retrieved from the NCBI database based on the Y-InDel site rs number. Primers were designed using Primer Premier version 3.0 based on the flanking sequences of 200 bases upstream and downstream of the core sequence of the Y-InDel site. The amplification product fragments of the Y-InDel site primers were all less than 200 bp in size; the primer annealing temperature range was 55-62℃, and the Tm value difference between primers did not exceed 2℃; the GC content of the primer sequences was 40%-60%.

[0021] In some embodiments of the present invention, at least one primer in the specific primers corresponding to each point or gene in the gene panel combination is labeled with a fluorescent dye at its 5' end.

[0022] In some embodiments of the present invention, the fluorescent dye includes at least one of 6-FAM, HEX, TAMRA, ROX, and L592.

[0023] In some embodiments of the present invention, the fluorescent dyes labeled in the specific primers corresponding to each point or gene in the gene panel combination are the same or different.

[0024] In some embodiments of the present invention, primer sequences of rs9285110, rs56142203, rs570435573, rs2261033, rs17207681, rs1250233, rs11104947, rs12006467, rs2920295, rs12130799, rs2851012, rs11841589, and rs722869 are labeled using 6-FAM; HEX labels are used. Record the primer sequences for rs9522149, rs1288367, rs28777, rs11034709, rs1063, rs10741584, rs6123723, rs534029120, rs4907251, rs6969817, and rs2642066; use TAMRA to label rs2024566, rs6078837, rs116783706, rs2042762, and rs759. Primer sequences 6027, rs12229892, rs464165, rs4704322, rs7745461, rs7554936, rs11224765, and rs79200067; ROX-labeled primer sequences rs17822931, rs17034666, rs17451739, rs6548616, rs3809161, rs1371048, rs11652805, and rs53531. Primer sequences for rs10497191, rs17599827, and rs12498138 were obtained; primer sequences for rs59950930, rs239031, rs7799912, rs174570, rs149768401, rs3217805, rs546642722, rs10008492, rs530357165, rs2717329, and rs434124 were obtained using L592.

[0025] The reagents provided by this invention include (1) an intercontinental specific locus set: carefully selected ancestral information loci covering five major intercontinental groups in Africa, Europe, East Asia, South Asia, and the Americas, ensuring accurate macro-level tracing. (2) an East Asian subgroup specific locus set: integrating characteristic loci for subgroups within East Asia, significantly improving the ability to distinguish between them. By integrating the above-mentioned intercontinental and East Asian specific loci, a multi-level biogeographical tracing capability is formed from macro-intercontinental groups to subgroups within East Asia. Using a machine learning-driven locus screening algorithm, specific ancestral information SNP loci are systematically mined from whole-genome data, significantly improving the identification accuracy of subgroups within East Asia.

[0026] The forensic ancestry inference system (i.e., the site combination described in the first aspect of this invention) constructed based on polymerase chain reaction combined with capillary electrophoresis platform has good performance, with high sensitivity and strong specificity, and is suitable for the analysis of forensic degraded samples.

[0027] A second aspect of the present invention provides a reagent kit comprising the reagent of the first aspect of the present invention.

[0028] In some embodiments of the present invention, the kit further includes reagents for PCR amplification and genotyping.

[0029] In some embodiments of the present invention, the reagents used for PCR amplification and genotyping include at least one of dNTPs, enzymes, buffers, KCl, MgCl2, BSA, or formamide.

[0030] In some embodiments of the present invention, the kit further includes a positive control and a negative control.

[0031] In some embodiments of the present invention, the positive control includes, but is not limited to, sex control samples, standard samples from five continental populations (Africa, Europe, East Asia, South Asia and the Americas), and standard samples from subgroups within East Asia.

[0032] The kit of this invention is designed to be compatible with existing capillary electrophoresis platforms in conventional forensic laboratories, enabling the entire detection process from DNA amplification and electrophoretic typing to data analysis under standard conditions. It provides an efficient, stable, and accurate technical solution for inferring the ancestral origin of biological samples in complex cases, helping to narrow down the range of suspects and clarify the direction of the investigation.

[0033] A third aspect of the present invention provides the use of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention in (1) or (2):

[0034] (1) Inference of ancestral information from forensic biogeography;

[0035] (2) Distinguish between the five continental groups and the East Asian group.

[0036] In some embodiments of the present invention, the five continental groups include African, European, East Asian, South Asian and American groups.

