High-coverage forensic medicine STR-SNP multi-marker joint detection system and application thereof

Abstract

The invention discloses a high-coverage forensic medicine STR-SNP (short tandem repeat-single nucleotide polymorphism) multi-marker joint detection system and application thereof, and belongs to the technical field of biotechnology and genetics. The detection system comprises amplification primers of 88 STRs (short tandem repeats) and 348 SNP (single nucleotide polymorphism) sites; the nucleotide sequence of the primer composition for amplifying the 88 STR sites is as shown in SEQ ID No. 1-176, and the nucleotide sequence of the primer composition for amplifying the 348 SNP sites is as shown in SEQ ID No. 177-872. 88 STR candidate markers and 348 SNP candidate markers are screened according to the background of Chinese population genetics, a method for detecting the STR and the SNP is designed, and the method is good in accuracy, large in provided information content and capable of being applied to forensic medicine individual recognition and genetic relationship identification.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and genetics, and in particular to a high-coverage forensic STR-SNP multi-marker combined detection system and its application. Background Technology

[0002] DNA analysis is a core technology in modern forensic science, providing crucial tools for individual identification and paternity testing. Short tandem repeats (STRs) have become widely used and important markers due to their high polymorphism, strong discriminative ability, and ease of standardization. Capillary electrophoresis-based STR (CE-STR) typing technology achieves high-accuracy typing based on fragment length and has long been considered the "gold standard" for forensic DNA analysis. However, the CE-STR method has certain limitations, including relatively low resolution, which may make it difficult to effectively distinguish STR loci with similar fragment lengths. Furthermore, the limited number of analyzable loci restricts the amount of genetic information obtained.

[0003] Next-generation sequencing (NGS) technology directly acquires sequence information, enabling the simultaneous detection of thousands of genetic markers in an integrated workflow, far exceeding the efficiency and resolution of traditional methods. While STRs remain the primary markers for individual identification, single nucleotide polymorphisms (SNPs) are needed as a supplement in certain scenarios. Although a single SNP has limited information due to its dimorphism, its ability to differentiate within the same group is strong. NGS technology can simultaneously detect STRs and SNPs, providing more comprehensive genetic information and effectively compensating for the limitations of CE technology.

[0004] Currently, several commercially available NGS testing kits are used in forensic identification, including the ForenSeq™ DNASignature Prep Kit, the Precision ID GlobalFiler™ MPS STR Panel v2, and the MGIEAasySignature Identification Library Prep Kit. However, these testing systems are mainly developed for the genetic characteristics of European and American populations, and generally suffer from technical problems such as uneven locus coverage and inconsistent STR nomenclature.

[0005] Therefore, developing a STR-SNP system that is adapted to the genetic characteristics of the Chinese population and has both high coverage and balanced detection is not only a key technological innovation, but also an urgent need for current forensic practice. Summary of the Invention

[0006] In view of this, the present invention aims to provide a high-coverage forensic STR-SNP multi-marker joint detection system and its application. Addressing the problems of insufficient detection site coverage, limited specificity for the Chinese population, and difficulty in balancing detection sensitivity and throughput in existing technologies, this invention constructs a primer system for STR and SNP detection suitable for the Chinese population by systematically integrating public databases, relevant literature data, and validated high-information-content forensic genetic markers. This system, along with its accompanying reagent kit, enables high-specificity, high-sensitivity, and high-throughput detection of target sites on an NGS platform, thereby significantly improving the accuracy and applicability of population genetic analysis and forensic identification.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a high-coverage forensic STR-SNP multi-marker locus combination, including 88 STRs and 348 SNP loci for the Chinese population; In a second aspect, the present invention provides a primer composition for detecting the above-mentioned STR and SNP sites, comprising amplification primers for 88 STR sites and 348 SNP sites; the nucleotide sequences of the primer composition for amplifying the 88 STR sites are shown in SEQ ID No. 1-176, and the nucleotide sequences of the primer composition for amplifying the 348 SNP sites are shown in SEQ ID No. 177-872.

