Forensic genetic marker detection kit based on high-throughput sequencing and use method

Abstract

The invention discloses a forensic genetic marker detection kit based on high-throughput sequencing and a use method, the kit comprises specific amplification primers of 199 genetic markers, and four forensic genetic markers of autosomes STR, Y chromosomes STR, X chromosomes STR and mitochondrial mtDNA hypervariable regions can be simultaneously detected. Compared with the prior art, the kit provided by the invention solves the problem that multiple forensic genetic markers such as a large number of autosomes STR, Y chromosomes STR, X chromosomes STR and mitochondria mtDNA cannot be detected at the same time, improves genetic information which can be obtained by one-time detection, provides more genetic information data for genetic relationship identification and difficult case detection, and has the advantages of high sensitivity, high sensitivity, high sensitivity, high sensitivity and the like. The requirements of actual case inspection, DNA database construction and paternity test can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic genetics, and relates to a kit for forensic identification of individual populations and identification of kinship, and specifically is a forensic genetic marker detection kit based on high-throughput sequencing and a method of use. Background Art

[0002] In forensic testing, short tandem repeats (STR) and mitochondrial mtDNA are two important technical means. STR, short tandem repeats, is a type of DNA sequence formed by repeated tandem with 2 to 6 bases as the core unit. STR is widely present in human loci and has a high degree of polymorphism, making it the most commonly used genetic marker in forensic genetic testing. mtDNA, mitochondrial DNA, is a genetic material present in the cytoplasm. Compared with nuclear DNA, mtDNA has the characteristics of a large number of copies and maternal inheritance. Based on these characteristics, mtDNA has unique advantages in detecting severely degraded DNA and identifying mother-child relationships. STR and mtDNA each have unique technical backgrounds and application values ​​in forensic testing, and together constitute an important part of the forensic identification system, providing strong technical support for judicial justice and case detection. However, in current forensic testing, length typing-based fluorescence composite amplification and capillary electrophoresis are still the mainstream technical means for detecting STR and mtDNA, which results in the inability to obtain a large amount of genetic marker information in one test, and it is almost impossible to detect STR and mtDNA simultaneously. However, with the development and application of high-throughput sequencing technology, it has become possible to detect a large number of STR and mtDNA simultaneously.

[0003] High-throughput sequencing, also known as "next-generation" sequencing technology (NGS) or massively parallel sequencing (MPS), is a gene analysis technology that can sequence millions to billions of DNA or RNA molecules simultaneously. Compared with capillary electrophoresis commonly used in forensic testing, high-throughput sequencing can simultaneously detect more loci, can detect not only locus length polymorphism but also locus sequence polymorphism, and can perform simultaneous testing of multiple samples. Summary of the invention

[0004] Technical problem to be solved: In order to overcome the deficiencies of the prior art and solve the problem that a large number of autosomal STR, Y chromosome STR, X chromosome STR, and mitochondrial mtDNA genetic markers cannot be detected simultaneously, the present invention provides a forensic genetic marker detection kit based on high-throughput sequencing and a method of use.

[0005] Technical solution: A forensic genetic marker detection kit based on high-throughput sequencing, the kit comprising specific amplification primers for amplifying 199 genetic markers; wherein the 199 genetic markers are 73 autosomal STR loci, 89 Y chromosome STR loci, 32 X chromosome STR loci, 2 gender indicator sites and 3 mitochondrial mtDNA hypervariable regions; specifically, the 73 autosomal STR loci are: D3S1358, D13S317, D7S820, D16S539, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D. D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253 , D6S477, D10S1435, SE33, D1S1627, D1S1677, D1GATA113, D2S1776, D3S4529 , D4S2408, D5S2500, D6S474, D6S1017, D9S1122, D11S4463, D12ATA63, D14S14 34. D17S1301, D18S853, D20S482, D7S3048, D9S925, D11S2368, D13S325, D18 S535, D22GATA198B05, D14S608, D7S1517, D4S2366, D17S1290, D20S470, D3S1 744, D21S1270, D5S2800, D10S2325, D9S2157, D8S1115, D4S2364, D3S3053, D2 S1360, D21S2055, D20S1082, D17S974, D18S1364, D1S2142, D3S1545, D20S85;The 89 Y chromosome STR loci are: DYS19, DYS385a / b, DYS389I, DYS389Ⅱ, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y_GATA_H4, DYS481, DYS533, DYS576, DYS643, DYS460, DYS549, DYF387S1a / b, DYS449, DYS518, DYS627, DYS570, DYS527a / b, DYS444, DYS447, DYS5 57. DYS596, DYS510, DYS622, DYS443, DYS587, DYS522, Y-GATA-A10, DYS520, DYS552, DYS593, DYS531, DYS459a / b, DYS508, DYS388, DYS617, DYS645, DYS630, DYF404S1a / b, DYS626, DYS612, DYS434, DYS722, DYS454, DYS450, DYS453, DYS435, DYS476, DYS485, DYS513, DYS590, DYS455, DYS565, DYS568 , DYS556, DYS641, DYS578, DYS638, DYS511, DYS472, DYS502, DYS530, DYS538, DYS541, DYS571, DYS572, DYS573, DYS585, DYS613, DYS640, DYS512, DYS616, DYS644, DYS561, DYS505, DYS446, DYS713, DYS526I, DYS526Ⅱ; the 32 X chromosome STR loci are: DXS8378, DXS7423, DXS6809, DXS10159, DXS10134, DXS7424, D XS9902, DXS981, DXS10079, DXS7132, DXS6789, GATA165B12, DXS101, DXS10103, HPRTB, DXS6800, GATA31E08, GATA172D05, DXS6803, DXS10074, DXS6810, DXS6795, DXS10075, DXS7133, DXS6807, DXS9895, DXS10101, DXS10135, DXS10162, DXS10164, DXS10148, DXS8377; 2 sex indicator loci: Amelogenin, SRY;The three highly variable regions of mitochondrial mtDNA are: HVS-I, HVS-Ⅱ, and HVS-III. ;