[0037] A fourth aspect of the present invention provides a method for forensic biogeographical ancestry information inference or a method for distinguishing between five continental populations and East Asian populations, comprising the step of detecting the genome of a sample to be tested using the reagents of the first aspect of the present invention or the kits of the second aspect of the present invention.

[0038] In some embodiments of the present invention, the method specifically includes the following steps:

[0039] The genome of the sample to be tested was amplified using the primers shown in SEQ ID NO:1-SEQ ID NO:180 to obtain the genotyping data of the sample to be tested.

[0040] The genotyping data was then analyzed in conjunction with the reference population data.

[0041] In some embodiments of the present invention, the amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0042] In some embodiments of the present invention, in the amplification reaction system, the final concentrations of the primers shown in SEQ ID NO:1-3 are 0.91 μM, the final concentrations of the primers shown in SEQ ID NO:4-6 are 0.57 μM, the final concentrations of the primers shown in SEQ ID NO:7-9 are 0.09 μM, the final concentrations of the primers shown in SEQ ID NO:10-12 are 1.36 μM, the final concentrations of the primers shown in SEQ ID NO:13-15 are 0.15 μM, the final concentrations of the primers shown in SEQ ID NO:16-18 are 0.19 μM, the final concentrations of the primers shown in SEQ ID NO:19-21 are 0.15 μM, the final concentrations of the primers shown in SEQ ID NO:22-24 are 0.11 μM, the final concentrations of the primers shown in SEQ ID NO:25-27 are 0.23 μM, and the final concentrations of the primers shown in SEQ ID NO:28-30 are 1.43 μM. The final concentrations of primers shown in SEQ ID NO:31-33 are 0.42 μM, the final concentrations of primers shown in SEQ ID NO:34-36 are 0.34 μM, the final concentrations of primers shown in SEQ ID NO:37-38 are 0.87 μM, the final concentrations of primers shown in SEQ ID NO:39-41 are 0.34 μM, the final concentrations of primers shown in SEQ ID NO:42-44 are 1.06 μM, the final concentrations of primers shown in SEQ ID NO:45-47 are 0.09 μM, the final concentrations of primers shown in SEQ ID NO:48-50 are 0.19 μM, the final concentrations of primers shown in SEQ ID NO:51-53 are 0.19 μM, the final concentrations of primers shown in SEQ ID NO:54-56 are 0.23 μM, the final concentrations of primers shown in SEQ ID NO:57-59 are 0.30 μM, and the final concentrations of primers shown in SEQ ID NO:60-62 are 0.34 μM. The final concentrations of primers shown in SEQ ID NO:63-65 are 0.36 μM, SEQ ID NO:66-68 are 0.11 μM, SEQ ID NO:69-71 are 0.34 μM, SEQ ID NO:72-73 are 0.11 μM, SEQ ID NO:74-76 are 0.19 μM, SEQ ID NO:77-79 are 0.64 μM, SEQ ID NO:80-82 are 0.30 μM, SEQ ID NO:83-85 are 0.26 μM, SEQ ID NO:86-88 are 1.17 μM, and SEQ ID NO:89-91 are 0.The final concentrations of the primers shown in SEQ ID NO: 92-94 are 0.57 μM, SEQ ID NO: 95-97 are 0.26 μM, SEQ ID NO: 98-100 are 0.11 μM, SEQ ID NO: 101-103 are 1.51 μM, SEQ ID NO: 104-106 are 1.13 μM, SEQ ID NO: 107-109 are 0.26 μM, SEQ ID NO: 110-112 are 0.42 μM, SEQ ID NO: 113-115 are 1.09 μM, SEQ ID NO: 116-117 are 0.19 μM, and SEQ ID NO: 118-120 are 0.68 μM. The final concentrations of primers NO:121-123 are 1.28 μM, SEQ ID NO:124-126 are 0.45 μM, SEQ ID NO:127-129 are 0.19 μM, SEQ ID NO:130-132 are 0.68 μM, SEQ ID NO:133-135 are 0.23 μM, SEQ ID NO:136-138 are 0.60 μM, SEQ ID NO:139-141 are 0.72 μM, SEQ ID NO:142-144 are 1.21 μM, SEQ ID NO:145-147 are 0.72 μM, and SEQ ID NO:148-150 are 0.11 μM. The final concentrations of primers shown in SEQ ID NO:151-153 are 0.11 μM; the final concentrations of primers shown in SEQ ID NO:154-156 are 0.98 μM; the final concentrations of primers shown in SEQ ID NO:157-159 are 0.75 μM; the final concentrations of primers shown in SEQ ID NO:160-162 are 0.19 μM; the final concentrations of primers shown in SEQ ID NO:163-165 are 0.19 μM; the final concentrations of primers shown in SEQ ID NO:166-168 are 0.19 μM; the final concentrations of primers shown in SEQ ID NO:169-171 are 0.15 μM; the final concentrations of primers shown in SEQ ID NO:172-174 are 0.34 μM; the final concentrations of primers shown in SEQ ID NO:175-177 are 0.23 μM; and the final concentrations of primers shown in SEQ ID NO:178-180 are 0.87 μM.