[0008] In a third aspect, the present invention provides a kit for detecting the above-mentioned STRs and SNPs, comprising the primer composition described above.

[0009] The present invention also provides a high-coverage forensic STR-SNP multi-marker combined detection system, comprising the primer composition described above.

[0010] The present invention also provides a method for detecting the above-mentioned STRs and SNPs, comprising the following steps: S1: Using sample genomic DNA (gDNA) as a template, perform multiplex PCR reactions to amplify multiple target regions on the genomic DNA; S2: Purification of multiplex PCR products using magnetic beads; S3: Add tag adapters to perform adapter sequence PCR reaction to amplify DNA library; S4: Purify the DNA library amplified in step S3 using magnetic beads; S5: The purified DNA library is mixed, denatured, and then sequenced. The sequencing results are then compared to determine the gene information.

[0011] Furthermore, the samples include blood, blood spots, saliva, oral swabs, fingernails, and hair with follicles.

[0012] Further, in step S1, the reaction system for the multiplex PCR reaction includes 5 μL of the primer composition described above, 10 μL of DNA polymerase mixture, 1 μL of gDNA, 2.5 μL of PCR enhancement buffer 1, 3.5 μL of PCR enhancement buffer 2, and 8 μL of nuclease-free water.

[0013] The DNA polymerase mixture consisted of: 60 mM Tris-HCl, 75 mM KCl, 4.5 mM MgSO4, 0.15% Tween-20, 0.6 mM dNTPs, 75 U / mL Taq polymerase, 15% glycerol, and pH 8.8.

[0014] PCR enhancement buffer 1 is 24 mM MgSO4; PCR enhancement buffer 2 contains 170 mM (NH4)2SO4, 45% DMSO, and 4.5 M Betaine.

[0015] Further, in step S1, the reaction conditions for multiplex PCR are: i) pre-denaturation at 95°C for 3 minutes and 30 seconds; ii) denaturation at 98°C for 20 seconds; annealing at 55°C for 1 minute; two extension cycles at 60°C for 1 minute and 65°C for 2 minutes; a total of 26 PCR cycles in this step; iii) final extension at 72°C for 5 minutes; iv) holding at 4°C.

[0016] Further, in step S3, the reaction system consists of 2 μL of premixed adapter primers (10 μM), 10 μL of DNA polymerase mixture, 15.5 μL of PCR product mixture, and 2.5 μL of PCR enhancement buffer 1; the reaction conditions are: i) pre-denaturation at 95°C for 3 minutes and 30 seconds; ii) denaturation at 98°C for 20 seconds; annealing at 58°C for 1 minute; extension at 72°C for 30 seconds; this step consists of 9 PCR cycles; iii) final extension at 72°C for 5 minutes; iv) holding at 4°C.

[0017] The present invention also provides the application of the above-described site combinations, or the above-described primer compositions, or the above-described kits in individual identification and / or kinship determination in the Chinese population.

[0018] The beneficial effects of this invention include at least the following: (1) This invention constructs a primer system suitable for the joint detection of STRs and SNPs in the Chinese population by systematically screening public databases and relevant literature data and combining them with validated high-information genetic marker loci in forensic medicine. This system and its matching kit can achieve high specificity, high sensitivity and high throughput detection of target loci on a next-generation sequencing platform, thereby significantly improving the accuracy and applicability of population genetic analysis and forensic identification.

[0019] (2) Based on next-generation sequencing technology, this invention has constructed a primer composition and kit containing amplification primers of 88 STRs and 348 SNPs. When selecting sites, different databases and relevant literature were referenced and genetic markers widely used in forensic medicine were combined. For the Chinese population, more genetic information and higher forensic efficacy can be obtained, which is conducive to forensic applications such as individual identification and kinship identification.

