A multi-source genetic marker composite detection system based on a high-throughput sequencing platform and its application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically discloses a multi-source genetic marker composite detection system based on a high-throughput sequencing platform and its application, wherein the detection system includes primers for amplifying the whole mitochondrial gene, 96 STR loci and 71 SNP loci, wherein the 96 STR loci include 29 autosomal STR loci, 32 X chromosome STR loci and 35 Y chromosome STR loci; and the 71 SNP loci are all Y chromosome SNP loci. The multi-source genetic marker composite detection system of the present invention can simultaneously obtain the genetic information of mitochondria, autosomes, X chromosomes and Y chromosomes using a high-throughput sequencing platform, has a wide coverage, reduces the operation steps, shortens the experimental time, and is suitable for the field of forensics.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and more specifically, relates to a multi-source genetic marker composite detection system based on a high-throughput sequencing platform and an application thereof. Background Art

[0002] Mitochondria are important organelles in eukaryotic cells. There are 1,000 to 10,000 mitochondria in each human cell, and each mitochondria has 2-10 copies of mitochondrial DNA (mtDNA). Human mitochondrial DNA is a double-stranded closed circular DNA with a total length of 16,569 bp. Mitochondrial DNA has many characteristics, such as multiple copies, no introns, maternal inheritance, high evolution rate, high heterogeneity, no gene recombination, etc. Mitochondrial DNA is often used to study genetic evolution and to assist forensic identification. Abnormal mitochondrial function can lead to a series of diseases, such as Parkinson's syndrome, diabetes, and Alzheimer's syndrome. Some studies have also found that mitochondrial DNA mutations are related to some cancers.

[0003] Short Tandom Repeat (STR), also known as microsatellite DNA or simple sequence repeat (SSR), is a type of repetitive sequence widely present in the human genome. It consists of 2-6 base pairs as the core sequence and is arranged in tandem repeats. STR is large in number in the human genome, widely distributed, highly genetically polymorphic, and has simple detection methods. Therefore, it is widely used in individual identification, paternity testing, and population genetics research.

[0004] Single Nucleotide Polymorphism (SNP) refers to the presence of transitions, transversions, insertions, deletions, and other changes at a specific nucleotide position in the DNA of the genome. As a third-generation genetic marker, SNP has certain advantages in forensic identification. First, it has a high density and a wide distribution. The sites of SNP are extremely abundant, almost throughout the entire genome. It is estimated that there is an average of one SNP per 1000bp in the genome. Second, it is relatively stable, and SNP has a relatively low spontaneous mutation rate (10 -8 ); Third, two alleles. SNP markers generally have only two alleles in a population or biological population, so they are also called bi-allelic markers, which also makes the analysis of typing results simple and easy; Fourth, the fragment is short. The amplification product of a single SNP site can be controlled below 150bp, which makes it easy to achieve simultaneous amplification of multiple sites and is conducive to the typing of degraded samples.

[0005] The second generation DNA sequencing technology is also known as high-throughput sequencing technology (HTS). With low cost and an accuracy of more than 99%, it can simultaneously perform rapid sequencing and analysis on hundreds of thousands to millions of DNA molecules from hundreds of thousands of samples. With the development and application of high-throughput sequencing technology, the number of STR loci detected in a single experiment can reach hundreds, hundreds or even more, and SNP genetic markers can be detected simultaneously to obtain more genetic information. However, the genetic information detected by the individual identification system based on the second generation sequencing is not rich enough, and it is often necessary to use multiple detection systems in combination to meet the wide application requirements in the forensic field, which limits its application in case detection. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present invention is to provide a multi-source genetic marker composite detection system based on a high-throughput sequencing platform and its application, aiming to solve the problem of less genetic information detected by the existing individual identification system based on second-generation sequencing.

[0007] To achieve the above-mentioned object, the present invention provides a multi-source genetic marker composite detection system based on a high-throughput sequencing platform, including primers for amplifying the whole mitochondrial gene, 96 STR loci and 71 SNP loci;