[0043] This invention designs a large number of primer sequences for repeated verification and screening, mixes primers for all sites at a certain concentration, and repeatedly adjusts the primer concentration ratio for each site according to the peak height of the PCR product electrophoresis pattern to ensure that each pair of primers in the multiplex amplification system still has high amplification efficiency, specificity and sensitivity, and gradually improves the balance of DNA typing patterns.

[0044] In some embodiments of the present invention, capillary electrophoresis is used to detect the amplified products. After capillary electrophoresis, GeneMapper® ID-X software is used to process the detected data to obtain the genotyping data of the sample to be tested.

[0045] In some embodiments of the present invention, the sample to be tested is the subject's bodily fluids / tissues, such as bloodstains, blood, saliva, oral swabs, hair roots with follicles, semen, muscle tissue, exfoliated cells, etc.

[0046] In some embodiments of the present invention, the analysis includes performing population genetic analysis on the genotyping data of the test sample and the reference population data, such as phylogenetic tree construction, principal component analysis (PCA), t-distributed random neighbor embedding (t-SNE), and Admixture ancestor component analysis.

[0047] In some embodiments of the present invention, the analysis further includes constructing a biogeographical origination model using machine learning algorithms (such as random forest, extreme gradient boosting (XGBoost), support vector machine, neural network, etc.) and evaluating its performance through ten-fold cross-validation.

[0048] A fifth aspect of the present invention provides a detection system for forensic biogeographical ancestry information inference or differentiation of five continental groups and East Asian groups, comprising:

[0049] Detection module: used to detect the genotyping data of the gene panel combination in the first aspect of the present invention in the sample to be tested, and output the genotyping data to the analysis module;

[0050] Analysis module: Analyzes the genotyping data with the reference population data, and infers the forensic biogeographical ancestry of the sample under test based on the analysis results or determines the intercontinental origin of the sample under test based on the analysis results.

[0051] In some embodiments of the present invention, the detection system may operate the method of the fourth aspect of the present invention.

[0052] The beneficial effects of this invention are:

[0053] This invention, based on machine learning algorithms, systematically screened and optimized a set of biogeographical ancestry markers across the entire genome. From 2135 candidate igneous SNPs, 58 high-ancestor information SNPs were selected, providing a core feature set for precise ancestry tracing. Furthermore, two Y-chromosome deletion / insertion sites and one enamel gene were screened for sex identification. This feature set can trace ancestry with high precision to five major continental populations: Africa, Europe, East Asia, South Asia, and the Americas (excluding mixed-race American ancestry). It particularly addresses the issue of insufficient resolution in ancestry tracing within East Asian populations, significantly improving the ability to distinguish ancestry within East Asian populations, making it more applicable and feasible in Chinese forensic practice. It achieves multi-level precise ancestry tracing from macro-continental populations to sub-groups within East Asia, providing efficient and stable technical support for forensic practice.

[0054] Based on six-color fluorescent labeling technology, this invention successfully established a multiplex PCR system (kit) capable of simultaneously, stably, specifically, and efficiently amplifying 58 AIM-SNP loci, 2 Y-InDel loci, and the Amelogenin gene. This system, with its large number of multiplexed detection sites, significantly improves the efficiency of inferring biogeographical ancestry information.

[0055] The method provided by this invention is simple, accurate, and efficient, requiring only PCR and a conventional capillary electrophoresis genetic analyzer to complete the composite detection. It offers an efficient, stable, and accurate technical solution for inferring the ancestral origin of biological samples in the investigation of complex cases, helping to narrow down the range of suspects and clarify the direction of the investigation. It is applicable to various human tissue and body fluid samples, facilitating its widespread application in grassroots forensic DNA laboratories. Attached Figure Description

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0057] Figure 1 This is a diagram showing the arrangement of 61 sites in the composite system described in Embodiment 1 of the present invention.