[0020] (3) This invention uses the AIdesign website (https: / / design.igenetech.com / ) for primer design, combines the thermodynamic stability algorithm for specific primer design, and adopts a two-step PCR amplification method to complete the amplification of the target region and library construction. The experimental process is simple and the experimental method is reliable. (4) The present invention can use a variety of sample types for library construction, such as gDNA from blood, blood spots, saliva, oral swabs, nails, hair with follicles, etc. Attached Figure Description

[0021] Figure 1 The results are the sensitivity test results of the second-generation sequencing detection system in Example 2 of this invention; Figure 2 The results of the second-generation sequencing detection system in case samples in Embodiment 3 of the present invention are shown. Figure 3 This is an agarose gel electrophoresis image of the degraded sample in Example 4 of the present invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0024] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings to provide a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0025] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0026] Example 1 This embodiment provides a primer composition and method for detecting STRs and SNPs based on next-generation sequencing technology. The specific steps for STR and SNP site screening, primer design, and detection method are as follows: 1. Site screening and primer design; 1) Autosomal STR loci include all extended CODIS core loci, STR loci recommended in the "Implementation Specifications for Biological Full Sibling Relationship Identification SF / Z JD0105002-2014", additional STR loci in commercially available STR kits on capillary electrophoresis platforms in China, and forensic extended STR loci.

[0027] 2) X and Y chromosome STR loci include the 26 X-STR loci from the Investigator Argus X-12 kit, GlobalFiler™ PCR Amplification Kit, and the AmpFlSTR YfilerPuls PCR Kit and the 33 Y-STR loci from the literature, which are commonly used in international forensic medicine.

[0028] 3) Autosomal SNP markers include: i) universal SNP markers with a minimum allele frequency (MAF) greater than 0.1 in the Han Chinese population, widely used in studies such as SNPforID 52-plex and KenKidd; ii) MAF greater than 0.3 in the Beijing Han Chinese population, conforming to Hardy-Weinberg equilibrium, and with a physical distance greater than 5 Mb from universal SNP markers and selected STRs; iii) MAF greater than 0.3 in the Beijing Han Chinese population from the HapMap database (http: / / hapmap.ncbi.nlm.nih.gov / ), conforming to Hardy-Weinberg equilibrium, located in an intron region; with a physical distance greater than 5 Mb from selected STRs and SNP markers and low genetic difference from other populations (Fst < 0.06); not located in a copy number variation region; and the ±15 bp flanking sequences of the SNP do not contain polybasic or insertion / deletion polymorphic markers and ±120 The GC content of the flanking sequences of bp is 30%-60%; iv) the physical distance between the selected SNP marker and the nearby functional gene is greater than 100 kb.

[0029] 4) X and Y chromosome SNP markers include some SNP markers from the literature and the research basis of our laboratory.

[0030] Detailed information such as the chromosomal location of the selected 88 STRs and 384 SNP marker sites on human reference genome 37 (GRCh37 / hg19) was uploaded to the AIdesign website (https: / / design.igenetech.com / ) for multiplex primer design. Multiplex PCR targeted capture technology was selected as the sequencing library construction method, and the amplicon was required to contain 50 bp sequences upstream and downstream of the target site.

[0031] The location of the amplification primers on the chromosome and the primer sequences described in this embodiment are shown in Table 1.

[0032] Table 1. Amplicon and primer sequences in the next-generation sequencing detection system

[0033] 2. Database creation; Multiplex PCR targeted capture technology was selected for target region amplification and library construction: multiplex PCR reaction, removal of unbound oligonucleotides, and addition of IGT. ® The UDI Primer 1-96 Kit tag adapters (premixed adapter primers) are used for DNA library amplification, magnetic bead purification, and library concentration measurement and quality control. The specific process is as follows: (1) Multiplex PCR reaction: Using 2 ng / μL gDNA as template, multiplex PCR reaction was performed. The total volume of the reaction system was 30 μL. The configuration table is shown in Table 2.