[0006] Preferably, the sequences of the specific amplification primers for amplifying 199 genetic markers are: the sequences of the specific amplification primers for 73 autosomal STR loci are: D3S1358, SEQ ID NOs: 1-2; D13S317, SEQ ID NOs: 3-4; D7S820, SEQ ID NOs: 5-6; D16S539, SEQ ID NOs: 7-8; Penta E, SEQ ID NOs: 9-10; TPOX, SEQ ID NOs: 11-12; TH01, SEQ ID NOs: 13-14; D2S1338, SEQ ID NOs: 15-16; CSF1PO, SEQ ID NOs: 17-18; Penta D, SEQ ID NOs: 19-20; D19S433, SEQ ID NOs: 21-22; vWA, SEQ ID NOs: 23-24; D21S11, SEQ ID NOs: 25-26; D18S51, SEQ ID NOs: NO: 27~28; D6S1043, SEQ ID NO: 29~30; D8S1179, SEQ ID NO: 31~32; D5S818, SEQ ID NO: 33~34; D12S391, SEQ ID NO: 35~36; FGA, SEQ ID NO: 37~38; D1S1656, SEQ ID NO:39~40; D2S441, SEQ ID NO:41~42; D22S1045, SEQ ID NO:43~44; D10S1248, SEQ ID NO:45~46; D8S1132, SEQ ID NO:47~48; D15S659, SEQ ID NO:49~50; D3S3045, SEQ ID NO:51~52; D19S253, SEQ ID NO:53~54; D6S477, SEQ ID NO:55~56; D10S1435, SEQ ID NO:57~58; SE33, SEQ ID NO:59~60; D1S1627, SEQ ID NO:61~62; D1S1677, SEQ ID NO:63~64; D1GATA113, SEQ ID NO:65~66; D2S1776, SEQ ID NO: 67~68; D3S4529, SEQ ID NO: 69~70; D4S2408, SEQ ID NO: 71~72; D5S2500, SEQ ID NO: 73~74; D6S474, SEQ ID NO: 75~76; D6S1017, SEQ ID NO: 77~78; D9S1122, SEQ ID NO:79~80;D11S4463, SEQ ID NO:81~82; D12ATA63, SEQ ID NO:83~84; D14S1434, SEQ ID NO:85~86; D17S1301, SEQ ID NO:87~88; D18S853, SEQ ID NO:89~90; D20S482, SEQ ID NO:91~92; D7S3048, SEQ ID NO:93~94; D9S925, SEQ ID NO:95~96; D11S2368, SEQ ID NO:97~98; D13S325, SEQ ID NO:99~100; D18S535, SEQ ID NO:101~102; D22GATA198B05, SEQ ID NO:103~104; D14S608, SEQ ID NO: 105~106; D7S1517, SEQ ID NO: 107~108; D4S2366, SEQ ID NO: 109~110; D17S1290, SEQ ID NO: 111~112; D20S470, SEQ ID NO: 113~114; D3S1744, SEQ ID NO: 115~116; D21S1270, SEQ ID NO: 117~118; D5S2800, SEQ ID NO: 119~120; D10S2325, SEQ ID NO: 121~122; D9S2157, SEQ ID NO: 123~124; D8S1115, SEQ ID NO:125~126; D4S2364, SEQ ID NO:127~128; D3S3053, SEQ ID NO:129-130; D2S1360, SEQ ID NO:131-132; D21S2055, SEQ ID NO:133-134; D20S1082, SEQ ID NO:135-136; D17S974, SEQ ID NO:137-138; D18S1364, SEQ ID NO:139-140; D1S2142, SEQ ID NO:141-142; D3S1545, SEQ ID NO:143-144; D20S85, SEQ ID NO:145-146; The sequences of the 89 Y chromosome STR loci specific amplification primers are: DYS19, SEQ ID NO:147-148; DYS385a / b, SEQ ID NO:149-150; DYS389I, SEQ ID NO:151-152; NO: 151~152; DYS389II, SEQ ID NO: 153~154;DYS390、SEQ ID NO:155~156;DYS391、SEQ ID NO:157~158;DYS392、SEQ IDNO:159~160;DYS393、SEQ ID NO:161~162;DYS437、SEQ ID NO:163~164;DYS438、SEQ IDNO:165~166;DYS439、SEQ ID NO:167~168;DYS448、SEQ ID NO:169~170;DYS456、SEQ IDNO:171~172;DYS458、SEQ ID NO:173~174;DYS635、SEQ ID NO:175~176;Y_GATA_H4、SEQID NO:177~178;DYS481、SEQ ID NO:179~180;DYS533、SEQ ID NO:181~182;DYS576、SEQID NO:183~184;DYS643、SEQ ID NO:185~186;DYS460、SEQ ID NO:187~188;DYS549、SEQID NO:189~190;DYF387S1a / b、SEQ ID NO:191~192;DYS449、SEQ ID NO:193~194;DYS518、SEQ ID NO:195~196;DYS627、SEQ ID NO:197~198;DYS570、SEQ ID NO:199~200;DYS527a / b、SEQ ID NO:201~202;DYS444、SEQ ID NO:203~204;DYS447、SEQ ID