[0058] Figure 2 This is the Allelic Ladder electrophoresis pattern described in Embodiment 2 of the present invention.

[0059] Figure 3 This is the electrophoretic pattern of standard 9948 as described in Embodiment 2 of the present invention.

[0060] Figure 4 The figure shows the biogeographical ancestry tracing efficiency evaluation results of the multiplex amplification system described in Example 3 of this invention; where A is the phylogenetic tree result of the five-continental reference population, B is the PCA dimensionality reduction visualization result of the five-continental reference population, C is the t-SNE dimensionality reduction visualization result of the five-continental reference population, and D is the ancestral component analysis result of the five-continental reference population.

[0061] Figure 5 The results are 10-fold cross-validation results of the biogeographical ancestor tracing model of the multiplex amplification system described in Embodiment 3 of the present invention; wherein, A is the standardized 10-fold cross-validation confusion matrix of a series of biogeographical ancestor tracing models constructed based on the five continental reference populations; B is the standardized 10-fold cross-validation confusion matrix of the biogeographical ancestor tracing model constructed based on the East Asian reference populations. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0063] Unless otherwise specified in the examples, standard conditions were followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Example 1: SNP site selection and primer design for multiplex amplification systems

[0066] (1) Selection of 58 AIM-SNP loci

[0067] Based on population data from the 1000 Genome Project phase III (1KGP) database, including 26 populations and 2504 individuals, 5 were from East Asia, 5 from Europe, 5 from South Asia, 7 from Africa, and 4 from the Americas. Plink2 was used to perform preliminary screening of loci in the 1000 Genome Project database across the entire genome. The basic criteria for AIM-SNP loci screening were as follows: 1) They must be autosomal DIP genetic markers and exhibit diallelic polymorphism. 2) Loci with a minimum allele frequency > 0.01 were selected. 3) Loci exhibiting LD imbalance were filtered out. R 2>0.4) loci, and all conformed to the Bonferroni-corrected Haven equilibrium law in all reference populations. 4) Gene frequency differences (δ values) among African, European, and East Asian populations were greater than 0.5; δ values ​​were greater than 0.3 between East Asian and South Asian and American populations, between South Asian and American populations, and between European and South Asian and American populations. 5) δ values ​​were greater than 0.25 among the five East Asian populations. 6) Loci with δ values ​​in the top 10 on each chromosome were obtained among the populations. 7) AIM-SNP loci were collected from published literature. Finally, after removing duplicate and linkage-disequilibrium AIM-SNP loci, a total of 2135 SNP loci with certain ancestry inference potential were obtained. In order to generate a small number of features for efficient classification and reduce model overfitting, we performed embedded feature selection using the random forest algorithm, and finally obtained 58 AIM-SNP loci.

[0068] The 58 autosomal SNP genetic polymorphism loci (i.e., AIM-SNP loci) obtained according to the screening criteria are as follows: rs9285110, rs56142203, rs570435573, rs2261033, rs17207681, rs1250233, rs11104947, rs12006467, rs2920295, rs12130799, rs2851012, and rs118415. 89. rs722869, rs9522149, rs1288367, rs28777, rs11034709, rs1063, rs10741584, rs6123723, rs534 029120, rs4907251, rs6969817, rs2642066, rs2024566, rs6078837, rs116783706, rs2042762, rs759 6027, rs12229892, rs464165, rs4704322, rs7745461, rs7554936, rs11224765, rs79200067, rs1782 2931, rs17034666, rs17451739, rs6548616, rs3809161, rs1371048, rs11652805, rs535319466, rs10 The loci are 497191, rs17599827, rs12498138, rs59950930, rs239031, rs7799912, rs174570, rs149768401, rs3217805, rs546642722, rs10008492, rs530357165, rs2717329, and rs434124, where the rs number indicates the locus number in the dbSNP database. In addition to the 58 AIM-SNP loci mentioned above, three more loci for sex determination have been added: two Y chromosome insertion / deletion loci (InDel, rs759551978 and rs199815934) and one sex-determining enamel gene, Amelogenin (Amel). In addition to the aforementioned 58 AIM-SNP sites, two Y chromosome insertion / deletion sites and one sex-specific Amelogenin gene (Amel) form a complex system.