[0034] Table 2. Multiplex PCR reaction system

[0035] Reaction conditions: i) Pre-denaturation 95℃, 3 min 30 sec; ii) Denaturation 98℃, 20 sec; Annealing 55℃, 1 min; Two extensions 60℃, 1 min; 65℃, 2 min; This step consists of 26 PCR cycles; iii) Final extension 72℃, 5 min; iv) Hold at 4℃.

[0036] (2) Purification of multiplex PCR products with magnetic beads: i) Add 27 μL of purified magnetic beads (SPB) to the above 30 μL reaction system, vortex to mix, incubate at room temperature for 5 minutes, briefly incubate and place on a magnetic rack until the solution is clear, and remove the supernatant; ii) Remove the PCR tube, add 50 μL of YF Buffer B, pipette to mix, incubate at room temperature for 5 minutes, briefly incubate and place on a magnetic rack until the solution is clear, and remove the supernatant; iii) Add 180 μL of 60% ethanol, incubate at room temperature for 30 seconds, remove the supernatant, and repeat twice; iv) Air dry for 5 minutes; v) Add 24 μL of nuclease-free water, remove the PCR tube from the magnetic rack, vortex to mix, briefly incubate and place at room temperature for 2 minutes; vi) Place on a magnetic rack until the solution is clear, and then aspirate 15.5 μL of supernatant to a new PCR tube.

[0037] (3) PCR reaction of adapter sequence for DNA library amplification: The PCR reaction system of adapter sequence is shown in Table 3. Table 3. PCR reaction system for adapter sequences

[0038] PCR reaction conditions: i) Pre-denaturation 95℃, 3 min 30 sec; ii) Denaturation 98℃, 20 sec; Annealing 58℃, 1 min; Extension 72℃, 30 sec; This step consists of 9 PCR cycles; iii) Final extension 72℃, 5 min; iv) Hold at 4℃.

[0039] (4) The DNA library was purified using magnetic beads, and then 1 μL of the library was used to purify it using Qubit. ® 3.0 The concentration was determined using a Fluorometer and the library concentration was recorded. A 1 μL sample of the library was taken and the length and purity of the library fragments were measured using an Agilent 2100 Bioanalyzer system (High Sensitivity DNA Kit).

[0040] (5) After mixing and denaturing the library, it was fed into the Illumina MiSeq sequencing platform for sequencing. The sequencing results were compared with the reference genome sequence. SNPs were found using software such as samtools and GATK. Then, ANNOVAR software was used to annotate the SNPs and Indel sites and determine the gene information corresponding to the mutation sites. STRinNGS v2.0 software was used to annotate the STR sites and determine the gene information of the short tandem repeat regions.

[0041] Example 2 This embodiment systematically evaluates the sequencing quality, sensitivity, and accuracy using the construction method of Example 1.

[0042] Genomic DNA from 90 unrelated individuals was used for library construction and sequencing according to the method described in Example 1. The results showed that the average depth of coverage (DoC) for 29 autosomal STRs, 26 X-STRs, and 33 Y-STRs were 4798×, 4676×, and 4678×, respectively; the average sequencing depths for 294 autosomal SNPs, 30 X-SNPs, and 24 Y-SNPs were 10816×, 11556×, and 10748×, respectively. These results indicate that the target sites are adequately covered, the sequencing depth is balanced, the library construction quality is excellent, and the sequencing performance is stable.

[0043] In terms of genotyping performance, when the DNA input was no less than 0.0625 ng, both the STR detection rate and genotyping accuracy reached 100%; when the input was no less than 0.03125 ng, the SNP detection rate and genotyping accuracy also reached 100%. Figure 1 As shown, this method maintains high sensitivity and accuracy even with extremely low template amounts. Furthermore, in 90 unrelated individuals, a total of 348 length polymorphic alleles and 683 sequence-specific alleles were detected. Based on SNP sequencing data, 22,320 expected genotyping results were obtained.

