CN107419017B - A method and system for inferring the origin of five intercontinental populations in individuals of unknown origin - Google Patents

Abstract

The invention discloses a SNP system for distinguishing five intercontinental populations and its application. The application provided by the present invention is specifically the application of 28 SNP sites in any of the following: (a) constructing a genotyping database of five intercontinental populations; (b) distinguishing five intercontinental populations. The system provided by the present invention can not only distinguish five intercontinental populations, but also has a certain ability to distinguish mixed populations, and the ancestral principal components and population matching of known source samples are consistent with their source information. And it can infer the composition of individual ancestral sources, which can be popularized and applied in actual cases.

Description

A method and system for inferring the origin of five intercontinental populations in individuals of unknown origin

technical field

The invention belongs to the field of biotechnology, and relates to a method and system for inferring the origin of five intercontinental ethnic groups for individuals of unknown origin.

Background technique

Ancestry informative marks (AIMs) can be detected by detecting DNA polymorphisms with large distribution differences among populations, which can infer the ethnic and geographic origin of DNA donors at crime scenes. Short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (Indels), etc. can be used as ancestry information sites (AIMs) for population inference, and play a good role. SNPs are supported by databases such as HapMap Project and 1000Genomes, and have become important genetic markers for screening AIMs loci in recent years. At present, a large number of AIMs systems have been reported for the differentiation of large intercontinental populations. For example, the 27-SNPs of this project group are used to infer three major populations in Africa, East Asia and Europe. Under the current conditions of forensic DNA laboratory detection technology, a well-performing SNPs population inference system should maintain a balanced ability to distinguish between populations on the premise of ensuring the effectiveness of population differentiation; the amount of locus information should be as high as possible, and the number of loci should be as large as possible Less; the detection method is simple and easy to implement.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method and system for inferring the origin of five intercontinental ethnic groups for individuals of unknown origin.

The present invention protects the application of 28 SNP sites in any of the following:

(a) Construction of a genotyping database for five intercontinental populations;

(b) distinguish between five intercontinental populations;

所述28个SNP位点分别为：rs10483251、rs12142199、rs1229984、rs12402499、rs12498138、rs12594144、rs1426654、rs1557553、rs16891982、rs17822931、rs1871534、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、 rs6054465, rs6437783, rs715605, rs8072587, rs8137373, rs9522149, rs9809818, rs9908046 (Table 2). The 28 SNP sites described below are the same.

The present invention protects a primer pair set for detecting 28 SNP sites in the human genome.

The primer pair set for detecting 28 SNP sites in the human genome provided by the present invention consists of the following (1)-(28): (1) primer pair 1 for detecting rs10483251, consisting of sequence 1 and It consists of two single-stranded DNAs shown in sequence 2; (2) primer pair 2 for detecting rs12142199 consists of two single-stranded DNAs shown in sequence 4 and sequence 5 in the sequence table; (3) for detecting rs1229984 The primer pair 3 is composed of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence listing; (4) primer pair 4 for detecting rs12402499 is composed of two single-stranded DNAs shown in sequence 10 and sequence 11 in the sequence listing. single-stranded DNA composition; (5) primer pair 5 for detecting rs12498138, consisting of two single-stranded DNAs shown in sequence 13 and sequence 14 in the sequence table; (6) primer pair 6 for detecting rs12594144, consisting of sequence It consists of two single-stranded DNAs shown in sequence 16 and sequence 17 in the list; (7) primer pair 7 for detecting rs1426654 consists of two single-stranded DNAs shown in sequence 19 and sequence 20 in the sequence list; (8) ) primer pair 8 for detecting rs1557553, consisting of two single-stranded DNAs shown in sequence 22 and sequence 23 in the sequence listing; (9) primer pair 9 for detecting rs16891982, consisting of sequence 25 and sequence 26 in the sequence listing (10) primer pair 10 for detecting rs17822931, consisting of two single-stranded DNAs shown in sequence 28 and sequence 29 in the sequence table; (11) primer for detecting rs1871534 Pair 11 is composed of two single-stranded DNAs shown in sequence 31 and sequence 32 in the sequence listing; (12) primer pair 12 for detecting rs2080161 is composed of two single-stranded DNAs shown in sequence 34 and sequence 35 in the sequence listing. DNA composition; (13) primer pair 13 for detecting rs2139931, consisting of two single-stranded DNAs shown in sequence 37 and sequence 38 in the sequence listing; (14) primer pair 14 for detecting rs2789823, from the sequence listing It consists of two single-stranded DNAs shown in sequence 40 and sequence 41; (15) primer pair 15 for detecting rs2814778 consists of two single-stranded DNAs shown in sequence 43 and sequence 44 in the sequence table; (16) use Primer pair 16 for detecting rs3751050 consists of two single-stranded DNAs shown in sequence 46 and sequence 47 in the sequence listing; (17) primer pair 17 for detecting rs3827760, which is shown in sequence 49 and sequence 50 in the sequence listing (18) primer pair 18 for detecting rs4657449, consisting of two single-stranded DNAs shown in sequence 52 and sequence 53 in the sequence table; (19) primer pair 19 for detecting rs4749305 , consisting of two single-stranded DNAs shown in sequence 55 and sequence 56 in the sequence listing; (20) use Primer pair 20 for detecting rs4792928, consisting of two single-stranded DNAs shown in sequence 58 and sequence 59 in the sequence listing; (21) primer pair 21 for detecting rs6054465, shown in sequence 61 and sequence 62 in the sequence listing (22) primer pair 22 for detecting rs6437783, consisting of two single-stranded DNAs shown in sequence 64 and sequence 65 in the sequence table; (23) primer pair 23 for detecting rs715605 , consisting of two single-stranded DNAs shown in Sequence 67 and Sequence 68 in the sequence listing; (24) Primer pair 24 for detecting rs8072587, consisting of two single-stranded DNAs shown in Sequence 70 and Sequence 71 in the sequence listing (25) primer pair 25 for detecting rs8137373, consisting of two single-stranded DNAs shown in sequence 73 and sequence 74 in the sequence listing; (26) primer pair 26 for detecting rs9522149, consisting of sequence 76 in the sequence listing and two single-stranded DNAs shown in sequence 77; (27) primer pair 27 for detecting rs9809818, consisting of two single-stranded DNAs shown in sequence 79 and sequence 80 in the sequence listing; (28) for detecting Primer pair 28 of rs9908046 consists of two single-stranded DNAs shown in SEQ ID NO: 82 and SEQ ID NO: 83 in the sequence listing.

In the primer pair set, the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6, the primer pair 7, the The primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair 13, the primer pair 14, the primer pair 15, the primer pair pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21, the primer pair 22, the primer pair 23, the primer pair 24 , the primer pair 25, the primer pair 26, the primer pair 27 and the primer pair 28 have a molar ratio of 0.8:0.6:1.5:2.7:3:0.8:2:1.3:1:1:1.5: 1:2.7:0.5:0.8:2.5:0.4:1.6:2.5:4:3:0.8:0.8:3:3:6:0.6:0.6. The molar ratio of both primers in each primer pair was 1:1.

The present invention protects a single-stranded DNA set for detecting 28 SNP sites in the human genome.