[0069] (2) Determination of primer sequences for AIM-SNP sites

[0070] The allele-specific primer design principle was to design three primers for each SNP site. Two specific upstream (downstream) primers had their 3' ends matched with mutant and wild-type bases, respectively. One primer had a 2-4 base mismatched nucleotide appended to its 5' end, resulting in a length difference in the amplified fragment and converting sequence polymorphism into length polymorphism, facilitating electrophoresis detection. A common downstream (upstream) primer divided the 58 SNP sites into five groups based on their fluorescent labeling groups. To effectively prevent non-specific extension, mismatched bases were artificially introduced at the penultimate or third-to-last position of the 3' end of the specific primers. The Tm value of all primers was set to (58 ± 0.5) °C, and the amplified product fragment size was less than 200 bp. For the confirmatory sequencing experiments, 58 pairs of sequencing primers were designed with a sequencing length of 300-400 bp as the standard. After primer design was completed, primer specificity was compared using Primer-Blast and UCSC In-Silico PCR Tool to select primer pairs with human specificity.

[0071] Temperature gradient tests were performed on each primer pair to determine the optimal annealing temperature. By varying primer lengths or base composition, the optimal annealing temperature for each locus was optimized to 58°C. To verify the accuracy of genotyping, five genotyped samples were randomly selected from each SNP locus and amplified using the designed sequencing primers, followed by Sanger sequencing. Homozygous genotype samples should amplify only a single peak, while heterozygous genotype samples should amplify two peaks with balanced peak heights. Only primer pairs meeting these criteria were used in subsequent experimental procedures, laying the foundation for the construction of the multiplex amplification system.

[0072] Based on the above principles and methods, the sequences of the specific amplification primers described in this invention are as follows: rs9285110: SEQ ID NO: 1-3; rs56142203: SEQ ID NO: 4-6; rs570435573: SEQ ID NO: 7-9; rs2261033: SEQ ID NO: 10-12; rs17207681: SEQ ID NO: 13-15; rs1250233: SEQ ID NO: 16-18; rs11104947: SEQ ID NO: 19-21; rs12006467: SEQ ID NO: 22-24; rs2920295: SEQ ID NO: 25-27; rs12130799: SEQ ID NO: 28-30; rs2851012: SEQ ID NO: 31-33; rs11841589: SEQ ID NO: 10 ...10-30; rs2851012: SEQ ID NO: 31-33; rs11841589: SEQ ID NO: 10-30; rs2851012: SEQ ID NO: 1 NO:34-36; rs759551978: SEQ ID NO:37-38; rs722869: SEQ ID NO:39-41; rs9522149: SEQ ID NO:42-44; rs1288367: SEQ ID NO:45-47; rs28777: SEQ ID NO:48-50; rs11034709: SEQ ID NO:51-53; rs1063: SEQ ID NO:54-56; rs10741584: SEQ ID NO:57-59; rs6123723: SEQ ID NO:60-62; rs534029120: SEQ ID NO:63-65; rs4907251: SEQ ID NO:66-68; rs6969817: SEQ ID NO:69-71; rs199815934: SEQ ID NO:72-73; rs2642066: SEQ ID NO:74-76; rs2024566: SEQ ID NO:77-79; rs6078837: SEQ ID NO:80-82; rs116783706: SEQ ID NO:83-85; rs2042762: SEQ ID NO:86-88; rs7596027: SEQ ID NO:89-91; rs12229892: SEQ ID NO:92-94; rs464165: SEQ ID NO:95-97; rs4704322: SEQ ID NO:98-100; rs7745461: SEQ ID NO:101-103; rs7554936: SEQ ID NO:104-106; rs11224765: SEQ ID NO:107-109;rs79200067: SEQ ID NO:110 - 112; rs17822931: SEQ ID NO:113 - 115; Amel: SEQ ID NO:116 - 117; rs17034666: SEQ ID NO:118 - 120; rs17451739: SEQ ID NO:121 - 123; rs6548616: SEQ ID NO:124 - 126; rs3809161: SEQ ID NO:127 - 129; rs1371048: SEQ ID NO:130 - 132; rs11652805: SEQ ID NO:133 - 135; rs535319466: SEQ ID NO:136 - 138; rs10497191: SEQ ID NO:139 - 141; rs17599827: SEQ ID NO:142 - 144; rs12498138: SEQ ID NO:145 - 147; rs59950930: SEQ ID NO:148 - 150; rs239031: SEQ ID NO:151 - 153; rs7799912: SEQ ID NO:154 - 156; rs174570: SEQ ID NO:157 - 159; rs149768401: SEQ ID NO:160 - 162; rs3217805: SEQ ID NO:163 - 165; rs546642722: SEQ ID NO:166 - 168; rs10008492: SEQ ID NO:169 - 171; rs530357165: SEQ ID NO:172 - 174; rs2717329: SEQ ID NO:175 - 177; rs434124: SEQ ID NO:178 - 180. The detailed sequences are shown in Table 1.;