[0044] Example 3 This embodiment demonstrates the application effect of the method provided in Embodiment 1 on various types of case samples.

[0045] Using the primer composition, kit, and method provided by this invention, blood, bloodstains, saliva, oral swabs, nails, and hair samples with follicles from one male and one female were analyzed. Results showed that all STRs and SNPs were fully genotyped in all 12 samples, with average Doc ranging from 3084× to 8075× (STRs) and 4244× to 18115× (SNPs), respectively. These results indicate that this method demonstrates good coverage and sequencing depth in samples from different types of cases, exhibiting excellent sample applicability. Figure 2 ).

[0046] Example 4 This embodiment demonstrates the application of the method provided in Example 1 in degrading samples.

[0047] The primer compositions, kits, and methods provided in this invention were used to detect six groups of degraded DNA samples digested with DNase I for 0, 5, 10, 20, 30, and 40 minutes, respectively. Library construction, sequencing, and data analysis were performed using the method provided in Example 1. The degree of sample degradation is as follows: Figure 3 As shown, the corresponding STR and SNP typing results and the average DoC are summarized in Table 4.

[0048] Table 4. STR and SNP genotyping and average Doc in degraded samples

[0049] The results showed that as the DNase I digestion time increased, the DNA fragments gradually degraded and shortened, with degradation products all below 500 bp. Simultaneously, the average DoC of STRs and SNPs also decreased. Despite this, the SNP typing accuracy remained at 100%, while the STR typing accuracy remained at 99.42% even after 40 minutes of digestion (fragment length approximately 100 bp). This indicates that the method retains high reliability even with highly degraded samples.

[0050] Based on the above embodiments, this invention establishes a detection system comprising 88 STRs and 348 SNPs, suitable for forensic individual identification and paternity testing. This system surpasses existing mainstream commercial NGS kits in multi-site coverage, providing richer genetic information and polymorphism levels, thereby effectively enhancing individual discrimination and kinship determination capabilities. Through short amplicon design, this method improves detection sensitivity in degraded samples and enhances detection accuracy by increasing the number of SNP sites, maintaining high genotyping accuracy even in severely fragmented degraded samples, demonstrating a significant competitive advantage. This invention provides forensic practice with a novel detection solution offering richer information, higher accuracy, and stronger system performance.