The single-stranded DNA set for detecting 28 SNP sites in the human genome provided by the present invention consists of the following (1)-(28): (1) primer pair 1 and extension primer 1 for detecting rs10483251; the Primer pair 1 is composed of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence listing; the extension primer 1 is the single-stranded DNA shown in sequence 3 in the sequence listing; (2) primer pair 2 for detecting rs12142199 and extension primer 2; the primer pair 2 is composed of two single-stranded DNAs shown in sequence 4 and sequence 5 in the sequence table; the extension primer 2 is the single-stranded DNA shown in sequence 6 in the sequence table; (3) use Primer pair 3 and extension primer 3 for detecting rs1229984; the primer pair 3 consists of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence table; the extension primer 3 is a single-stranded DNA shown in sequence 9 in the sequence table. stranded DNA; (4) primer pair 4 and extension primer 4 for detecting rs12402499; the primer pair 4 consists of two single-stranded DNAs shown in sequence 10 and sequence 11 in the sequence listing; the extension primer 4 is sequence The single-stranded DNA shown in sequence 12 in the list; (5) primer pair 5 and extension primer 5 for detecting rs12498138; the primer pair 5 is composed of two single-stranded DNAs shown in sequence 13 and sequence 14 in the sequence listing; The extension primer 5 is the single-stranded DNA shown in the sequence 15 in the sequence table; (6) the primer pair 6 and extension primer 6 for detecting rs12594144; the primer pair 6 is shown in the sequence table 16 and 17 in the sequence table. It consists of two single-stranded DNAs; the extension primer 6 is the single-stranded DNA shown in sequence 18 in the sequence table; (7) primer pair 7 and extension primer 7 for detecting rs1426654; the primer pair 7 consists of the sequence in the sequence table. 19 and two single-stranded DNAs shown in sequence 20; the extension primer 7 is the single-stranded DNA shown in sequence 21 in the sequence table; (8) primer pair 8 and extension primer 8 for detecting rs1557553; the primers Pair 8 is composed of two single-stranded DNAs shown in sequence 22 and sequence 23 in the sequence listing; the extension primer 8 is the single-stranded DNA shown in sequence 24 in the sequence listing; (9) primer pairs 9 and 24 for detecting rs16891982 Extension primer 9; the primer pair 9 consists of two single-stranded DNAs shown in sequence 25 and sequence 26 in the sequence listing; the extension primer 9 is the single-stranded DNA shown in sequence 27 in the sequence listing; (10) used for Primer pair 10 and extension primer 10 for detecting rs17822931; the primer pair 10 is composed of two single-stranded DNAs shown in sequence 28 and sequence 29 in the sequence listing; the extension primer 10 is the single-stranded DNA shown in sequence 30 in the sequence listing DNA; (11) primer pair 11 and extension primer 11 for detecting rs1871534; the primer pair 11 consists of two single-stranded DNAs shown in sequence 31 and sequence 32 in the sequence listing; the extension primer 11 is the sequence listing Single-stranded DNA shown in sequence 33; (12) used to detect rs20 Primer pair 12 and extension primer 12 of 80161; the primer pair 12 consists of two single-stranded DNAs shown in sequence 34 and sequence 35 in the sequence listing; the extension primer 12 is the single-stranded DNA shown in sequence 36 in the sequence listing (13) primer pair 13 and extension primer 13 for detecting rs2139931; described primer pair 13 is made up of two single-stranded DNAs shown in sequence 37 and sequence 38 in the sequence table; the extension primer 13 is in the sequence table single-stranded DNA shown in sequence 39; (14) primer pair 14 and extension primer 14 for detecting rs2789823; the primer pair 14 is composed of two single-stranded DNAs shown in sequence 40 and sequence 41 in the sequence listing; the The extension primer 14 is the single-stranded DNA shown in the sequence 42 in the sequence listing; (15) the primer pair 15 and extension primer 15 used to detect rs2814778; the primer pair 15 consists of the two sequences shown in the sequence 43 and the sequence 44 in the sequence listing. Single-stranded DNA composition; the extension primer 15 is the single-stranded DNA shown in sequence 45 in the sequence listing; (16) primer pair 16 and extension primer 16 for detecting rs3751050; the primer pair 16 consists of sequence 46 and 46 in the sequence listing. It consists of two single-stranded DNAs shown in sequence 47; the extension primer 16 is the single-stranded DNA shown in sequence 48 in the sequence listing; (17) primer pair 17 and extension primer 17 for detecting rs3827760; the primer pair 17 It consists of two single-stranded DNAs shown in sequence 49 and sequence 50 in the sequence listing; the extension primer 17 is the single-stranded DNA shown in sequence 51 in the sequence listing; (18) Primer pair 18 and extension primer for detecting rs4657449 18; the primer pair 18 is composed of two single-stranded DNAs shown in the sequence 52 and the sequence 53 in the sequence listing; the extension primer 18 is the single-stranded DNA shown in the sequence 54 in the sequence listing; (19) for detecting rs4749305 The primer pair 19 and the extension primer 19 are composed of the two single-stranded DNAs shown in the sequence 55 and the sequence 56 in the sequence listing; the extension primer 19 is the single-stranded DNA shown in the sequence 57 in the sequence listing; (20) a primer pair 20 and an extension primer 20 for detecting rs4792928; the primer pair 20 is composed of two single-stranded DNAs shown in sequence 58 and sequence 59 in the sequence table; the extension primer 20 is the sequence in the sequence table. The single-stranded DNA shown in 60; (21) a primer pair 21 and an extension primer 21 for detecting rs6054465; the primer pair 21 is composed of two single-stranded DNAs shown in sequence 61 and sequence 62 in the sequence listing; the extension Primer 21 is the single-stranded DNA shown in sequence 63 in the sequence listing; (22) primer pair 22 and extension primer 22 for detecting rs6437783; the primer pair 22 consists of two single-stranded DNAs shown in sequence 64 and sequence 65 in the sequence listing. Strand DNA composition; the extension primer 22 is the single-stranded DNA shown in sequence 66 in the sequence table; (23) primer pair 23 for detecting rs715605 and extension primer 23; the primer pair 23 is composed of two single-stranded DNAs shown in sequence 67 and sequence 68 in the sequence listing; the extension primer 23 is the single-stranded DNA shown in sequence 69 in the sequence listing; (24) use Primer pair 24 and extension primer 24 for detecting rs8072587; the primer pair 24 consists of two single-stranded DNAs shown in SEQ ID NO: 70 and SEQ ID NO: 71 in the sequence listing; stranded DNA; (25) primer pair 25 and extension primer 25 for detecting rs8137373; the primer pair 25 consists of two single-stranded DNAs shown in sequence 73 and sequence 74 in the sequence listing; the extension primer 25 is the sequence The single-stranded DNA shown in sequence 75 in the list; (26) primer pair 26 and extension primer 26 for detecting rs9522149; the primer pair 26 is composed of two single-stranded DNAs shown in sequence 76 and sequence 77 in the sequence listing; The extension primer 26 is the single-stranded DNA shown in the sequence 78 in the sequence listing; (27) the primer pair 27 and extension primer 27 for detecting rs9809818; the primer pair 27 is shown in the sequence 79 and the sequence 80 in the sequence listing. It consists of two single-stranded DNAs; the extension primer 27 is the single-stranded DNA shown in sequence 81 in the sequence listing; (28) a primer pair 28 and an extension primer 28 for detecting rs9908046; the primer pair 28 consists of the sequence in the sequence listing. 82 and two single-stranded DNAs shown in sequence 83; the extension primer 28 is the single-stranded DNA shown in sequence 84 in the sequence listing.