[0008] The 96 STR loci are D2S441, D1S1656, TPOX, D2S1338, D3S1358, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, TH01, vWA, D12S391, D13S317, PentaE, D16S539, D18S51, D19S433, D21S11, PentaD, D10S1248, D22S1045, D8S1132, D15S 659, D3S3045, D19S253, D6S477, D10S1435, DYS19, DYS385a, DYS385b, DYS389Ⅰ, DYS389Ⅱ, DYS390, DYS391, DYS392 , DYS437, DYS438, DYS439, DYS448, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, DYS393, DYS456, DYS458, DYS460 , DYS643, DYF387S1, DYS549, DYS570, DYS449, DYS518, DYS447, DYS444, DYS557, DYS596, DYS627, DYS508, DYS527 a. DYS527b, DXS10101, DXS10134, DXS10148, DXS10159, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809 , DXS10135, DXS10074, DXS10079, DXS101, DXS6789, DXS6810, DXS10103, DXS7132, DXS7423, DXS8378, HPRTB, DXSGATA31E08, DXS9895, DXS7133, DXS7424, DXS9902, DXS8377, GATA165B12, DXS981, GATA172D05, DXS6797, and DXS6804;

[0009] The 71 SNP sites are rs13447361, rs52812045, rs2075181, SRY8299, M134, rs9786247, rs2267801, rs9786707, rs3848982, rs4589047, M119, rs17250121, rs16980426, rs3906, rs16980363, rs2032650, rs3898, rs17269816, rs1 7269928, rs2032674, rs16980601, rs9786502, rs2032665, rs2032652, P31, rs16981340, rs17269396, rs9306 841, M117, rs17276358, rs17276338, rs3900, rs2032678, rs11575897, rs9786479, rs9786401, rs2075640, rs1 6980391, rs17316592, rs9341278, rs17276345, rs2032602, rs16980396, rs11096432, rs17316543, rs178423 87. rs2032624, rs35284970, rs17323322, rs9785908, M148, rs16981290, rs9306848, rs917759, rs17316007, r s7067458, rs9786095, rs13447354, rs16980641, rs2032645, rs9786394, rs11096433, rs16980610, M122, rs2196155, rs17174528, rs2032631, rs3853054, rs9306845, M111 and rs17276777, where the numbers with rs indicate the numbers of the loci in the dbSNP database;

[0010] The primer sequences used to amplify the whole mitochondrial gene are shown in SEQ ID NO.1 to SEQ ID NO.198;

[0011] The primer sequences for amplifying the 96 STR loci and the 71 SNP loci are shown as SEQ ID NO.199 to SEQ ID NO.526.

[0012] According to another aspect of the present invention, a method for DNA typing detection is provided, which uses the above-mentioned multi-source genetic marker composite detection system to type and detect DNA.

[0013] Preferably, the method for DNA typing detection comprises the following steps:

[0014] S1, the mitochondrial full gene is amplified in at least one first PCR amplification reaction system, and the 96 STR loci and the 71 SNP loci are amplified in at least one second PCR amplification system;

[0015] S2, combining all the products amplified in step S1 and purifying them;

[0016] S3, connecting the product purified in step S2 to connector I5 and connector I7 to obtain a DNA library;

[0017] S4, purifying the DNA library;

[0018] S5, sequencing and data analysis.

[0019] Preferably, in step S1, the first PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of a first primer mixture, 2 μL of amplification buffer, 3 μL of a DNA template and 10 μL of deionized water, and the first primer mixture is at least one pair of primers used to amplify the entire mitochondrial gene.

[0020] Preferably, the amplification program of the first PCR amplification reaction system is: 95°C, 3.5 min; 23 cycles, 98°C, 20s, 60°C, 3.5 min; 72°C, 5 min.

[0021] Preferably, in step S1, the second PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of a second primer mixture, 6 μL of amplification buffer, 3 μL of a DNA template and 10 μL of deionized water, and the second primer mixture is at least one pair of primers for amplifying the 96 STR sites and the 71 SNP sites.

[0022] Preferably, the amplification program of the second PCR amplification reaction system is: 95°C, 3.5 min; 27 cycles, 98°C, 20s, 60°C, 4 min; 72°C, 5 min.

[0023] Preferably, in step S2 and step S4, the purification is performed by a magnetic bead method.

[0024] According to another aspect of the present invention, a kit is also provided, comprising the above-mentioned multi-source genetic marker composite detection system.

[0025] Preferably, the kit further comprises DNA polymerase, amplification buffer, linker I5 and linker I7, and deionized water.

[0026] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0027] The detection system of the present invention includes primer sequences designed for the whole mitochondrial gene, 96 STR loci and 71 SNP loci, with wide locus coverage. Only one amplification is required to simultaneously obtain genetic marker information on autosomes, X chromosomes and Y chromosomes, thereby reducing the number of operation steps and shortening the experimental time. The present invention combines multi-source genetic markers with the inherent high sensitivity of the second-generation sequencing system, which is beneficial for forensic typing of trace, old and degraded samples. In addition, practical applications show that the detection system of the present invention has strong human specificity, can eliminate the interference of animal DNA, and has more accurate detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram of the quality inspection results of the mitochondrial system library in an embodiment of the present invention.