[0073] All primers were grouped according to the expected amplified fragment length and assigned to five different fluorescent dyes: 6-FAM, HEX, TAMRA, ROX, and L592. The first group of SNP primers (SEQ ID NO:1 to SEQ ID NO:41) was labeled with 6-FAM fluorescent dye; the second group (SEQ ID NO:42 to SEQ ID NO:76) was labeled with HEX fluorescent dye; the third group (SEQ ID NO:77 to SEQ ID NO:112) was labeled with TAMRA fluorescent dye; the fourth group (SEQ ID NO:113 to SEQ ID NO:147) was labeled with ROX fluorescent dye; and the fifth group (SEQ ID NO:148 to SEQ ID NO:180) was labeled with L592 fluorescent dye. The distribution diagram of the 58 AIM-SNPs and 3 sex loci is shown below. Figure 1 As shown.

[0074] Table 1 Primer information for 61 sites

[0075]

[0076] (3) Determination of primer concentration for AIM-SNP sites

[0077] The amplification product fragment size and primer amplification efficiency vary significantly at each site. Based on the amplification detection results of single genes, a multiplex amplification strategy was used to systematically validate and screen primer pairs for all sites. This process involved the detection and analysis of multiple samples, aiming to gradually adjust the concentration of primers for each gene through a series of experimental optimizations to achieve the best state of amplification balance, and ultimately determine the optimal concentration of each primer pair in the multiplex detection system.

[0078] The final concentrations of each primer at the 61 sites described in this invention are shown in Table 1.

[0079] Example 2: Construction of a multiplex amplification system and detection of amplification products

[0080] Based on the successful construction of a single-site amplification system, primers for new sites were gradually introduced into the system for testing. Through a series of repeated experiments, key parameters in the multiplex amplification system were adjusted, including the number of PCR cycles, annealing temperature, final extension time, amount of polymerase, total volume of the multiplex amplification reaction system, and amount of DNA, to ensure that the multiplex system could achieve stable and balanced AIM-SNP ancestry typing results. After comprehensive optimization, the optimal total volume of the multiplex amplification reaction was finally determined to be 10 μL. The specific components of this system and their corresponding sample volumes are detailed in Table 2. The specific multiplex amplification procedure is shown in Table 3.

[0081] Table 2. Multiplex PCR amplification reaction system

[0082]

[0083] The Master Mix contains dNTPs, Taq polymerase, Tris-HCl buffer, KCl, MgCl2, and BSA (bovine serum albumin). The human biological sample to be tested can be genomic DNA extracted from human bodily fluids / tissues (such as bloodstains, blood, saliva, oral swabs, hair roots with follicles, semen, muscle tissue, exfoliated cells, etc.), and can be extracted and quantified using methods such as the Chelex-100 method, phenol-chloroform method, and magnetic bead method. It can also directly amplify various samples (such as blood filter paper, blood gauze, FTA cards, saliva cards, etc.) without extraction.

[0084] Table 3. Multiplex PCR amplification reaction procedure

[0085]

[0086] PCR amplification products were separated and detected by array capillary electrophoresis using an ABI 3500 genetic analyzer (Applied Biosystems, USA). A 36cm array was used, with an injection time of 13s at 1.2 kV. 1 μL of amplification product was mixed with 10 μL of deionized formamide and 0.3 μL of AGCU Marker SIZ-500 standard (AGCU ScienTech, Jiangsu, China). The mixture was denatured at 94℃ for 3 min, followed by an ice bath for 3 min, and then transferred to the genetic analyzer for electrophoresis. Genotyping was performed using GeneMapper ID-X software v1.5 (Applied Biosystems, USA). A Bin file was created for each marker based on the identified loci. Inserted alleles at InDel loci were named 1, and deleted alleles were named 0. All relevant Bin files were combined into a single Panel file. The Panel file was imported into the analysis method for automatic identification and interpretation of loci in the electrophoresis results.