In the single-stranded DNA group, the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6, the primer pair 7, The primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair 13, the primer pair 14, the primer pair 15, the primer pair primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21, the primer pair 22, the primer pair 23, the primer pair 24. The molar ratio of the primer pair 25, the primer pair 26, the primer pair 27 and the primer pair 28 is 0.8:0.6:1.5:2.7:3:0.8:2:1.3:1:1:1.5 : 1: 2.7: 0.5: 0.8: 2.5: 0.4: 1.6: 2.5: 4: 3: 0.8: 0.8: 3: 3: 6: 0.6: 0.6; the molar ratio of both primers in each primer pair is 1: 1. The extension primer 1, the extension primer 2, the extension primer 3, the extension primer 4, the extension primer 5, the extension primer 6, the extension primer 7, the extension primer 8, the extension primer extension primer 9, the extension primer 10, the extension primer 11, the extension primer 12, the extension primer 13, the extension primer 14, the extension primer 15, the extension primer 16, the extension primer 17. The extension primer 18, the extension primer 19, the extension primer 20, the extension primer 21, the extension primer 22, the extension primer 23, the extension primer 24, the extension primer 25, The molar ratio of the extension primer 26, the extension primer 27 and the extension primer 28 is 0.45:0.35:1.2:1.7:4:1:3:0.8:1:0.8:1.8:1.1:1.1:1.1:0.8 :1.4:1:0.5:1.3:1.8:1.6:0.9:0.9:2:2.3:3:1:0.6.

The present invention also protects a kit for distinguishing between five continents.

The kit for distinguishing five intercontinental populations provided by the present invention contains the aforementioned primer pair set or single-stranded DNA set.

According to needs, the kit may further contain at least one of the following substances: dNTP, DNA polymerase, alkaline phosphatase.

The present invention also protects the use of a substance for detecting 28 SNP sites in any of the following:

(a) Construction of a genotyping database for five intercontinental populations;

(b) Distinguish between five intercontinental populations.

Wherein, the substance for detecting 28 SNP sites may be the primer pair set or single-stranded DNA set or kit described above.

The present invention also protects a method for constructing a genotyping database of five intercontinental populations.

The method for constructing a genotyping database of five intercontinental populations provided by the present invention may specifically include the following steps:

(a1) Selecting the 28 SNP loci from the 1000 Genomes Project and the Human Genome Diversity Project for typing of five intercontinental populations to form an original typing library;

(a2) Perform structure cluster analysis on all the samples in the original genotyping database, and select the part with the ancestral principal component greater than 90% to constitute the genotyping database of the five intercontinental populations.

Structure is a free, multi-platform (Windows, Mac, Linux), open source, classical bioinformatics that implements model-based clustering methods using multi-locus genotype data consisting of unlinked markers to infer population composition structure It is widely used in human genetics, population genetics, forensic genetics and other fields. The parameters used are: 10000burnins, 10000repetitions, mixed model; the running result can know the proportion of ancestral components of each sample.

The present invention also protects a method of distinguishing between five intercontinental populations.

The method for distinguishing five intercontinental populations provided by the present invention may specifically include the following steps:

(b1) Constructing a genotyping database of five intercontinental populations according to the method described above;

(b2) extracting the genomic DNA of the subject to be tested, and performing the detection of 28 SNP sites to obtain the original genotype data of the subject to be tested on the 28 SNP sites;

(b3) Comparing the original genotype data of the test subject at the 28 SNP sites with the five-continental population genotyping database by an analytical method, so as to determine that the test subject belongs to five continents which one in the crowd.

In the above two methods, when the detection of the 28 SNP sites is carried out, the primer pair set or single-stranded DNA set or kit described above is used; 28-fold PCR amplification is carried out, and the annealing temperature is 55°C.

The five continents mentioned above are East Asia, Europe, Africa, Oceania and the Americas.

In order to realize the distinction of five intercontinental populations (East Asia, Europe, Africa, Oceania and America), the present invention screened out 28 SNP sites, constructed a composite detection system, and used the system to detect 712 samples from 16 populations. A total of 2804 individual genotyping data were combined from 20 populations in the 1000 Genomes and 2 populations in the CEPH database. Cluster analysis and principal component analysis were used to evaluate the system efficacy. Select samples with an ancestral principal component greater than 90% to construct a reference population genotyping database, perform population matching probability and individual principal component analysis on 140 individuals with known ancestral origins to infer population origin, and evaluate the population of the system in actual samples. Source discrimination ability. The results show that the system can not only distinguish five intercontinental populations, but also has a certain ability to distinguish mixed populations. The ancestral principal components and population matching of samples from known sources are consistent with their source information. And it can infer the composition of individual ancestral sources, which can be popularized and applied in actual cases.

Description of drawings

Figure 1 shows the genotyping diagram of samples with a DNA concentration of 5 ng/μL (28-plex SNP detection results). In the figure, 1-28 represent 28 SNPs (corresponding to the numbers in Table 2), and each number is followed by the corresponding typing data (since some detect complementary strands, the typing data displayed after the number is the complementary base in Table 2).

Figure 2 is a principal component analysis of genotype frequency based on 28SNPs in 38 populations. A: African individuals; B: American individuals; C: East Asian individuals; D: European individuals; E: mixed population; F: Oceania individuals.

Figure 3 shows the results of Structure analysis of 28 SNPs on 38 populations.

Figure 4 is a graph of the population classification analysis of the test samples. A: African individuals; B: American individuals; C: East Asian individuals (Guangxi Han); D: East Asian individuals (Henan Han); E: European individuals; F: Uighurs; G: Oceania individuals.

Fig. 5 is a graph showing the detection results of the "9947" and "HWQ" samples of the present invention and the article system. A: "9947" sample article system test result; B: "HWQ" sample article article system test result; C: "9947" sample article system test result; D: "HWQ" sample article system test result. In A-D, 1-28 represent 28 SNPs (corresponding to the numbers in the "Number" column in Table 6), and each number is followed by the corresponding typing data.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the construction and application of the five intercontinental population genotyping database of the present invention

1. Materials and methods

1. Sample information

The present invention selects 2000 samples from 20 populations in the public database Thousand Genomes (1000genomes) and 92 samples from 2 populations in the Human Genome Diversity Project (HGDP-CEPH), and the detection samples include 712 samples from 16 populations. 2804 individuals from 38 populations were used as validation samples. See Table 1 for details.

Table 1 Population sample information table

Note: Italics indicate the test population, and brackets indicate the number of selected test samples.

2. Source of SNPs

By analyzing "The Global AIMs Nano set", 31 AIMs were deleted (de la Puente M, Santos C, Fondevila M, et al. The Global AIMs Nano set: A 31-plex SNaPshot assay of ancestry-informative SNPs [J ]. Forensic Science International: Genetics, 2016, 22: 81-88.), the 3 triallelic SNP sites were used, and the remaining 28 biallelic SNPs sites were used to construct a composite system. For site information, see Table 2.