NO:205~206;DYS557、SEQ ID NO:207~208;DYS596、SEQ ID NO:209~210;DYS510、SEQ IDNO:211~212;DYS622、SEQ ID NO:213~214;DYS443、SEQ ID NO:215~216;DYS587、SEQ IDNO:217~218;DYS522、SEQ ID NO:219~220;Y-GATA-A10、SEQ ID NO:221~222;DYS520、SEQ ID NO:223~224;DYS552、SEQ ID NO:225~226;DYS593、SEQ ID NO:227~228;DYS531、SEQ ID NO:229~230;DYS459a / b、SEQ ID NO:231~232;DYS508、SEQ ID NO:233~234;DYS388、SEQ ID NO:235~236;DYS617、SEQ ID NO:237~238;DYS645、SEQ ID NO:239~240;DYS630、SEQ ID NO:241~242;DYF404S1a / b、SEQ ID NO:243~244;DYS626、SEQ IDNO:245~246;DYS612、SEQ ID NO:247~248;DYS434、SEQ ID NO:249~250;DYS722、SEQ IDNO:251~252;DYS454、SEQ ID NO:253~254;DYS450、SEQ ID NO:255~256;DYS453、SEQ IDNO:257~258;DYS435、SEQ ID NO:259~260;DYS476、SEQ ID NO:261~262;DYS485、SEQ IDNO:263~264;DYS513、SEQ ID NO:265~266;DYS590、SEQ ID NO:267~268;DYS455、SEQ IDNO:269~270;DYS565、SEQ ID NO:271~272;DYS568、SEQ ID NO:273~274;DYS556、SEQ IDNO:275~276;DYS641、SEQ ID NO:277~278;DYS578、SEQ ID NO:279~280;DYS638、SEQ IDNO:281~282;DYS511、SEQ ID NO:283~284;DYS472、SEQ ID NO:285~286;DYS502、SEQ IDNO:287~288;DYS530、SEQ ID NO:289~290;DYS538、SEQ ID NO:291~292;DYS541、SEQ IDNO:293~294;DYS571、SEQ ID NO:295~296;DYS572、SEQ ID NO:297~298;DYS573、SEQ IDNO:299~300;DYS585、SEQ ID NO:301~302;DYS613、SEQ ID NO:303~304;DYS640、SEQ IDNO:305~306;DYS512、SEQ ID NO:307~308;DYS616、SEQ ID NO:309~310;DYS644、SEQ IDNO:311~312;The sequences of the primers for amplifying the specific STR loci of the 32 X chromosomes are: DXS8378, SEQ ID NOs: 325-326; DXS7423, SEQ ID NOs: 327-328; DXS6809, SEQ ID NOs: 329-330; DXS10159, SEQ ID NOs: 331-332; DXS10134, SEQ ID NOs: 333-334; DXS7424, SEQ ID NOs: 335-336; DXS8378, SEQ ID NOs: 336-337; DXS8378, SEQ ID NOs: 337-338; DXS8378, SEQ ID NOs: 338-339; DXS8378, SEQ ID NOs: 339-340; DXS8378, SEQ ID NOs: 341-342; DXS8378, SEQ ID NOs: 343-343; DXS8378, SEQ ID NOs: 344-344; DXS8378, SEQ ID NOs: 345-346; DXS8378, SEQ ID NOs: 347-348; DXS8378, SEQ ID NOs: 349-350; DXS8378, SEQ ID NOs: 348-351; DXS8378, SEQ ID NOs: 349-352 NO:335~336; DXS9902, SEQ ID NO:337~338; DXS981, SEQ ID NO:339~340; DXS10079, SEQ ID NO:341~342; DXS7132, SEQ ID NO:343~344; DXS6789, SEQ ID NO:345~346; GATA165B12, SEQ ID NO:347~348; DXS101, SEQ ID NO:349~350; DXS10103, SEQ ID NO:351~352; HPRTB, SEQ ID NO:353~354; DXS6800, SEQ ID NO:355~356; GATA31E08, SEQ ID NO:357~358; GATA172D05, SEQ ID NO:359~360; DXS6803, SEQ ID NO:361~362; DXS10074, SEQ ID NO:363~364; DXS6810, SEQ ID NO:365~366; DXS6795, SEQ ID NO:367~368; DXS10075, SEQID NO:369~370; DXS7133, SEQ ID NO:371~372; DXS6807, SEQ ID NO:373~374; DXS9895, SEQ ID NO:375~376; DXS10101, SEQ ID NO:377~378; DXS10135, SEQ ID NO:379~380; DXS10162, SEQ ID NO:381~382; DXS10164, SEQ ID NO:383~384; DXS10148, SEQ ID NO:385~386;DXS8377, SEQ ID NO: 387-388; the sequences of two sex-indicating site-specific amplification primers are: Amelogenin, SEQ ID NO: 389-390; SRY, SEQ ID NO: 391-392; the sequences of three mitochondrial mtDNA hypervariable region-specific amplification primers are: HVS-I, SEQ ID NO: 393-394; HVS-II, SEQ ID NO: 395-396; HVS-III, SEQ ID NO: 397-398. ;