[0087] This invention also prepares an allelic genotyping standard for capillary electrophoresis genotyping. The preparation process is as follows: First, using DNA samples containing specific homozygous alleles, confirmed by sequencing, as templates, PCR amplification is performed separately to obtain single products of each target allele. Next, the amplification product of each homozygous allele is independently diluted to the desired concentration. Then, according to a preset ratio, the diluted PCR products of each allele are mixed. The peak pattern of this mixture is analyzed using capillary electrophoresis, and the proportion of each allele in the mixture is finely adjusted based on the height of each peak. This adjustment step aims to ensure that the signal intensity of all alleles is uniform. The final mixture constitutes the allelic genotyping standard for that locus.

[0088] Allelic typing standards are composed of a mixture of the products amplified by primers at each locus, respectively, of the corresponding allelic fragments. Figure 2 This is the Allelic Ladder electrophoresis pattern of this system. This invention further tested the detection performance of the multiplex amplification system in standard 9948. Figure 3 This is the genotyping map obtained using 1 ng DNA (standard 9948). As shown in the genotyping map, all sites in the multiplex amplification system produced normal peaks, and the peak heights between sites were well balanced, indicating that the above system can effectively detect DNA samples.

[0089] Example 3: Evaluation and Model Construction of Biogeographical Ancestry Tracing Performance of 58 AIM-SNP Loci

[0090] To evaluate the ancestral information inference efficiency of the selected 58 AIM-SNP loci, population data from five continents within the 1000 Genomes dataset were used, employing a combination of population genetic analysis methods, including phylogenetic tree construction, principal component analysis (PCA), t-distributed random neighbor embedding (t-SNE), and Admixture ancestral component analysis. Based on the population differentiation index among 26 populations (…),… F ST Phylogenetic trees constructed (e.g.) Figure 4 (A) clearly shows the branching structure of the five continental populations, and further identifies three sub-branches within the East Asian population. The dimensionality reduction visualization results of PCA and t-SNE (…) Figure 4 The results of the 58 AIM-SNP loci multiplex amplification system described in this invention (TCB) indicate that it can preliminarily distinguish four intercontinental populations (Africa, Europe, East Asia, and South Asia) besides the American population. Preliminary clustering trends are also observed within the East Asian population in the t-SNE plot. Admixture ancestral component analysis results (…) Figure 4 The results (D) show that this system can effectively reveal the differences in genetic structure between different intercontinental populations and within East Asian populations at the five-continental population level, and determine the optimal ancestral component number (D). K The value is 6.

[0091] Based on the 58 AIM-SNP locus system and the 1000-person genome dataset described in this invention, this embodiment employs various machine learning algorithms (including random forest, extreme gradient boosting (XGBoost), support vector machine, and neural network) to construct a biogeographical origination model, and evaluates its performance through 10-fold cross-validation. The confusion matrix results of the 10-fold cross-validation model show that the 58 AIM-SNP locus system achieves an average accuracy exceeding 92% in classification tasks across five continental populations (Africa, Europe, East Asia, South Asia, and the Americas). Figure 5 (A). In fine-grained classification tasks within East Asian populations, the average accuracy rate exceeded 80%. Figure 5 Table 4 summarizes the performance metrics of each machine learning model on the test set after 10-fold cross-validation for classification tasks with different biogeographical ancestry ranges. The results show that the multiplex amplification system based on 58 AIM-SNP loci described in this invention exhibits good classification efficiency at both the intercontinental population level and the intra-East Asian population level. This fully demonstrates that the multiplex amplification system described in this invention can be effectively used for biogeographical ancestry tracing.

[0092] Table 4. Results of 10-fold cross-validation of the biogeographical ancestry tracing test set for 58 AIM-SNP loci.

[0093]

[0094] The above population genetic analysis results show that the 58 AIM-SNP loci of this invention have good distinguishing ability for individuals from five continental populations, and can further subdivide the ancestral origin of populations within East Asia.