Table 2 Detailed information of 28 SNP sites

3. PCR compound amplification

plusDNA聚合酶(QIAGEN公司，德国)0.1μL，5ng/μL模板DNA 1μL，水补足至5μL。PCR反应条件：95℃10min后；95℃30s，55℃40s，72℃1min，循环40次；最后延伸72℃20min。纯化反应体系为7.5μL，内含扩增产物5μL，H₂O 1μL，ExoΙ(10U/μL)0.2μL，SAP(1U/μL)1μL，10×SAP buffer0.3μL。充分振荡混匀后37℃孵育45min，85℃15min灭活酶活性。The PCR compound amplification reaction system is 5μL, containing 0.6μL of 10×PCR buffer (containing Mg ²⁺ 15mmol/L), 0.9μL of 25mmol/LMgCl ₂ , 0.1μL of 10mmol/L dNTP, 0.7μL of compound amplification primers, 5U/L μL

plus DNA polymerase (QIAGEN, Germany) 0.1 μL, 5 ng/μL template DNA 1 μL, and water to make up to 5 μL. PCR reaction conditions: 95°C for 10min; 95°C for 30s, 55°C for 40s, 72°C for 1min, cycle 40 times; final extension at 72°C for 20min. The purification reaction system was 7.5 μL, containing 5 μL of amplification product, 1 μL of H ₂ O, 0.2 μL of ExoI (10 U/μL), 1 μL of SAP (1 U/μL), and 0.3 μL of 10×SAP buffer. After fully shaking and mixing, incubate at 37°C for 45min, and at 85°C for 15min to inactivate the enzyme activity.

Multiplex Kit(ABI公司，美国)进行单碱基延伸反应，采用5.5μL体系，内含纯化后PCR产物2μL，复合延伸引物1μL，SNaPshot mix 2.5μL。PCR反应条件：96℃10s后；59℃5s，60℃30s，循环33次。之后向反应产物中加入1μL SAP(1U/μL)，37℃孵育80min，85℃15min进行纯化处理，去除多余的引物和dNTP。use

Multiplex Kit (ABI, USA) was used for single-base extension reaction, using a 5.5 μL system, containing 2 μL of purified PCR product, 1 μL of composite extension primers, and 2.5 μL of SNaPshot mix. PCR reaction conditions: 96°C for 10s; 59°C for 5s, 60°C for 30s, cycle 33 times. Then, 1 μL of SAP (1 U/μL) was added to the reaction product, incubated at 37° C. for 80 min, and purified at 85° C. for 15 min to remove excess primers and dNTPs.

Capillary electrophoresis was performed on the above-mentioned extension purified products on a 3130-XL Genetic Analyzer (ABI, USA). The detection system was 10 μL, including 1 μL of the purified product after single-base extension, 9 μL of formamide and a mixture of internal standard GeneScanLiz-120 (38:1 volume ratio) (ABI Company, USA). Electrophoresis parameter settings: injection time 18s, injection voltage 3kV, electrophoresis voltage 13.4kV, and electrophoresis time 15min. Genotyping analysis was performed according to Genmapper ID v3.2 software.

Among them, the primer sequences and concentrations used to detect each SNP site are shown in Table 3.

Table 3 Primer sequences and their concentrations used to detect each SNP site

site label Amplification primer (F) Amplification primer (R) Amplification primer concentration (μM) extension primer number of bases Extension primer concentration (μM) rs10483251 EX-28*22 sequence 1 sequence 2 0.8 sequence 3 48 0.45 rs12142199 EX-28*23 sequence 4 sequence 5 0.6 sequence 6 63 0.35 rs1229984 EX-28*2 sequence 7 sequence 8 1.5 sequence 9 73 1.2 rs12402499 EX-28*24 sequence 10 sequence 11 2.7 sequence 12 51 1.7 rs12498138 EX-28*25 sequence 13 sequence 14 3 sequence 15 63 4 rs12594144 EX-28*26 sequence 16 sequence 17 0.8 sequence 18 40 1 rs1426654 EX-28*3 sequence 19 sequence 20 2 sequence 21 67 3 rs1557553 EX-28*4 sequence 22 sequence 23 1.3 sequence 24 93 0.8 rs16891982 EX-28*27 sequence 25 sequence 26 1 sequence 27 80 1 rs17822931 EX-28*28 sequence 28 sequence 29 1 sequence 30 59 0.8 rs1871534 EX-28*5 sequence 31 sequence 32 1.5 sequence 33 105 1.8 rs2080161 EX-28*6 sequence 34 sequence 35 1 sequence 36 48 1.1 rs2139931 EX-28*7 sequence 37 sequence 38 2.7 sequence 39 109 1.1 rs2789823 EX-28*8 sequence 40 sequence 41 0.5 sequence 42 51 1.1 rs2814778 EX-28*9 sequence 43 sequence 44 0.8 sequence 45 89 0.8 rs3751050 EX-28*10 sequence 46 sequence 47 2.5 sequence 48 44 1.4 rs3827760 EX-28*11 sequence 49 sequence 50 0.4 sequence 51 89 1 rs4657449 EX-28*12 sequence 52 sequence 53 1.6 sequence 54 93 0.5 rs4749305 EX-28*13 sequence 55 sequence 56 2.5 sequence 57 67 1.3 rs4792928 EX-28*14 sequence 58 sequence 59 4 sequence 60 99 1.8 rs6054465 EX-28*15 sequence 61 sequence 62 3 sequence 63 84 1.6 rs6437783 EX-28*16 sequence 64 sequence 65 0.8 sequence 66 45 0.9 rs715605 EX-28*1 sequence 67 sequence 68 0.8 sequence 69 113 0.9 rs8072587 EX-28*17 sequence 70 sequence 71 3 sequence 72 96 2 rs8137373 EX-28*18 sequence 73 sequence 74 3 sequence 75 100 2.3 rs9522149 EX-28*19 sequence 76 sequence 77 6 sequence 78 72 3 rs9809818 EX-28*20 sequence 79 sequence 80 0.6 sequence 81 55 1 rs9908046 EX-28*21 sequence 82 sequence 83 0.6 sequence 84 109 0.6

2. Software and Analysis Methods

1. Principal Component Analysis (PCA)

Use Rv3.2.3 software to conduct principal component analysis: a. Divide 38 populations, including public database samples and test samples, into Africa, America, East Asia, Europe, Oceania, mixed populations (Central Asia, South Asia, South Asia), and perform population principal component analysis based on gene frequency; b. Randomly select one sample from each of the seven test populations (Table 1) for individual principal component analysis (perform principal component analysis with R v3.3.2, and use R v3.3.2 for principal component analysis, and use The R package ggplot2 draws racial categorization graphs).

2. Cluster analysis

For the 38 populations in Table 1, use Structure.v2.3.4 software to perform clustering analysis (K takes 3-7), analyze the genetic structure of each population, and use Distruct 1.1 to draw a population clustering result map; The 7 samples of principal component analysis were used for the statistics of individual ancestry components.