[0007] Preferably, the concentrations of the specific amplification primers are as follows: the concentrations of the specific amplification primers for 73 autosomal STR loci, 2 sex indicator loci and 3 mitochondrial mtDNA hypervariable regions are all 0.05-0.2 μM; the concentrations of the specific amplification primers for 89 Y STR loci and 32 X chromosome STR loci are all 0.1-0.3 μM.

[0008] The specific information of the specific amplification primers for amplifying 199 genetic markers of the present invention is shown in Table 1:

[0009] Table 1 Primer information

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] Preferably, the kit includes a reaction buffer premixed with PCR amplification enzyme, positive standard 9948, water, sequencing tag adapter primers, and a library purification kit.

[0028] Preferably, the reaction buffer of the premixed PCR amplification enzyme includes MgCl25mM, Tris-HCl 150mM, KCl150mM, TC 30mM, dNTPs 1mM, trehalose 2mM, BSA 2.5g / L, and hot start Taq enzyme 2U / μL.

[0029] Preferably, the library purification kit comprises purification magnetic beads, anhydrous ethanol, water and TE Buffer.

[0030] Preferably, the sequencing tag adapter primers include 8 P5 primers and 12 P7 primers, the 8 P5 primers are P5-1, P5-2, P5-3, P5-4, P5-5, P5-6, P5-7, P5-8, and the nucleotide sequences are shown in SEQ ID No.399-406, and the 12 P7 primers are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7, P7-8, P7-9, P7-10, P7-11, P7-12, and the nucleotide sequences are shown in SEQ ID No.407-418. P5 primers and P7 primers can be combined in pairs to form 96 different tag combinations for distinguishing different samples.

[0031] The specific information of the sequencing index adapter primers is shown in Table 2.

[0032] Table 2 Sequencing index adapter primer information

[0033]

[0034]

[0035] A method for using any of the forensic genetic marker detection kits based on high-throughput sequencing, the method comprising two rounds of amplification and two rounds of purification, the specific steps being:

[0036] S1, first round amplification: targeted enrichment, using specific amplification primers to amplify the sample and enrich the targeted sequence;

[0037] S2, the first round of purification: target purification, use the library purification kit to purify the amplified product, remove excess primers, reaction buffer, template, and normalize the amplified product;

[0038] S3, second round of amplification: sequencing tag connection, using PCR method to connect the sequencing tag to the purified amplification product to form a complete library;

[0039] S4, second round of purification: Use a library purification kit to purify the library in S3, remove excess sequencing index primers, reaction buffer, and template, and obtain a homogenized library.

[0040] Application of any of the above-mentioned forensic genetic marker detection kits based on high-throughput sequencing in forensic identification of individual individuals in the population.

[0041] Application of any of the above-mentioned forensic genetic marker detection kits based on high-throughput sequencing in kinship identification.

[0042] Beneficial effects: (1) The kit of the present invention can simultaneously detect 73 autosomal STRs, 89 Y chromosome STRs, 32 X chromosomes, 2 sex indicator sites and 3 mitochondrial mtDNA hypervariable regions, a total of 199 genetic markers; (2) The kit of the present invention can detect a large number of genetic markers of various types, and can also perform base sequence-specific analysis, thereby providing more genetic information data for kinship identification and actual case detection; (3) The primers in the kit of the present invention have the advantages of strong specificity, high sensitivity and accurate typing results, and can fully meet the needs of actual case inspection, DNA database construction and paternity testing. DETAILED DESCRIPTION

[0043] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0044] Example 1 Construction of the kit of the present invention

[0045] The construction of the kit of the present invention includes the selection of genetic markers and the screening of the system, wherein the selected genetic markers mainly refer to the genetic markers involved in the national standard GB / T41009-2021 "Loci and data structures selected for forensic DNA databases", and also include some genetic markers used in commercial capillary electrophoresis detection kits. The basic information of the genetic markers included in the kit is shown in Table 3.

[0046] Table 3 Basic information of genetic markers in the kit

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Based on Table 3, specific amplification primers were designed and screened, and the primer information is shown in Table 1.

[0055] Table 1 Primer information

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] The kit comprises a reaction buffer premixed with PCR amplification enzyme, a positive standard 9948, water, a sequencing tag adapter primer, and a library purification kit.

[0074] The reaction buffer of the premixed PCR amplification enzyme includes MgCl25mM, Tris-HCl 150mM, KCl 150mM, TC30mM, dNTPs 1mM, trehalose 2mM, BSA 2.5g / L, and hot start Taq enzyme 2U / μL.

[0075] The library purification kit includes purification magnetic beads, anhydrous ethanol, water and TE Buffer.

[0076] The sequencing tag adapter primers include 8 P5 primers and 12 P7 primers, the 8 P5 primers are P5-1, P5-2, P5-3, P5-4, P5-5, P5-6, P5-7, P5-8, and the nucleotide sequences are shown in SEQ ID No.399-406, and the 12 P7 primers are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7, P7-8, P7-9, P7-10, P7-11, and P7-12, and the nucleotide sequences are shown in SEQ ID No.407-418. P5 primers and P7 primers can be combined in pairs to form 96 different tag combinations for distinguishing different samples.

[0077] The specific information of the sequencing index adapter primers is shown in Table 2.