[0029] Figure 2 This is a distribution diagram of base quality values ​​of the mitochondrial system library in an embodiment of the present invention.

[0030] Figure 3 This is a diagram showing the quality inspection results of the nuclear chromosome system library in an embodiment of the present invention.

[0031] Figure 4 Graph showing the base quality distribution of the nuclear chromosome system library in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The present invention provides a multi-source genetic marker composite detection system based on a high-throughput sequencing platform, including primers for amplifying the whole mitochondrial gene, 96 STR loci and 71 SNP loci;

[0034] The 96 STR loci are D2S441, D1S1656, TPOX, D2S1338, D3S1358, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, TH01, vWA, D12S391, D13S317, PentaE, D16S539, D18S51, D19S433, D21S11, PentaD, D10S1248, D22S1045, D8S1132, D15S 659, D3S3045, D19S253, D6S477, D10S1435, DYS19, DYS385a, DYS385b, DYS389Ⅰ, DYS389Ⅱ, DYS390, DYS391, DYS392 , DYS437, DYS438, DYS439, DYS448, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, DYS393, DYS456, DYS458, DYS460 , DYS643, DYF387S1, DYS549, DYS570, DYS449, DYS518, DYS447, DYS444, DYS557, DYS596, DYS627, DYS508, DYS527 a. DYS527b, DXS10101, DXS10134, DXS10148, DXS10159, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809 , DXS10135, DXS10074, DXS10079, DXS101, DXS6789, DXS6810, DXS10103, DXS7132, DXS7423, DXS8378, HPRTB, DXSGATA31E08, DXS9895, DXS7133, DXS7424, DXS9902, DXS8377, GATA165B12, DXS981, GATA172D05, DXS6797, and DXS6804;

[0035] The 71 SNP sites are rs13447361, rs52812045, rs2075181, SRY8299, M134, rs9786247, rs2267801, rs9786707, rs3848982, rs4589047, M119, rs17250121, rs16980426, rs3906, rs16980363, rs2032650, rs3898, rs17269816, rs1 7269928, rs2032674, rs16980601, rs9786502, rs2032665, rs2032652, P31, rs16981340, rs17269396, rs9306 841, M117, rs17276358, rs17276338, rs3900, rs2032678, rs11575897, rs9786479, rs9786401, rs2075640, rs1 6980391, rs17316592, rs9341278, rs17276345, rs2032602, rs16980396, rs11096432, rs17316543, rs178423 87. rs2032624, rs35284970, rs17323322, rs9785908, M148, rs16981290, rs9306848, rs917759, rs17316007, r s7067458, rs9786095, rs13447354, rs16980641, rs2032645, rs9786394, rs11096433, rs16980610, M122, rs2196155, rs17174528, rs2032631, rs3853054, rs9306845, M111 and rs17276777, where the numbers with rs indicate the number of the site in the dbSNP database.

[0036] Among them, the 96 STR loci include 29 autosomal STR loci, 32 X chromosome STR loci and 35 Y chromosome STR loci, and the 71 SNP loci are all Y chromosome SNP loci. A pair of primers is designed for each locus, and a pair of primers is used to simultaneously amplify the DNA fragments of the corresponding loci from both ends. Specifically, the primer sequences for amplifying the whole mitochondrial gene are shown in Table 1, and the primer sequences for amplifying the 96 STR loci and the 71 SNP loci are shown in Table 2.

[0037] Table 1 Primer sequences for mitochondrial full gene amplification

[0038]

[0039]

[0040]

[0041]

[0042] Table 2 Primer sequences corresponding to 96 STR loci and 71 SNP loci

[0043]

[0044]

[0045]

[0046]

[0047] The present invention also provides a method for DNA typing detection, which uses the above-mentioned multi-source genetic marker composite detection system to type and detect DNA. The DNA sample can be prepared from fresh blood, blood spots, dried blood cards, hair with hair follicles, oral swabs, etc. The detection limit of the DNA content in the sample during PCR amplification can be as low as 100pg / μL fresh blood.