Structure is a free, multi-platform (Windows, Mac, Linux), open source, classical bioinformatics that implements model-based clustering methods using multi-locus genotype data consisting of unlinked markers to infer population composition structure It is widely used in human genetics, population genetics, forensic genetics and other fields. The parameters used are: 10000burnins, 10000repetitions, mixed model; the running result can know the proportion of ancestral components of each sample.

3. Probability of random population matching

Forensic intelligence software was used to calculate random population matching for 140 randomly selected test samples (marked in Table 1) from 7 populations.

3. Experimental results

1. 28-plex SNP test results

The results are shown in Figure 1. It can be seen from the figure that: 28 AIMs meet the following criteria: 1) maintain a good balance in the discrimination ability of five intercontinental populations; 2) the interval between loci is at least 1Mb, which reduces the chance of linked inheritance. The system adopts the relatively popular SNaPshot detection and typing technology, and alleles of 28 loci can be obviously judged for typing.

2. Evaluation of system differentiation efficiency

(1) Principal Component Analysis

The results are shown in Figure 2. It can be seen from the figure that Principal Component 1 (PC1) and Principal Component 2 (PC2) explain 61.3% of the difference, and 28 loci can clearly distinguish 38 populations into six major parts. In Table 1, we found that the more concentrated populations are from the same intercontinental ancestry, and 30 populations from five continents (Africa, Europe, East Asia, America, and Oceania) were clearly divided into five major parts. In principal component 1, Central America , East Asia, Oceania, Africa, and Europe are divided in turn. Among them, the East Asian population and the European population are relatively concentrated, and the mixed population (8) is relatively scattered, but they are all located between the East Asian population and Europe. This part belongs to In the mixed populations of South Asia and Eurasian, the non-centralized distribution further illustrates the complexity of their genetic structure and needs further research; in principal component 2, Oceania and America populations are distinguished.

(2) Cluster analysis

Using the 28 AIMs, a total of 2804 samples from the above 38 populations were analyzed for genetic structure. The results are shown in Figure 3. It can be seen from the figure that when K = 3, the population is clustered into three parts: Africa and Europe are separated, the American, Oceanian and East Asian populations show the same ancestral composition, and the Uighurs are mixed. The genetic component of the population presents a continuous distribution of European and East Asian ancestral components. As the K value increases, new ancestral principal components appear successively in the Americas and Oceania. When the K value is 6, all individuals correspond to 6 population groups: Africa, America, Oceania, East Asia, mixed populations, and Europe, and mixed populations appear. The independent ancestral component shows that the mixed race of Uyghur and South Asia has become a transitional race with relatively stable genetic components after long-term fusion and evolution. When the K value increased to 7, a new component appeared in the mixed population of South Asia, indicating that it was different from the mixed population of Central Asia in ancestral origin and genetic structure. When the K value continued to increase, there was no further stratification in the East Asian population, indicating that the system could not further differentiate the subpopulations in the East Asian region.

(3) Population-derived inference test for individual individuals

In order to avoid the appearance of mixed components and achieve the maximum distinguishing ability at the same time, K=6 is selected as the ideal distinguishing ability of the system; at the same time, in order to be further applied to actual forensic cases and improve the accuracy of inference, the present invention selects the ancestral principal component greater than 90% of the individuals (2201 samples in total) were used as reference samples to construct a reference population genotyping database, and the following statistical methods were used to evaluate the individual inference ability of the system.

A. Likelihood ratio

The probability of random population matching was calculated for 140 samples of known individual origin based on the reference database (excluding 140 test samples), and the statistics of possible intercontinental population sources were performed based on the likelihood ratio. The population matching probability is the random matching probability. Simply put, it is the estimated probability that a specific type of a combination of loci may appear in the population. It can also be understood as a random sample drawn from the population. type of theoretical probability. The calculation of the LR value is as follows: the matching probability of the group with the largest unknown individual probability is the denominator, the matching probability of other groups is the numerator, and the likelihood ratios of different groups are obtained in turn. The results are shown in Table 4. Among the 140 test samples, 137 have the same ancestry source inferred with the sample information; the other 3 samples from the Uyghur ethnic group are from mixed population, East Asia and Europe, but their likelihood ratios are all less than 100. , the inference results do not exclude sample information. To sum up, the absolute accuracy rate of the system inferring the ancestral source of the test sample is 97.86%, and another 2.14% cannot exclude the source of sample information. Combining with Table 4, it can be seen that the system has high accuracy in inferring the origin of five intercontinental populations, while inferring mixed populations requires comprehensive analysis of ancestry components and MP values. In addition, race and ethnicity are not the same concept. The Uighurs are located at the border of Asia and Europe, and belong to the mixed race of Europe and Asia. There is no obvious boundary between the integration of different races, and the ethnic information in the household registration is not completely consistent with the race. Three Although each Uyghur sample did not meet the criteria for discrimination, the inferred results were in line with its geographical distribution. In the future, various aspects should be considered comprehensively in the differentiation of this population.

Table 4 Test sample matching probability results

B. Population Origin Classification of Test Subjects

From the above 140 test samples, one sample was randomly selected from each test population to conduct individual principal component analysis based on the constructed reference database to infer its population origin, as shown in Figure 4. The calculation statistics of the ancestral components of the seven samples are shown in the table. 5. From the results of individual principal component analysis (Figure 4), it can be seen that the seven known individual samples can all fall into the corresponding ancestral population. In the calculation of individual ancestry components (Table 5), we can see that the ancestral main components of the seven test samples The ingredients are all over 94%.

Table 5 Analysis results of ancestry origin of seven test samples

Based on the results of this example, it can be seen that the SNP composite system provided by the present invention can effectively carry out genetic structure analysis and individual ancestral origin inference of five major populations and mixed populations in East Asia, Europe, Africa, America and Oceania. Considering the frequent and wide flow of population in today's society, the system uses fewer sites to distinguish between five intercontinental populations, compared to our previously established 27-Plex SNP for three intercontinental populations of Europe, Asia, and Africa. The coverage is wider, and the two systems can confirm each other's ancestral origin inference for unknown samples in forensic DNA testing, providing more and more accurate investigative clues for cases.

Embodiment 2, the comparison of the present invention and prior art

In this example, standard European DNA samples (9947) and East Asian DNA samples (HWQ, Asians in the inventor's laboratory) were used to further verify the 28 systems of the present invention and 31 AIMs (de la Puente M, Santos C , Fondevila M, et al. The Global AIMs Nano set: A 31-plex SNaPshot assay ofancestry-informative SNPs [J]. Forensic Science International: Genetics, 2016, 22: 81-88.) The pros and cons of both.

The 28-system group of the present invention: operate according to the relevant steps in Example 1.

Article 31 AIMs set: According to "de la Puente M, Santos C, Fondevila M, et al. TheGlobal AIMs Nano set:A 31-plex SNaPshot assay of ancestry-informative SNPs[J].Forensic Science International:Genetics,2016, 22:81-88." The operation is carried out as described in the text, that is, the amplification primers and extension primers are matched according to the concentrations in the article (three trialleles are removed).