[0078] Table 2 Sequencing index adapter primer information

[0079]

[0080]

[0081] Example 2

[0082] The practical application of the kit described in Example 1 of the present invention, taking the result of positive standard 9948 as an example, the specific steps are as follows:

[0083] Collect genomic DNA, target enrichment, target purification, sequencing index connection, library purification, sequencing, and result analysis.

[0084] 1. Collect genomic DNA

[0085] The sample sources used in forensic identification, paternity testing or DNA family tree construction include human genomic DNA extracted by Chelex-100 method, magnetic bead extraction method or organic extraction method; or human blood or oral cells collected by any carrier such as filter paper, FTA card, cotton swab or gauze without extraction. The sources of samples include human blood, blood stains, semen, saliva, body fluids, hair, muscle or tissue organs.

[0086] 2. Targeted Enrichment

[0087] ① In this step, the following reagents are prepared in the kit of the present invention: reaction buffer premixed with PCR amplification enzyme, specific amplification primer mixture, and water.

[0088] ② For each sample to be tested, an amplification system is prepared according to the following reaction system: reaction buffer premixed with PCR amplification enzyme, 10 μL; specific amplification primer mixture, 5 μL; sample to be tested and water, 10 μL.

[0089] ③After the amplification system is prepared, put it into the PCR instrument for amplification. The amplification program is as follows: 95℃, 2min; (95℃, 15s; 60℃, 120s; 72℃, 30s)*20cycles; 72℃, 5min.

[0090] 3. Target purification

[0091] This step requires the preparation of the library purification kit in the kit of the present invention, which contains the following reagents: water, purification magnetic beads, and anhydrous ethanol. The specific operation is as follows:

[0092] ① Take out the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use. Pipette 60μL of purified magnetic beads and 25μL of water into new tubes respectively, and transfer the targeted enriched PCR products to the new tubes containing purified magnetic beads and water. Use a pipette to quickly blow 8 to 10 times to mix evenly, and avoid bubbles during the process.

[0093] ②Incubate at room temperature for 10 minutes.

[0094] ③ Place on a magnetic stand and let stand for 5 minutes until the liquid becomes clear, then carefully aspirate and discard the supernatant with a pipette.

[0095] ④ Keep the centrifuge tube or 96-well plate on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube wall, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0096] ⑤ Repeat step ④ and try to drain the liquid in the tube. If there is still liquid left in the tube, centrifuge briefly and use a 20μL pipette tip to suck out the remaining liquid at the bottom.

[0097] ⑥ Keep the centrifuge tube or 96-well plate fixed on the magnetic rack and dry at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0098] ⑦ Remove the centrifuge tube or 96-well plate from the magnetic rack and add 20 μL of water to elute the DNA. Make sure that the added water fully soaks the magnetic beads to prevent the magnetic beads from drying out too much and causing loss of PCR products.

[0099] ⑧Let stand at room temperature for 5 minutes. Prepare for next step.

[0100] 4. Sequencing tag connection

[0101] ① In this step, the following reagents in the kit of the present invention are prepared: reaction buffer premixed with PCR amplification enzyme, sequencing tag combination pair of primers (each pair of sequencing tags contains different P5 and P7 primers).

[0102] ② For each sample to be tested, the amplification system is prepared according to the following reaction system: reaction buffer premixed with PCR amplification enzyme, 10 μL; sequencing label combination primer pair, 2 μL; product after purification in the previous step, 13 μL.

[0103] ③After the amplification system is prepared, put it into the PCR instrument for amplification. The amplification program is as follows: 95℃, 2min; (95℃, 15s; 60℃, 30s; 72℃, 30s)*8cycles; 72℃, 5min.

[0104] 5. Library purification

[0105] This step requires the preparation of the library purification kit in the kit of the present invention, which contains the following reagents: water, purification magnetic beads, anhydrous ethanol, and TE Buffer. The specific operation is as follows:

[0106] ① Take out the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use. Pipette 35μL of purified magnetic beads and 25μL of water into new tubes respectively. Transfer the PCR product connected with the sequencing tag to the new tube containing purified magnetic beads and water. Use a pipette to quickly blow 8 to 10 times to mix evenly. Avoid bubbles during the process.

[0107] ②Incubate at room temperature for 10 minutes.

[0108] ③ Place on a magnetic stand and let stand for 5 minutes until the liquid becomes clear, then carefully aspirate and discard the supernatant with a pipette.

[0109] ④ Keep the centrifuge tube or 96-well plate on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube wall, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0110] ⑤ Repeat step ④ and try to drain the liquid in the tube. If there is still liquid left in the tube, centrifuge briefly and use a 20μL pipette tip to drain the remaining liquid at the bottom.

[0111] ⑥ Keep the centrifuge tube or 96-well plate fixed on the magnetic rack and dry at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0112] ⑦ Remove the centrifuge tube or 96-well plate from the magnetic stand and add 22μL TE Buffer to elute the DNA. Make sure that the added TE Buffer fully soaks the magnetic beads to prevent the magnetic beads from drying out too much and causing loss of PCR products.

[0113] ⑧Leave at room temperature for 5 minutes.

[0114] ⑨ Place the centrifuge tube or 96-well plate on the magnetic rack and let it stand for 5 minutes until the liquid becomes clear. Transfer 20 μL of the supernatant to a new centrifuge tube or 96-well plate for storage.

[0115] 6. Sequencing

[0116] According to the sequencing requirements of the GeneMind FASTASeq 300 gene sequencer, the prepared library was quantified, pooled, and processed, and then sequenced according to the sequencing steps of the sequencer.