[0048] Specifically, the method for DNA typing detection comprises the following steps:

[0049] S1, the mitochondrial full gene is amplified in at least one first PCR amplification reaction system, and the 96 STR loci and the 71 SNP loci are amplified in at least one second PCR amplification system;

[0050] S2, combining all the products amplified in step S1 and purifying them;

[0051] S3, connecting the product purified in step S2 to connector I5 and connector I7 to obtain a DNA library;

[0052] S4, purifying the DNA library;

[0053] S5, sequencing and data analysis.

[0054] After multiple rounds of experiments, the optimal PCR amplification reaction system and amplification conditions for the multi-source genetic marker composite detection system were obtained. Preferably, in step S1, the first PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of the first primer mixture, 2 μL of amplification buffer, 3 μL of DNA template and 10 μL of deionized water, and the first primer mixture is at least one pair of primers used to amplify the whole mitochondrial gene. The amplification program of the first PCR amplification reaction system is: 95°C, 3.5min; 23 cycles, 98°C, 20s, 60°C, 3.5min; 72°C, 5min.

[0055] The second PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of second primer mixture, 6 μL of amplification buffer, 3 μL of DNA template and 10 μL of deionized water, wherein the second primer mixture is at least one pair of primers for amplifying the 96 STR loci and the 71 SNP loci. The amplification program of the second PCR amplification reaction system is: 95°C, 3.5 min; 27 cycles, 98°C, 20 s, 60°C, 4 min; 72°C, 5 min.

[0056] In step S2 and step S4 of some embodiments, the purification is performed using a magnetic bead method.

[0057] In step S3 of some embodiments, the linker I5 sequence and the linker I7 sequence can be selected from the corresponding sequences in Table 3.

[0058] Table 3 Sequences of linkers I5 and I7

[0059] serial number Adapter sequence serial number Adapter sequence I5-01 AGGAGTAG I7-03 TGACACTC I5-02 CTTACCGC I7-04 CTCACACA I5-03 TACGATAG I7-05 GCAAGAGA I5-04 GCCTTTCT I7-06 ACAGTACT I5-05 AGGAGCTC I7-07 TCCGCTTA I5-06 CCTACATG I7-08 CTGCGGCT I5-07 GTCCCCTC I7-09 AGTCTAGC I5-08 CAGCTCTC I7-10 TATGCATT I5-09 TCCGCAGG I7-11 AGTACGAG I5-10 ATATGTAA I7-12 AGTGTCAG I5-11 TCTGGGAT I7-13 GAGCACTA I5-12 ATATCCTC I7-14 TATATGCC I5-13 TTTTCAGC I7-15 CTGTGCAG I5-14 GCATACCT I7-16 TAAGACGT I5-15 GCGGTAAG I7-17 ACGTCAGG I5-16 AGAAAGGC I7-18 CGTGTCCC I7-01 ATCGTCGC I7-19 CCTGACGT I7-02 ACTGGTCA I7-20 GGAGGTAA

[0060] In addition, the present invention also provides a kit, comprising the above-mentioned multi-source genetic marker composite detection system, DNA polymerase, amplification buffer (preferably enhanced buffer), connector I5 and connector I7, and deionized water. Wherein, the concentration of each primer in the composite detection system is 0.1 μM, and the concentration of each connector I5 and connector I7 is 10 μM. The kit can be used for individual identification, kinship identification, maternal blood typing, mixed complex sample typing, second-generation DNA file establishment, blood type identification, etc.

[0061] The above technical solution is described in detail below in conjunction with specific embodiments.

[0062] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0063] This example uses the detection system provided by the present invention to simultaneously detect four DNA samples (fresh blood donated by four volunteers), and the specific operation steps are as follows:

[0064] 1. Multiplex Amplification

[0065] 1. Amplification system: Each DNA sample is divided into two reaction tubes, where:

[0066] 1.1 Add 10 μL of DNA polymerase, 5 μL of mitochondrial primer mix, 2 μL of enhancement buffer, 3 μL of DNA template and 10 μL of deionized water to reaction tube 1 of each sample;

[0067] 1.2 Add 10 μL of DNA polymerase, 5 μL of STR and SNP primer mixture, 6 μL of enhancement buffer, 3 μL of DNA template and 10 μL of deionized water to reaction tube 2 of each sample;

[0068] 2. Amplification Procedure

[0069] 2.1 After mixing and instant centrifugation, place the reaction tube 1 of each sample in a PCR instrument and perform the following reaction: 95°C, 3.5 min; 23 cycles (98°C, 20 s; 60°C, 3.5 min); 72°C, 5 min;

[0070] 2.2 Place the reaction tube 2 of each sample into the PCR instrument and perform the following reaction: 95°C, 3.5 min; 27 cycles (98°C, 20 s; 60°C, 4 min); 72°C, 5 min.

[0071] 2. Merge and Purify

[0072] 1. Take one reaction tube for each DNA sample, take 3 μL of the amplified product from reaction tube 1 in the previous step, take 27 μL of the amplified product from reaction tube 2, combine them, add 27 μL of purified magnetic beads, pipette 20 times to mix thoroughly, and place at room temperature for 5 minutes;

[0073] 2. Place on the magnetic rack for 3 minutes until clear and remove the supernatant;

[0074] 3. Add 180 μL of freshly prepared 80% ethanol, keep on the magnetic stand, and place at room temperature for 30 seconds; remove the supernatant;

[0075] 4. Repeat step 3 once;

[0076] 5. Keep the PCR tube on the magnetic rack, discard the residual ethanol with a 10μL pipette tip, open the lid and air-dry the magnetic beads for 3 minutes until cracks just appear;

[0077] 6. Remove the PCR tube from the magnetic stand, add 24 μL of water, mix thoroughly, and leave at room temperature for 3 minutes;

[0078] 7. Place on the magnetic rack for 3 minutes to clarify, pipette 13.5 μL into a new PCR tube and mark it with the corresponding number.

[0079] 3. Adapter Amplification

[0080] 1. Amplification system: Add 10 μL of DNA polymerase, 1 μL of adapter I5, 1 μL of adapter I7, 2.5 μL of enhancement buffer and 2 μL of deionized water to each reaction tube containing 13.5 μL of purified product; all samples use I5-01 adapter, and samples 1 to 4 use I7-01 to I7-04 adapters respectively;

[0081] 2. Amplification procedure: After mixing and centrifuging briefly, place the mixture in a PCR instrument and perform the following reaction: 95°C, 3.5 min; 9 cycles (98°C, 20 s; 58°C, 1 min; 72°C, 30 s); 72°C, 5 min.

[0082] 4. Library Purification

[0083] 1. Add 27 μL of purified magnetic beads to 30 μL of amplified product, pipette 20 times to mix thoroughly, and place at room temperature for 5 minutes;

[0084] 2. Place on the magnetic rack for 3 minutes until clear and remove the supernatant;

[0085] 3. Add 180 μL of freshly prepared 80% ethanol, keep on the magnetic stand, and place at room temperature for 30 seconds; remove the supernatant;

[0086] 4. Repeat step 3 once;

[0087] 5. Keep the PCR tube on the magnetic rack, discard the residual ethanol with a 10μL pipette tip, open the lid and air-dry the magnetic beads for 3 minutes until cracks just appear;

[0088] 6. Remove the PCR tube from the magnetic stand, add 24 μL of water, mix thoroughly, and leave at room temperature for 3 minutes;

[0089] 7. Place on the magnetic rack for 3 minutes to clarify, pipette 20 μL into a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library, which should be stored at -25°C to -15°C.

[0090] 5. Sequencing and Data Analysis

[0091] Take the prepared library, use Illumina HiSeq 4000 for sequencing according to the instructions, and analyze the data results.

[0092] The sequencing analysis results of the library constructed by the mitochondrial system are shown in Table 4 and Figure 1 , Figure 2 As shown, the coverage and uniformity of the library of this system are excellent, and the capture rate (capture rate) and uniformity (20% × mean depth coverage rate) are both greater than 97%. The number and frequency of some mutation sites detected in the library constructed by the mitochondrial system are consistent, and are all verified to be correct by first-generation sequencing, indicating that the mutation site results detected by this system are accurate.

[0093] Table 4 Main data of mitochondrial system library construction

[0094]

[0095] The sequencing analysis results of the library constructed by the nuclear chromosome system are shown in Table 5 and Figure 3 , Figure 4 As shown, the coverage and uniformity of the library of this system are good, with a capture rate (capture rate) > 90% and a uniformity (20% × mean depth coverage rate) > 95%. Some of the Y-STR sites detected are consistent with the first-generation sequencing results, indicating that its capture efficiency and accuracy are good.