The results are shown in Table 6 and Figure 5. For the standard European DNA sample of "9947", the identification results using the 28 system of the present invention and the article system are consistent; and for the East Asian DNA sample "HWQ", the identification results using the 28 system of the present invention and the article system are used. There were two inconsistencies (see the bold underlined part in Table 6 for details), and the results confirmed that the detection results of the 28 system of the present invention were correct by single amplification and sequencing of the inconsistent sites. It can be seen that, compared with the 31 AIMs in the article, the 28 system of the present invention further improves the accuracy of the detection results while reducing the number of SNP sites. The detection results are more accurate.

It can be seen from Figure 5 that the amplification primers and extension primers are matched according to the concentrations in the article (excluding three trialleles), and the following problems mainly occur: the 31 AIMs groups in the article have incomplete locus peaks and incomplete locus peaks. Equilibrium (heterozygous peak heights differ greatly), and peak overlap between certain sites has an impact on the type. The 28 system of the present invention effectively solves the above problems: 1. The solution for "incomplete peaks at sites": adjust the ratio between primers. 2. The solution to "imbalance of site peaks (heterozygote peak heights are quite different)": reduce the annealing temperature while ensuring the specificity of the peaks. 3. The solution for "overlapping peaks between certain sites and affecting the type": adjust the length of the extension primer. The 28 system of the present invention finally achieves accurate typing through the above adjustments.

Table 6 The detection results of the two detection systems of the present invention and the article to "9947" and "HWQ"

<110> Ministry of Public Security Material Evidence Identification Center

<120> A method and system for inferring the origin of five intercontinental populations for individuals of unknown origin

<130> GNCLN171048

<160> 84

<170> PatentIn version 3.5

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gcacgttctt aaccttggct at 22

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ttctgaatat cccacccaca a 21

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gtgccacgtc gtgaaagtct gacaaggaaa aagttatgtg accagatt 48

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aacaactgac taaactaggt gccacgtcgt gaaagtctga caatcaaaca tgttcctctg 60

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caactgacta aactaggtgc cacgtcgtga aagtctgaca agggaatcct gttattcaca 60

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taatacaaga gccgcctgga 20

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taaactaggt gccacgtcgt gaaagtctga caacaactga ctaaactagg tgccacgtcg 60

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gaataaagtg aggaaaacac ggagt 25

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gtttctcatc tacgaaagag gagtc 25

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cacgtcgtga aagtctgaca acaactgact aaactaggtg ccacgtcgtg aaagtctgac 60

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cctagagtcc cccaaacctc 20

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acatcctgca gaccttcctg 20

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cagaccttgg gcgtcagat 19

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ctgacaacaa ctgactaaac taggtgccac gtcgtgaaag tctgacaaca actgactaaa 60

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ctcctagtca tggttgatgt gg 22

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acaacaactg actaaactag gtgccacgtc gtgaaagtct gacaacaact gactaaacta 60

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agtctgacaa ctaggtgcca cgtcgtgaaa gtctgacaac taggtgccac gtcgtgaaag 60

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actaggtgcc acgtcgtgaa agtctgacaa caactgacta aactaggtgc cacgtcgtga 60

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aaactaggtg ccacgtcgtg aaagtctgac aacaactgac taaactaggt gccacgtcgt 60

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caactgacta aactaggtgc cacgtcgtga aagtctgaca acaactgact aaactaggtg 60

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tgacaacaac tgactaaact aggtgccacg tcgtgaaagt ctgacaacaa ctgactaaac 60

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gaaagtctga caacaactga ctaaactagg tgccacgtcg tgaaagtctg acaacaactg 60

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aactgactaa actaggtgcc acgtcgtgaa agtctgacaa caactgacta aactaggtgc 60

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acaactgact aaactaggtg ccacgtcgtg aaagtctgac aacaactgac taaactaggt 60

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agtctgacaa caactgacta aactaggtgc cacgtcgtga aagtctgaca acaactgact 60

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Claims (11)