[0117] 7. Results Analysis

[0118] The sequencing data of GeneMind FASTASeq 300 gene sequencer was used to obtain all the typing of 199 genetic markers in the kit of the present invention and their corresponding sequence structures through bioinformatics data analysis software.

[0119] In this example, the typing of all genetic markers of positive standard 9948 and their corresponding sequence structures are obtained, as shown in Table 4:

[0120] Table 4 Genetic marker typing and corresponding sequence structure of positive standard 9948

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

Claims

1. A forensic genetic marker detection kit based on high-throughput sequencing, characterized in that: The kit includes specific amplification primers for amplifying 199 genetic markers; wherein the 199 genetic markers are 73 autosomal STR loci, 89 Y chromosome STR loci, 32 X chromosome STR loci, 2 gender indicator sites and 3 mitochondrial mtDNA hypervariable regions; specifically, the 73 autosomal STR loci are: D3S1358, D13S317, D7S820, D16S539, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D. D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253 , D6S477, D10S1435, SE33, D1S1627, D1S1677, D1GATA113, D2S1776, D3S4529 , D4S2408, D5S2500, D6S474, D6S1017, D9S1122, D11S4463, D12ATA63, D14S14 34. D17S1301, D18S853, D20S482, D7S3048, D9S925, D11S2368, D13S325, D18 S535, D22GATA198B05, D14S608, D7S1517, D4S2366, D17S1290, D20S470, D3S1 744, D21S1270, D5S2800, D10S2325, D9S2157, D8S1115, D4S2364, D3S3053, D2 S1360, D21S2055, D20S1082, D17S974, D18S1364, D1S2142, D3S1545, D20S85;The 89 Y chromosome STR loci are: DYS19, DYS385a / b, DYS389I, DYS389Ⅱ, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y_GATA_H4, DYS481, DYS533, DYS576, DYS643, DYS460, DYS549, DYF387S1a / b, DYS449, DYS518, DYS627, DYS570, DYS527a / b, DYS444, DYS447, DYS5 57. DYS596, DYS510, DYS622, DYS443, DYS587, DYS522, Y-GATA-A10, DYS520, DYS552, DYS593, DYS531, DYS459a / b, DYS508, DYS388, DYS617, DYS645, DYS630, DYF404S1a / b, DYS626, DYS612, DYS434, DYS722, DYS454, DYS450, DYS453, DYS435, DYS476, DYS485, DYS513, DYS590, DYS455, DYS565, DYS568 , DYS556, DYS641, DYS578, DYS638, DYS511, DYS472, DYS502, DYS530, DYS538, DYS541, DYS571, DYS572, DYS573, DYS585, DYS613, DYS640, DYS512, DYS616, DYS644, DYS561, DYS505, DYS446, DYS713, DYS526I, DYS526Ⅱ; the 32 X chromosome STR loci are: DXS8378, DXS7423, DXS6809, DXS10159, DXS10134, DXS7424, D XS9902, DXS981, DXS10079, DXS7132, DXS6789, GATA165B12, DXS101, DXS10103, HPRTB, DXS6800, GATA31E08, GATA172D05, DXS6803, DXS10074, DXS6810, DXS6795, DXS10075, DXS7133, DXS6807, DXS9895, DXS10101, DXS10135, DXS10162, DXS10164, DXS10148, DXS8377; 2 sex indicator loci: Amelogenin, SRY;The three highly variable regions of mitochondrial mtDNA are: HVS-I, HVS-Ⅱ, and HVS-III. ; 2. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 1, characterized in that: The sequences of the specific amplification primers for amplifying 199 genetic markers are as follows: the sequences of the specific amplification primers for 73 autosomal STR loci are as follows: D3S1358, SEQ ID NOs: 1-2; D13S317, SEQ ID NOs: 3-4; D7S820, SEQ ID NOs: 5-6; D16S539, SEQ ID NOs: 7-8; Penta E, SEQ ID NOs: 9-10; TPOX, SEQ ID NOs: 11-12; TH01, SEQ ID NOs: 13-14; D2S1338, SEQ ID NOs: 15-16; CSF1PO, SEQ ID NOs: 17-18; PentaD, SEQ ID NOs: 19-20; D19S433, SEQ ID NOs: 21-22; vWA, SEQ ID NOs: 23-24; D21S11, SEQ ID NOs: 25-26; D18S51, SEQ ID NOs: 27-28; NO: 27~28; D6S1043, SEQ ID NO: 29~30; D8S1179, SEQ ID NO: 31~32; D5S818, SEQ ID NO: 33~34; D12S391, SEQ ID NO: 35~36; FGA, SEQ ID NO: 37~38; D1S1656, SEQ ID NO:39~40; D2S441, SEQ ID NO:41~42; D22S1045, SEQ ID NO:43~44; D10S1248, SEQ ID NO:45~46; D8S1132, SEQ ID NO:47~48; D15S659, SEQ ID NO:49~50; D3S3045, SEQ ID NO:51~52; D19S253, SEQ ID NO:53~54; D6S477, SEQ ID NO:55~56; D10S1435, SEQ ID NO:57~58; SE33, SEQ ID NO:59~60; D1S1627, SEQ ID NO:61~62; D1S1677, SEQ ID NO:63~64; D1GATA113, SEQ ID NO:65~66; D2S1776, SEQ ID NO:67~68; D3S4529, SEQ ID NO:69~70; D4S2408, SEQ ID NO:71~72; D5S2500, SEQ ID NO:73~74; D6S474, SEQ ID NO:75~76; D6S1017, SEQ ID NO:77~78; D9S1122, SEQ ID NO:79~80;D11S4463、SEQ ID NO:81~82;D12ATA63、SEQ ID NO:83~84;D14S1434、SEQ ID NO:85~86;D17S1301、SEQ ID NO:87~88;D18S853、SEQ ID NO:89~90;D20S482、SEQ ID NO:91~92;D7S3048、SEQ ID NO:93~94;D9S925、SEQ ID NO:95~96;D11S2368、SEQ ID NO:97~98;D13S325、SEQ ID NO:99~100;D18S535、SEQ ID NO:101~102; D22GATA198B05, SEQ ID NO: 103 - 104; D14S608, SEQ ID NO: 105 - 106; D7S1517, SEQ ID NO: 107 - 108; D4S2366, SEQ ID NO: 109 - 110; D17S1290, SEQ ID NO: 111 - 112; D20S470, SEQ ID NO: 113 - 114; D3S1744, SEQ ID NO: 115 - 116; D21S1270, SEQ ID NO: 117 - 118; D5S2800, SEQ ID NO: 119 - 120; D10S2325, SEQ ID NO: 121 - 122; D9S2157, SEQ ID NO: 123 - 124; D8S1115, SEQ ID NO: 125 - 126; D4S2364, SEQ ID NO: 127 - 128; D3S3053, SEQ ID NO: 129 - 130; D2S1360, SEQ ID NO: 131 - 132; D21S2055, SEQ ID NO: 133 - 134; D20S1082, SEQ ID NO: 135 - 136; D17S974, SEQ ID NO: 137 - 138; D18S1364, SEQ ID NO: 139 - 140; D1S2142, SEQ ID NO: 141 - 142; D3S1545, SEQ ID NO: 143 - 144; D20S85, SEQ ID NO: 145 - 146; The sequences of the specific amplification primers for 89 Y - chromosome STR loci are: DYS19, SEQ ID NO: 147 - 148; DYS385a / b, SEQ ID NO: 149 - 150; DYS389I, SEQ ID NO: 151 - 152; DYS389Ⅱ, SEQ ID NO: 153 - 154; DYS390, SEQ ID NO: 155 - 156; DYS391, SEQ ID NO: 157 - 158; DYS392, SEQ ID NO: 159 - 160; DYS393, SEQ ID NO: 161 - 162; DYS437, SEQ ID NO: 163 - 164; DYS438, SEQ ID NO: 165 - 166; DYS439, SEQ ID NO: 167 - 168; DYS448, SEQ ID NO: 169 - 170; DYS456, SEQ ID NO: 171 - 172; DYS458, SEQ ID NO: 173 - 174; DYS635, SEQ ID NO: 175 - 176;Y_GATA_H4、SEQ ID NO:177~178;DYS481、SEQID NO:179~180;DYS533、SEQ ID NO:181~182;DYS576、SEQ ID NO:183~184;DYS643、SEQID NO:185~186;DYS460、SEQ ID NO:187~188;DYS549、SEQ ID NO:189~190;DYF387S1a / b、SEQ ID NO:191~192;DYS449、SEQ ID NO:193~194;DYS518、SEQ ID NO:195~196;DYS627、SEQ ID NO:197~198;DYS570、SEQ ID NO:199~200;DYS527a / b、SEQ ID NO:201~202;DYS444、SEQ ID NO:203~204;DYS447、SEQ ID NO:205~206;DYS557、SEQ ID NO:207~208;DYS596、SEQ ID NO:209~210;DYS510、SEQ ID NO:211~212;DYS622、SEQ ID NO:213~214;DYS443、SEQ ID NO:215~216;DYS587、SEQ ID NO:217~218;DYS522、SEQ IDNO:219~220;Y-GATA-A10、SEQ ID NO:221~222;DYS520、SEQ ID NO:223~224;DYS552、SEQ ID NO:225~226;DYS593、SEQ ID NO:227~228;DYS531、SEQ ID NO:229~230;DYS459a / b、SEQ ID NO:231~232;DYS508、SEQ ID NO:233~234;DYS388、SEQ ID NO:235~236;DYS617、SEQ ID NO:237~238;DYS645、SEQ ID NO:239~240;DYS630、SEQ ID NO:241~242;DYF404S1a / b、SEQ ID NO:243~244;DYS626、SEQ ID NO:245~246;DYS612、SEQ IDNO:247~248;DYS434、SEQ ID NO:249~250;DYS722、SEQ ID NO:251~252;DYS454、SEQ IDNO:253~254;DYS450, SEQ ID NO: 255 - 256; DYS453, SEQ ID NO: 257 - 258; DYS435, SEQ ID NO: 259 - 260; DYS476, SEQ ID NO: 261 - 262; DYS485, SEQ ID NO: 263 - 264; DYS513, SEQ ID NO: 265 - 266; DYS590, SEQ ID NO: 267 - 268; DYS455, SEQ ID NO: 269 - 270; DYS565, SEQ ID NO: 271 - 272; DYS568, SEQ ID NO: 273 - 274; DYS556, SEQ ID NO: 275 - 276; DYS641, SEQ ID NO: 277 - 278; DYS578, SEQ ID NO: 279 - 280; DYS638, SEQ ID NO: 281 - 282; DYS511, SEQ ID NO: 283 - 284; DYS472, SEQ ID NO: 285 - 286; DYS502, SEQ ID NO: 287 - 288; DYS530, SEQ ID NO: 289 - 290; DYS538, SEQ ID NO: 291 - 292; DYS541, SEQ ID NO: 293 - 294; DYS571, SEQ ID NO: 295 - 296; DYS572, SEQ ID NO: 297 - 298; DYS573, SEQ ID NO: 299 - 300; DYS585, SEQ ID NO: 301 - 302; DYS613, SEQ ID NO: 303 - 304; DYS640, SEQ ID NO: 305 - 306; DYS512, SEQ ID NO: 307 - 308; DYS616, SEQ ID NO: 309 - 310; DYS644, SEQ ID NO: 311 - 312; DYS561, SEQ ID NO: 313 - 314; DYS505, SEQ ID NO: 315 - 316; DYS446, SEQ ID NO: 317 - 318; DYS713, SEQ ID NO: 319 - 320; DYS526I, SEQ ID NO: 321 - 322; DYS526II, SEQ ID NO: 323 - 324; The sequences of the specific amplification primers for 32 X - chromosome STR loci are: DXS8378, SEQ ID NO: 325 - 326; DXS7423, SEQ ID NO: 327 - 328; DXS6809, SEQ ID NO: 329 - 330; DXS10159, SEQ ID NO: 331 - 332;DXS10134, SEQ ID NO:333~334; DXS7424, SEQ ID NO:335~336; DXS9902, SEQ ID NO:337~338; DXS981, SEQ ID NO:339~340; DXS10079, SEQ ID NO:341~342; DXS7132, SEQ ID NO:343~344; DXS6789, SEQ ID NO:345~346; GATA165B12, SEQ ID NO:347~348; DXS101, SEQ ID NO:349~350; DXS10103, SEQ ID NO:351~352; HPRTB, SEQ ID NO:353~354; DXS6800, SEQ ID NO:355~356; GATA31E08, SEQ ID NO:357~358; GATA172D05, SEQ ID NO:359~360; DXS6803, SEQ ID NO:361~362; DXS10074, SEQ ID NO:363~364; DXS6810, SEQ ID NO:365~366; DXS6795, SEQ ID NO:367~368; DXS10075, SEQ ID NO:369~370; DXS7133, SEQ ID NO:371~372; DXS6807, SEQ ID NO:373~374; DXS9895, SEQ ID NO:375~376; DXS10101, SEQ ID NO:377~378; DXS10135, SEQ ID NO:379~380; DXS10162, SEQ ID NO:381-382; DXS10164, SEQ ID NO:383-384; DXS10148, SEQ ID NO:385-386; DXS8377, SEQ ID NO:387-388; the sequences of the two sex-indicating site-specific amplification primers are: Amelogenin, SEQ ID NO:389-390; SRY, SEQ ID NO:391-392; the sequences of the three mitochondrial mtDNA hypervariable region-specific amplification primers are: HVS-I, SEQ ID NO:393-394; HVS-Ⅱ, SEQ ID NO:395-396; HVS-III, SEQ ID NO:397-398. ; 3. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 1, characterized in that: The concentrations of the specific amplification primers are as follows: the concentrations of the specific amplification primers for 73 autosomal STR loci, 2 sex indicator loci and 3 mitochondrial mtDNA hypervariable regions are all 0.05-0.2 μM; the concentrations of the specific amplification primers for 89 Y STR loci and 32 X chromosome STR loci are all 0.1-0.3 μM.

4. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 1, characterized in that: The kit comprises a reaction buffer premixed with PCR amplification enzyme, a positive standard 9948, water, a sequencing tag adapter primer, and a library purification kit.

5. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 4, characterized in that: The reaction buffer of the premixed PCR amplification enzyme includes MgCl25mM, Tris-HCl 150mM, KCl 150mM, TC 30mM, dNTPs 1mM, trehalose 2mM, BSA 2.5g / L, and hot start Taq enzyme 2U / μL.

6. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 4, characterized in that: The library purification kit includes purification magnetic beads, anhydrous ethanol, water and TE Buffer.

7. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 4, characterized in that: The sequencing tag adapter primers include 8 P5 primers and 12 P7 primers, the 8 P5 primers are P5-1, P5-2, P5-3, P5-4, P5-5, P5-6, P5-7, and P5-8, and the nucleotide sequences are shown in SEQ ID No.399-406; the 12 P7 primers are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7, P7-8, P7-9, P7-10, P7-11, and P7-12, and the nucleotide sequences are shown in SEQ ID No.407-418.

8. The method for using the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 1 to 7, characterized in that: The method comprises two rounds of amplification and two rounds of purification, and the specific steps are: S1, first round amplification: targeted enrichment, using specific amplification primers to amplify the sample and enrich the targeted sequence; S2, the first round of purification: target purification, use the library purification kit to purify the amplified product, remove excess primers, reaction buffer, template, and normalize the amplified product; S3, second round of amplification: sequencing tag connection, using PCR method to connect the sequencing tag to the purified amplification product to form a complete library; S4, second round of purification: Use a library purification kit to purify the library in S3, remove excess sequencing index primers, reaction buffer, and template, and obtain a homogenized library.

9. Use of the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 1 to 7 in forensic identification of individuals in a population.

10. Use of the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 1 to 7 in kinship identification.

Images