[0095] Example 4: Application verification of the multiplex amplification system described in this invention

[0096] Genotyping was performed on two individuals with known biogeographical ancestry using the composite system described in Example 1, following the method (i.e., multiplex amplification system) and procedure of Example 2. DNA samples of standard 9948 were used as positive controls. The biogeographical ancestry of these individuals was inferred using the XGBoost method, and the system's biogeographical ancestry tracing efficacy for real samples was evaluated.

[0097] The genotyping results of two individuals from known sources are shown in Table 5. The predicted probabilities of the samples at 58 AIM-SNP loci are shown in Table 6.

[0098] Table 5. Genotyping results of two individuals from different known sources.

[0099]

[0100] Table 6. Predicted probability results of one sample at 58 AIM-SNP loci

[0101]

[0102] The above results indicate that genotyping could be completely detected at all loci in both real samples, and both were correctly inferred to be of true biogeographical ancestral origin. This shows that the composite system described in this invention has good detection capability and biogeographical ancestral tracing capability for real samples, and the composite amplification system and method described in this invention can be used for effective individual origin inference.

[0103] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent for detecting gene panel combinations, characterized in that, The gene panel combination includes 58 autosomal SNP sites, 2 Y chromosome deletion / insertion sites, and 1 tooth enamel gene; The 58 autosomal SNP loci include rs9285110, rs56142203, rs570435573, rs2261033, rs17207681, rs1250233, rs11104947, rs12006467, rs2920295, rs12130799, rs2851012, rs11841589, rs722869, rs 9522149, rs1288367, rs28777, rs11034709, rs1063, rs10741584, rs6123723, rs534029120, rs4907251, rs6969817, rs2642066, rs2024566, rs6078837, rs116783706, rs2042762, rs7596 027, rs12229892, rs464165, rs4704322, rs7745461, rs7554936, rs11224765, rs79200067, r s17822931, rs17034666, rs17451739, rs6548616, rs3809161, rs1371048, rs11652805, rs535 319466, rs10497191, rs17599827, rs12498138, rs59950930, rs239031, rs7799912, rs174570, rs149768401, rs3217805, rs546642722, rs10008492, rs530357165, rs2717329 and rs434124; The two Y chromosome deletion / insertion sites include rs759551978 and rs199815934; the one enamel gene is the Amelogenin gene; The reagents include specific primers.

2. The reagent according to claim 1, characterized in that, The gene panel combination is used for forensic biogeographical ancestry information inference and to differentiate between the five continental groups and the East Asian group.

3. The reagent according to claim 2, characterized in that, The sequences of the specific primers are shown in SEQ ID NO:1-SEQ ID NO:

180.

4. The reagent according to claim 3, characterized in that, At least one primer in the specific primers corresponding to each point or gene in the gene panel combination is labeled with a fluorescent dye at its 5' end.

5. The reagent according to claim 4, characterized in that, The fluorescent dye includes at least one of 6-FAM, HEX, TAMRA, ROX, and L592.

6. A kit comprising the reagent according to any one of claims 1-5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes reagents for PCR amplification and genotyping.

8. The reagent kit according to claim 7, characterized in that, The reagents used for PCR amplification and genotyping include at least one of dNTPs, enzymes, buffer solutions, KCl, MgCl2, BSA, or formamide.

9. The use of the reagent of any one of claims 1-5 or the kit of any one of claims 6-8 in (1) or (2): (1) Inference of ancestral information from forensic biogeography; (2) Distinguish between the five continental groups and the East Asian group.

10. A method comprising the step of detecting the genome of a sample to be tested using the reagent of any one of claims 1-5 or the kit of any one of claims 6-8; The method described is either a method for inferring ancestral information from forensic biogeography or a method for distinguishing between the five continental groups and the East Asian group.

11. The method according to claim 10, characterized in that, The method specifically includes the following steps: The genome of the sample to be tested was amplified using the primers shown in SEQ ID NO:1-SEQ ID NO:180 to obtain the genotyping data of the sample to be tested. The genotyping data was then analyzed in conjunction with the reference population data.

12. A detection system for inferring or differentiating between five continental populations and East Asian populations using forensic biogeographical ancestry information, comprising: Detection module: used to detect the genotyping data of the gene panel combination described in any one of claims 1-5 in the sample to be tested, and output the genotyping data to the analysis module; Analysis module: Analyzes the genotyping data with the reference population data, and infers the forensic biogeographical ancestry of the sample under test based on the analysis results or determines the intercontinental origin of the sample under test based on the analysis results.

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