1. The application of 28 SNP site combinations in any of the following: (a) Construction of a genotyping database for five intercontinental populations; (b) distinguish between five intercontinental populations; 所述28个SNP位点分别为：rs10483251、rs12142199、rs1229984、rs12402499、rs12498138、rs12594144、rs1426654、rs1557553、rs16891982、rs17822931、rs1871534、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、 rs6054465, rs6437783, rs715605, rs8072587, rs8137373, rs9522149, rs9809818, and rs9908046. 2. A primer pair set for detecting 28 SNP sites in the human genome; the 28 SNP sites are: rs10483251, rs12142199, rs1229984, rs12402499, rs12498138, rs12594144, rs1426654, rs1557553, rs16891982, 3534829 rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、rs6054465、rs6437783、rs715605、rs8072587、rs8137373、rs9522149、rs9809818和rs9908046；所述引物对组由如下(1)-(28)组成： (1) Primer pair 1 for detecting rs10483251, consisting of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence listing; (2) Primer pair 2 for detecting rs12142199, consisting of two single-stranded DNAs shown in sequence 4 and sequence 5 in the sequence listing; (3) Primer pair 3 for detecting rs1229984, consisting of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence listing; (4) Primer pair 4 for detecting rs12402499, consisting of two single-stranded DNAs shown in sequence 10 and sequence 11 in the sequence listing; (5) Primer pair 5 for detecting rs12498138, consisting of two single-stranded DNAs shown in sequence 13 and sequence 14 in the sequence listing; (6) Primer pair 6 for detecting rs12594144, consisting of two single-stranded DNAs shown in sequence 16 and sequence 17 in the sequence listing; (7) Primer pair 7 for detecting rs1426654, consisting of two single-stranded DNAs shown in sequence 19 and sequence 20 in the sequence listing; (8) Primer pair 8 for detecting rs1557553, consisting of two single-stranded DNAs shown in sequence 22 and sequence 23 in the sequence listing; (9) primer pair 9 for detecting rs16891982, consisting of two single-stranded DNAs shown in sequence 25 and sequence 26 in the sequence listing; (10) primer pair 10 for detecting rs17822931, consisting of two single-stranded DNAs shown in sequence 28 and sequence 29 in the sequence listing; (11) Primer pair 11 for detecting rs1871534, consisting of two single-stranded DNAs shown in sequence 31 and sequence 32 in the sequence listing; (12) primer pair 12 for detecting rs2080161, consisting of two single-stranded DNAs shown in sequence 34 and sequence 35 in the sequence listing; (13) primer pair 13 for detecting rs2139931, consisting of two single-stranded DNAs shown in sequence 37 and sequence 38 in the sequence listing; (14) primer pair 14 for detecting rs2789823, consisting of two single-stranded DNAs shown in sequence 40 and sequence 41 in the sequence listing; (15) primer pair 15 for detecting rs2814778, consisting of two single-stranded DNAs shown in sequence 43 and sequence 44 in the sequence listing; (16) primer pair 16 for detecting rs3751050, consisting of two single-stranded DNAs shown in sequence 46 and sequence 47 in the sequence listing; (17) primer pair 17 for detecting rs3827760, consisting of two single-stranded DNAs shown in sequence 49 and sequence 50 in the sequence listing; (18) primer pair 18 for detecting rs4657449, consisting of two single-stranded DNAs shown in sequence 52 and sequence 53 in the sequence listing; (19) primer pair 19 for detecting rs4749305, consisting of two single-stranded DNAs shown in sequence 55 and sequence 56 in the sequence listing; (20) primer pair 20 for detecting rs4792928, consisting of two single-stranded DNAs shown in sequence 58 and sequence 59 in the sequence listing; (21) primer pair 21 for detecting rs6054465, consisting of two single-stranded DNAs shown in sequence 61 and sequence 62 in the sequence listing; (22) primer pair 22 for detecting rs6437783, consisting of two single-stranded DNAs shown in sequence 64 and sequence 65 in the sequence listing; (23) primer pair 23 for detecting rs715605, consisting of two single-stranded DNAs shown in sequence 67 and sequence 68 in the sequence listing; (24) primer pair 24 for detecting rs8072587, consisting of two single-stranded DNAs shown in sequence 70 and sequence 71 in the sequence listing; (25) primer pair 25 for detecting rs8137373, consisting of two single-stranded DNAs shown in sequence 73 and sequence 74 in the sequence listing; (26) primer pair 26 for detecting rs9522149, consisting of two single-stranded DNAs shown in sequence 76 and sequence 77 in the sequence listing; (27) primer pair 27 for detecting rs9809818, consisting of two single-stranded DNAs shown in sequence 79 and sequence 80 in the sequence listing; (28) Primer pair 28 for detecting rs9908046, consisting of two single-stranded DNAs shown in SEQ ID NO: 82 and SEQ ID NO: 83 in the sequence listing. 3. The primer pair set according to claim 2, wherein: in the primer pair set, the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 4, the primer pair primer pair 5, the primer pair 6, the primer pair 7, the primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair 13. The primer pair 14, the primer pair 15, the primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21, The molar ratio of the primer pair 22, the primer pair 23, the primer pair 24, the primer pair 25, the primer pair 26, the primer pair 27 and the primer pair 28 is 0.8:0.6:1.5 :2.7:3:0.8:2:1.3:1:1:1.5:1:2.7:0.5:0.8:2.5:0.4:1.6:2.5:4:3:0.8:0.8:3:3:6:0.6:0.6 ; The molar ratio of both primers in each primer pair was 1:1. 4. Single-stranded DNA set for detecting 28 SNP sites in the human genome; the 28 SNP sites are: rs10483251, rs12142199, rs1229984, rs12402499, rs12498138, rs12594144, rs14276654, rs1557553, rs16891982, rs15348222 、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、rs6054465、rs6437783、rs715605、rs8072587、rs8137373、rs9522149、rs9809818和rs9908046；所述单链DNA组由如下(1)-(28) composition: (1) Primer pair 1 and extension primer 1 for detecting rs10483251; the primer pair 1 consists of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence table; the extension primer 1 is the sequence in the sequence table. 3 shows the single-stranded DNA; (2) Primer pair 2 and extension primer 2 for detecting rs12142199; the primer pair 2 consists of two single-stranded DNAs shown in sequence 4 and sequence 5 in the sequence table; the extension primer 2 is the sequence in the sequence table. The single-stranded DNA shown in 6; (3) primer pair 3 and extension primer 3 for detecting rs1229984; the primer pair 3 is composed of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence table; the extension primer 3 is the sequence in the sequence table. The single-stranded DNA shown in 9; (4) Primer pair 4 and extension primer 4 for detecting rs12402499; the primer pair 4 is composed of two single-stranded DNAs shown in sequence 10 and sequence 11 in the sequence table; the extension primer 4 is the sequence in the sequence table. The single-stranded DNA shown in 12; (5) Primer pair 5 and extension primer 5 for detecting rs12498138; the primer pair 5 is composed of two single-stranded DNAs shown in sequence 13 and sequence 14 in the sequence table; the extension primer 5 is the sequence in the sequence table. Single-stranded DNA shown in 15; (6) primer pair 6 and extension primer 6 for detecting rs12594144; the primer pair 6 is composed of two single-stranded DNAs shown in sequence 16 and sequence 17 in the sequence table; the extension primer 6 is the sequence in the sequence table. The single-stranded DNA shown in 18; (7) Primer pair 7 and extension primer 7 for detecting rs1426654; the primer pair 7 is composed of two single-stranded DNAs shown in sequence 19 and sequence 20 in the sequence table; the extension primer 7 is the sequence in the sequence table. The single-stranded DNA shown in 21; (8) Primer pair 8 and extension primer 8 for detecting rs1557553; the primer pair 8 is composed of two single-stranded DNAs shown in sequence 22 and sequence 23 in the sequence table; the extension primer 8 is the sequence in the sequence table. Single-stranded DNA shown in 24; (9) Primer pair 9 and extension primer 9 for detecting rs16891982; the primer pair 9 is composed of two single-stranded DNAs shown in sequence 25 and sequence 26 in the sequence table; the extension primer 9 is the sequence in the sequence table. The single-stranded DNA shown in 27; (10) A primer pair 10 and an extension primer 10 for detecting rs17822931; the primer pair 10 is composed of two single-stranded DNAs shown in sequence 28 and sequence 29 in the sequence listing; the extension primer 10 is the sequence in the sequence listing. Single-stranded DNA shown in 30; (11) Primer pair 11 and extension primer 11 for detecting rs1871534; the primer pair 11 consists of two single-stranded DNAs shown in sequence 31 and sequence 32 in the sequence table; the extension primer 11 is the sequence in the sequence table. The single-stranded DNA shown in 33; (12) Primer pair 12 and extension primer 12 for detecting rs2080161; the primer pair 12 is composed of two single-stranded DNAs shown in sequence 34 and sequence 35 in the sequence listing; the extension primer 12 is the sequence in the sequence listing. Single-stranded DNA shown in 36; (13) Primer pair 13 and