[0051] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0052] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A high-coverage forensic STR-SNP multi-marker locus combination, characterized in that, Includes 88 STRs and 348 SNPs; The 88 STR loci are D1S1656, D2S1338, D2S441, TPOX, D3S1358, D3S3045, FGA, CSF1PO, D5S818, D6S1043, D6S477, D7S820, D8S1132, D8S1179, D10S1248, D10S1435, TH01, D12S391, vWA, D13S317, D15S659, PentaD, PentaE, D16S539, D18S51, D19S253, D19S433, D21S11, D22S1045, DXS10074, DXS10079, DXS101, DXS1010 1. DXS10103, DXS10135, DXS10159, DXS10162, DXS6789, DXS6795, DXS6800, DXS6807, DXS6809, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS981, DXS9895, DXS9902, GATA165B12, GATA172D05, GATA31E08, HPRTB, DYF387S1a, DYS19, DYS385a, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, D YS437, DYS438, DYS439, DYS444, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS 527a, DYS527b, DYS533, DYS549, DYS557, DYS570, DYS576, DYS596, DYS627, DYS635, DYS643, Y-GATA-H4; The 348 SNP sites are respectively rs10430105, rs10495407, rs10863256, rs10888979, rs10889636, rs10914803, rs1294331, rs1325298, rs1413212, rs1490413, rs2022858, rs3767382, rs4274013, rs4847034, rs560681, rs6687760, rs6697503, rs681968, rs7520386, rs859400, rs874718, rs891700, rs1013972, rs1109037, rs12622958, rs12997453, rs13019672, rs13034774, rs13411044, rs164605, rs1706868, rs1914267, rs2033849, rs2068834, rs2113418, rs2561892, rs4411759, rs7597826, rs876724, rs907100, rs993934, rs11128935, rs12714757, rs1355366, rs1357617, rs1872575, rs1900861, rs2254931, rs2399332, rs३4309901, rs4364205, rs4678083, rs6439466, rs6444724, rs6810064, rs9809528, rs9866013, rs9872936, rs10003686, rs10005781, rs10034991, rs13134862, rs1396009, rs1401907, rs1405154, rs1554472, rs1979255, rs2046361, rs2048961, rs279844, rs4484354, rs6811238, rs6857303, rs7668855, rs9307465, rs10079086, rs1156358, rs13182883, rs158803, rs159606, rs2162739, rs251934, rs2672757, rs2972312, rs299081, rs315791, rs338882, rs3763070, rs717302, rs7704770, rs8४1933, rs1022690, rs13218440, rs1336071, rs1358856, rs1478829, rs214955, rs2206597 It should be noted that in the original text, "rs३4309901" seems to have a wrong character. It is translated as it is here. You may want to check and correct it if necessary.rs2272998、rs2503107、rs2811231、rs6455989、rs727811、rs9392060、rs998828、rs1015570、rs1019029、rs10257353、rs10499822、rs12112961、rs12701077、rs2189011、rs2237427、rs2813838、rs321198、rs362633、rs606056、rs6955448、rs6967307、rs730437、rs740136、rs10092491、rs10098647、rs1380896、rs2056277、rs2056665、rs3735973、rs3802268、rs3808378、rs4074183、rs4288409、rs4606077、rs6473257、rs6474513、rs763869、rs1000709、rs1001389、rs1003416、rs1015250、rs10746873、rs10756250、rs10776839、rs10973637、rs1220054、rs1360288、rs1463729、rs1557004、rs2270529、rs334355、rs7041158、rs7849853、rs10437508、rs10444205、rs1410059、rs1418407、rs1668172、rs2386626、rs2435344、rs3780962、rs4414169、rs4415704、rs4749273、rs4750494、rs7088884、rs735155、rs740598、rs7907488、rs826472、rs964681、rs10488710、rs10500617、rs1498553、rs1873059、rs2076848、rs2711823、rs4237677、rs4939141、rs590162、rs6591147、rs7104420、rs7937238、rs901398、rs1007469、rs10506847、rs10773760、rs11178999、rs2107612、rs2111980、rs2255301、rs2269355、rs2730648、rs2920816、rs6581429、rs734075、rs7962675、rs959897、rs1058083、rs11617748、rs1335873、rs1413577、rs1886510、rs354439、rs9527220、rs9546538、rs9580523、rs1004667、rs12431779、rs12432727、rs1454361、rs4530059、rs7141285、rs722290、rs873196、rs10519137、rs12442886、rs1528460、rs1821380、rs2016276、rs266380、rs28887925、rs6495751、rs698500、rs8037429、rs1382387、rs2342747、rs430046、rs4315319、rs4781878、rs7205345、rs729172、rs8052258、rs9928480、rs1004357、rs1015308、rs1027895、rs116187、rs11869379、rs2291395、rs295886、rs3744163、rs3744338、rs4796362、rs740910、rs8070085、rs8070506、rs8078417、rs938283、rs9905977、rs1024116、rs11081448、rs11665111、rs1469328、rs1493232、rs1736442、rs521861、rs7229946、rs9635999、rs985492、rs9951171、rs9956753、rs1014440、rs10413687、rs1108414、rs576261、rs719366、rs7259841、rs8103778、rs8105430、rs1005533、rs1010870、rs1024283、rs1031825、rs12480506、rs1523537、rs2009308、rs2567608、rs445251、rs6063600、rs1004663、rs221956、rs2830795、rs2831700、rs2833736、rs464663、rs722098、rs914165、rs1028528、rs112603、rs2040411、rs2073383、rs5746846、rs5769852、rs733164、rs987640、rs12688347、rs12844754、rs12846665、rs1458178、rs1887801、rs2209420、rs2214174、rs2430212、rs2768595、rs2808742、rs28900、rs4826248、rs5908324、rs5915052、rs5916781、rs5917032、rs5925134、rs5931302、rs594031、rs5980274、rs5984589、rs5986751、rs6529455、rs6631828、rs6639398、rs6643690、rs6649211、rs7060326、rs7471388、rs9781645、rs11096432、rs11096433、rs13447354、rs16980426、rs16980601、rs16980711、rs17269816、rs17276345、rs17276358、rs17316592、rs17323322、rs2032645、rs2032650、rs2032652、rs2075640、rs3848982、rs3900、rs78149062、rs9306845、rs9786043、rs9786247、rs9786401、rs9786479、rs9786707。、 2. A primer composition for detecting the STR and SNP sites of claim 1, characterized in that, The primers include amplification primers for 88 STR sites and 348 SNP sites; the nucleotide sequences of the primer compositions for amplifying the 88 STR sites are shown in SEQ ID No. 1-176, and the nucleotide sequences of the primer compositions for amplifying the 348 SNP sites are shown in SEQ ID No. 177-872.