[0096] Table 5 Main data of nuclear chromosome system library construction (partial Y-STR loci)

[0097]

[0098] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-source genetic marker composite detection system based on a high-throughput sequencing platform, characterized in that: Includes primers for amplifying the entire mitochondrial genome, 96 STR loci, and 71 SNP loci; The 96 STR loci are D2S441, D1S1656, TPOX, D2S1338, D3S1358, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, TH01, vWA, D12S391, D13S317, PentaE, D16S539, D18S51, D19S433, D21S11, PentaD, D10S1248, D22S1045, D8S1132, D15S 659, D3S3045, D19S253, D6S477, D10S1435, DYS19, DYS385a, DYS385b, DYS389Ⅰ, DYS389Ⅱ, DYS390, DYS391, DYS392 , DYS437, DYS438, DYS439, DYS448, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, DYS393, DYS456, DYS458, DYS460 , DYS643, DYF387S1, DYS549, DYS570, DYS449, DYS518, DYS447, DYS444, DYS557, DYS596, DYS627, DYS508, DYS527 a. DYS527b, DXS10101, DXS10134, DXS10148, DXS10159, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809 , DXS10135, DXS10074, DXS10079, DXS101, DXS6789, DXS6810, DXS10103, DXS7132, DXS7423, DXS8378, HPRTB, DXSGATA31E08, DXS9895, DXS7133, DXS7424, DXS9902, DXS8377, GATA165B12, DXS981, GATA172D05, DXS6797, and DXS6804; The 71 SNP sites are rs13447361, rs52812045, rs2075181, SRY8299, M134, rs9786247, rs2267801, rs9786707, rs3848982, rs4589047, M119, rs17250121, rs16980426, rs3906, rs16980363, rs2032650, rs3898, rs17269816, rs1 7269928, rs2032674, rs16980601, rs9786502, rs2032665, rs2032652, P31, rs16981340, rs17269396, rs9306 841, M117, rs17276358, rs17276338, rs3900, rs2032678, rs11575897, rs9786479, rs9786401, rs2075640, rs1 6980391, rs17316592, rs9341278, rs17276345, rs2032602, rs16980396, rs11096432, rs17316543, rs178423 87. rs2032624, rs35284970, rs17323322, rs9785908, M148, rs16981290, rs9306848, rs917759, rs17316007, r s7067458, rs9786095, rs13447354, rs16980641, rs2032645, rs9786394, rs11096433, rs16980610, M122, rs2196155, rs17174528, rs2032631, rs3853054, rs9306845, M111 and rs17276777, where the numbers with rs indicate the numbers of the loci in the dbSNP database; The primer sequences used to amplify the whole mitochondrial gene in the first PCR amplification reaction system are shown in SEQ ID NO.1 to SEQ ID NO.198; The primer sequences used to amplify the 96 STR loci and the 71 SNP loci in the second PCR amplification reaction system are shown as SEQ ID NO.199 to SEQ ID NO.

526.

2. A method for DNA typing detection for non-diagnostic purposes, characterized in that: The multi-source genetic marker composite detection system according to claim 1 is used to detect DNA typing.

3. The method according to claim 2, characterized in that The steps include: S1, the whole mitochondrial gene is amplified in a first PCR amplification reaction system, and the 96 STR loci and the 71 SNP loci are amplified in a second PCR amplification reaction system; S2, combining all the products amplified in step S1 and purifying them; S3, connecting the product purified in step S2 to connector I5 and connector I7 to obtain a DNA library; S4, purifying the DNA library; S5, sequencing and data analysis.

4. The method according to claim 3, characterized in that: In step S1, the first PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of a first primer mixture, 2 μL of an amplification buffer, 3 μL of a DNA template and 10 μL of deionized water, and the first primer mixture is the primer for amplifying the whole mitochondrial gene.

5. The method according to claim 4, characterized in that: The amplification program of the first PCR amplification reaction system is: 95°C, 3.5min; 23 cycles, 98°C, 20s, 60°C, 3.5min; 72°C, 5min.

6. The method according to claim 3, characterized in that: In step S1, the second PCR amplification reaction system includes 10 μL of DNA polymerase, 5 μL of a second primer mixture, 6 μL of an amplification buffer, 3 μL of a DNA template and 10 μL of deionized water, and the second primer mixture is a primer for amplifying the 96 STR sites and the 71 SNP sites.

7. The method according to claim 6, characterized in that: The amplification program of the second PCR amplification reaction system is: 95°C, 3.5min; 27 cycles, 98°C, 20s, 60°C, 4min; 72°C, 5min.

8. The method according to claim 3, characterized in that: In step S2 and step S4, the purification is performed by magnetic bead method.

9. A kit, characterized in that: It includes the multi-source genetic marker composite detection system described in claim 1.

10. The kit according to claim 9, characterized in that: Also included are DNA polymerase, amplification buffer, adapter I5 and adapter I7, and deionized water.

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