extension primer 13 for detecting rs2139931; the primer pair 13 consists of two single-stranded DNAs shown in sequence 37 and sequence 38 in the sequence listing; the extension primer 13 is the sequence in the sequence listing. The single-stranded DNA shown in 39; (14) Primer pair 14 and extension primer 14 for detecting rs2789823; the primer pair 14 is composed of two single-stranded DNAs shown in sequence 40 and sequence 41 in the sequence listing; the extension primer 14 is the sequence in the sequence listing. Single-stranded DNA shown in 42; (15) Primer pair 15 and extension primer 15 for detecting rs2814778; the primer pair 15 consists of two single-stranded DNAs shown in sequence 43 and sequence 44 in the sequence listing; the extension primer 15 is the sequence in the sequence listing Single-stranded DNA shown in 45; (16) primer pair 16 and extension primer 16 for detecting rs3751050; the primer pair 16 is composed of two single-stranded DNAs shown in sequence 46 and sequence 47 in the sequence listing; the extension primer 16 is the sequence in the sequence listing Single-stranded DNA shown in 48; (17) Primer pair 17 and extension primer 17 for detecting rs3827760; the primer pair 17 consists of two single-stranded DNAs shown in sequence 49 and sequence 50 in the sequence listing; the extension primer 17 is the sequence in the sequence listing. Single-stranded DNA shown in 51; (18) Primer pair 18 and extension primer 18 for detecting rs4657449; the primer pair 18 consists of two single-stranded DNAs shown in sequence 52 and sequence 53 in the sequence listing; the extension primer 18 is the sequence in the sequence listing Single-stranded DNA shown in 54; (19) Primer pair 19 and extension primer 19 for detecting rs4749305; the primer pair 19 is composed of two single-stranded DNAs shown in sequence 55 and sequence 56 in the sequence listing; the extension primer 19 is the sequence in the sequence listing. Single-stranded DNA shown in 57; (20) a primer pair 20 and an extension primer 20 for detecting rs4792928; the primer pair 20 is composed of two single-stranded DNAs shown in sequence 58 and sequence 59 in the sequence table; the extension primer 20 is the sequence in the sequence table. Single-stranded DNA shown in 60; (21) A primer pair 21 and an extension primer 21 for detecting rs6054465; the primer pair 21 is composed of two single-stranded DNAs shown in sequence 61 and sequence 62 in the sequence listing; the extension primer 21 is the sequence in the sequence listing. Single-stranded DNA shown in 63; (22) A primer pair 22 and an extension primer 22 for detecting rs6437783; the primer pair 22 is composed of two single-stranded DNAs shown in sequence 64 and sequence 65 in the sequence listing; the extension primer 22 is the sequence in the sequence listing. Single-stranded DNA shown in 66; (23) primer pair 23 and extension primer 23 for detecting rs715605; the primer pair 23 is composed of two single-stranded DNAs shown in sequence 67 and sequence 68 in the sequence listing; the extension primer 23 is the sequence in the sequence listing Single-stranded DNA shown in 69; (24) primer pair 24 and extension primer 24 for detecting rs8072587; the primer pair 24 is composed of two single-stranded DNAs shown in sequence 70 and sequence 71 in the sequence listing; the extension primer 24 is the sequence in the sequence listing Single-stranded DNA shown in 72; (25) primer pair 25 and extension primer 25 for detecting rs8137373; the primer pair 25 is composed of two single-stranded DNAs shown in sequence 73 and sequence 74 in the sequence listing; the extension primer 25 is the sequence in the sequence listing Single-stranded DNA shown in 75; (26) A primer pair 26 and an extension primer 26 for detecting rs9522149; the primer pair 26 is composed of two single-stranded DNAs shown in sequence 76 and sequence 77 in the sequence listing; the extension primer 26 is the sequence in the sequence listing. Single-stranded DNA shown in 78; (27) Primer pair 27 and extension primer 27 for detecting rs9809818; the primer pair 27 is composed of two single-stranded DNAs shown in sequence 79 and sequence 80 in the sequence listing; the extension primer 27 is the sequence in the sequence listing. Single-stranded DNA shown in 81; (28) primer pair 28 and extension primer 28 for detecting rs9908046; the primer pair 28 is composed of two single-stranded DNAs shown in sequence 82 and sequence 83 in the sequence listing; the extension primer 28 is the sequence in the sequence listing 84 single-stranded DNA. 5. The single-stranded DNA group according to claim 4, wherein: in the single-stranded DNA group, the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, The primer pair 5, the primer pair 6, the primer pair 7, the primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair primer pair 13, the primer pair 14, the primer pair 15, the primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21. The molar ratio of the primer pair 22, the primer pair 23, the primer pair 24, the primer pair 25, the primer pair 26, the primer pair 27 and the primer pair 28 is 0.8:0.6 :1.5:2.7:3:0.8:2:1.3:1:1:1.5:1:2.7:0.5:0.8:2.5:0.4:1.6:2.5:4:3:0.8:0.8:3:3:6:0.6 : 0.6; the molar ratio of the two primers in each primer pair is 1:1; The extension primer 1, the extension primer 2, the extension primer 3, the extension primer 4, the extension primer 5, the extension primer 6, the extension primer 7, the extension primer 8, the extension primer extension primer 9, the extension primer 10, the extension primer 11, the extension primer 12, the extension primer 13, the extension primer 14, the extension primer 15, the extension primer 16, the extension primer 17. The extension primer 18, the extension primer 19, the extension primer 20, the extension primer 21, the extension primer 22, the extension primer 23, the extension primer 24, the extension primer 25, The molar ratio of the extension primer 26, the extension primer 27 and the extension primer 28 is 0.45:0.35:1.2:1.7:4:1:3:0.8:1:0.8:1.8:1.1:1.1:1.1:0.8 :1.4:1:0.5:1.3:1.8:1.6:0.9:0.9:2:2.3:3:1:0.6. 6. A test kit for distinguishing five intercontinental populations, comprising the primer pair group described in claim 2 or 3 or the single-stranded DNA group described in claim 4 or 5, and at least one of the following substances: dNTP, DNA polymerase, alkaline phosphatase. 7. Use of a substance for detecting a combination of 28 SNP sites in any of the following: (a) Construction of a genotyping database for five intercontinental populations; (b) distinguish between five intercontinental populations; 所述28个SNP位点分别为：rs10483251、rs12142199、rs1229984、rs12402499、rs12498138、rs12594144、rs1426654、rs1557553、rs16891982、rs17822931、rs1871534、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、 rs6054465, rs6437783, rs715605, rs8072587, rs8137373, rs9522149, rs9809818, and rs9908046. 8. The application according to claim 7, characterized in that: the substance used to detect the combination of 28 SNP sites is the primer pair set described in claim 2 or 3 or the single described in claim 4 or 5. stranded DNA set or the kit of claim 6. 9. A method for constructing a genotyping database of five intercontinental populations, comprising the steps of: (a1) Select 28 SNP loci from the 1000 Genomes Project and the Human Genome Diversity Project for typing of five intercontinental populations to form an original typing library; (a2) Perform structure clustering analysis on all the samples in the original genotyping database, and select the part with the ancestral principal component greater than 90% to constitute the genotyping database of five intercontinental populations; 所述28个SNP位点分别为：rs10483251、rs12142199、rs1229984、rs12402499、rs12498138、rs12594144、rs1426654、rs1557553、rs16891982、rs17822931、rs1871534、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、 rs6054465, rs6437783, rs715605, rs8072587, rs8137373, rs9522149, rs9809818, and rs9908046. 10. A method of distinguishing five intercontinental populations, comprising the steps of: (b1) constructing a genotyping database of five intercontinental populations according to the method of claim 9; (b2) extracting the genomic DNA of the subject to be tested, and performing the detection of 28 SNP sites to obtain the original genotype data of the subject to be tested on the 28 SNP sites; (b3) comparing the original genotype data of the subject at the 28 SNP sites with the genotyping database of the five intercontinental populations, so as to determine that the subject belongs to the five intercontinental populations What kind; 所述28个SNP位点分别为：rs10483251、rs12142199、rs1229984、rs12402499、rs12498138、rs12594144、rs1426654、rs1557553、rs16891982、rs17822931、rs1871534、rs2080161、rs2139931、rs2789823、rs2814778、rs3751050、rs3827760、rs4657449、rs4749305、rs4792928、 rs6054465, rs6437783, rs715605, rs8072587, rs8137373, rs9522149, rs9809818, and rs9908046. 11. method according to claim 10, is characterized in that: when carrying out the detection of described 28 SNP sites, what adopts is the primer pair set described in claim 2 or 3 or the primer pair described in claim 4 or 5 The single-stranded DNA group or the kit of claim 6; 28-fold PCR amplification is performed, and the annealing temperature is 55°C.

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