3. A kit for detecting the STR and SNP of claim 1, characterized in that, Includes the primer composition of claim 2.

4. A high-coverage forensic STR-SNP multi-marker combined detection system, characterized in that, Includes the primer composition of claim 2.

5. A method for detecting the STR and SNP as described in claim 1, characterized in that, Includes the following steps: S1: Using the sample's genomic DNA as a template, perform multiplex PCR reactions to amplify multiple target regions on the genomic DNA; S2: Purification of multiplex PCR products using magnetic beads; S3: Add tag adapters to perform adapter sequence PCR reaction to amplify DNA library; S4: Purify the DNA library amplified in step S3 using magnetic beads; S5: The purified DNA library is mixed, denatured, and then sequenced. The sequencing results are then compared to determine the gene information.

6. The method according to claim 5, characterized in that, The samples include blood, bloodstains, saliva, oral swabs, fingernails, and hair with follicles.

7. The method according to claim 5, characterized in that, In step S1, the reaction system for the multiplex PCR reaction includes 5 μL of the primer composition described in claim 2, 10 μL of DNA polymerase mixture, 1 μL of gDNA, 2.5 μL of PCR enhancement buffer 1, 3.5 μL of PCR enhancement buffer 2, and 8 μL of nuclease-free water.

8. The method according to claim 5, characterized in that, In step S1, the reaction conditions for multiplex PCR are as follows: i) pre-denaturation at 95°C for 3 minutes and 30 seconds; ii) denaturation at 98°C for 20 seconds; annealing at 55°C for 1 minute; two extension cycles at 60°C for 1 minute and 65°C for 2 minutes; a total of 26 PCR cycles are performed in this step; iii) final extension at 72°C for 5 minutes; iv) hold at 4°C.

9. The method according to claim 5, characterized in that, In step S3, the reaction system consisted of 2 μL of premixed adapter primers, 10 μL of DNA polymerase mixture, 15.5 μL of PCR product mixture, and 2.5 μL of PCR enhancement buffer 1. The reaction conditions were: i) pre-denaturation at 95°C for 3 minutes and 30 seconds; ii) denaturation at 98°C for 20 seconds; annealing at 58°C for 1 minute; extension at 72°C for 30 seconds; a total of 9 PCR cycles were performed in this step; iii) final extension at 72°C for 5 minutes; and iv) holding at 4°C.

10. The application of the STR-SNP multi-marker combined detection system of claim 1, or the primer composition of claim 2, or the kit of claim 3 in individual identification and / or kinship determination in the Chinese population.

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