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

Abstract

The present invention discloses a forensic genetic marker detection kit based on high-throughput sequencing and its use method. The kit contains specific amplification primers for 199 genetic markers and can simultaneously detect four types of forensic genetic markers: autosomal STRs, Y-chromosome STRs, X-chromosome STRs, and mitochondrial mtDNA hypervariable regions. Compared with existing technologies, the kit of the present invention solves the problem of being unable to simultaneously detect a large number of forensic genetic markers, including autosomal STRs, Y-chromosome STRs, X-chromosome STRs, and mitochondrial mtDNA. It increases the genetic information that can be obtained in a single test, provides more genetic information data for the identification of kinship and the detection of difficult cases, and can meet the needs of actual case inspection, DNA database construction, and paternity testing.

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 people and identification of kinship, specifically 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 (STRs) and mitochondrial DNA (mtDNA) are two important techniques. STRs, or short tandem repeats, are a type of DNA sequence consisting of 2-6 bases repeated in series. Due to their widespread presence and high polymorphism across human loci, STRs are the most commonly used genetic marker in forensic genetic testing. Mitochondrial DNA (mtDNA), also known as mitochondrial DNA, is a type of genetic material found in the cytoplasm. Compared to nuclear DNA, mtDNA has a higher copy number and follows maternal inheritance. These characteristics give mtDNA unique advantages in detecting severely degraded DNA and determining maternal-child relationships. STRs and mtDNA each possess unique technical backgrounds and application value in forensic testing. Together, they constitute a vital component of the forensic identification system, providing strong technical support for judicial justice and case resolution. However, current forensic testing still relies on length typing-based fluorescence multiplex amplification and capillary electrophoresis as the mainstream techniques for detecting STRs and mtDNA. This results in a lack of information on a large number of genetic markers in a single test, making simultaneous detection of STRs and mtDNA nearly impossible. However, with the development and application of high-throughput sequencing technology, simultaneous detection of large numbers of STRs and mtDNAs has become possible.

[0003] High-throughput sequencing (also known as next-generation sequencing technology (NGS) or massively parallel sequencing (MPS)) is a genetic analysis technique capable of simultaneously sequencing millions to billions of DNA or RNA molecules. Compared to capillary electrophoresis, commonly used in forensic testing, high-throughput sequencing can simultaneously examine a greater number of loci, detect both length and sequence polymorphisms, and perform simultaneous testing on multiple samples. Summary of the Invention

[0004] Technical Problem Solved: In order to overcome the shortcomings of the existing technology and solve the problem that a large number of autosomal STRs, Y chromosome STRs, X chromosome STRs, 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, 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 sex 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, and DYS551. 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 hypervariable 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 as follows: 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 32 X-chromosome STR loci 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. 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 NOs: 387-388; two sex-indicating locus-specific amplification primers: Amelogenin, SEQ ID NOs: 389-390; SRY, SEQ ID NOs: 391-392; three mitochondrial mtDNA hypervariable region-specific amplification primers: HVS-I, SEQ ID NOs: 393-394; HVS-II, SEQ ID NOs: 395-396; HVS-III, SEQ ID NOs: 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 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 index 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 their nucleotide sequences are shown in SEQ ID Nos. 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 their nucleotide sequences are shown in SEQ ID Nos. 407-418. P5 primers and P7 primers can be combined in pairs to form 96 different index combinations for distinguishing different samples.

[0031] The detailed 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 above-mentioned forensic genetic marker detection kits based on high-throughput sequencing, comprising two rounds of amplification and two rounds of purification, specifically comprising the following steps:

[0036] S1. First round of amplification: Target enrichment, using specific amplification primers to amplify the sample and enrich the target sequence;

[0037] S2. First round of purification: Target purification: Use a 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 ligation, using PCR to connect the sequencing tags to the purified amplification products to form a complete library;

[0039] S4, second round of purification: Use a library purification kit to purify the library in S3 to remove excess sequencing index primers, reaction buffer, and template to 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 loci and 3 mitochondrial mtDNA hypervariable regions, totaling 199 genetic markers; (2) The kit of the present invention can detect a rich variety of genetic markers in large quantities and can simultaneously 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 detection, DNA database construction and paternity testing. DETAILED DESCRIPTION

[0043] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques 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 described herein involves the selection of genetic markers and system screening. The selected genetic markers primarily reference the genetic markers specified in the national standard GB / T41009-2021, "Loci and Data Structures for Forensic DNA Database Selection," and also include some genetic markers used in commercial capillary electrophoresis detection kits. Basic information about 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. 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 includes a reaction buffer premixed with PCR amplification enzyme, positive standard 9948, water, sequencing tag adapter primers, 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 index 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 their nucleotide sequences are shown in SEQ ID Nos. 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 their nucleotide sequences are shown in SEQ ID Nos. 407-418. P5 primers and P7 primers can be combined in pairs to form 96 different index combinations for distinguishing different samples.

[0077] The detailed 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 results 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] Sample sources used in forensic identification, paternity testing, or DNA family tree construction include human genomic DNA extracted using the Chelex-100 method, magnetic bead extraction, or organic extraction methods; or human blood or buccal cells collected using any of the following carriers: filter paper, FTA cards, cotton swabs, or gauze. Sample sources include human blood, bloodstains, semen, saliva, body fluids, hair, muscle, or tissues.

[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] ③ After the amplification system is prepared, place it in 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 preparation of the library purification kit in the kit of the present invention. The library purification kit contains the following reagents: water, purification magnetic beads, and anhydrous ethanol. The specific steps are as follows:

[0092] ① Remove the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex thoroughly before use. Pipette 60 μL of purified magnetic beads and 25 μL of water into a new tube. Transfer the target-enriched PCR product to the new tube containing the purified magnetic beads and water. Mix thoroughly by pipetting 8-10 times quickly, avoiding bubbles.

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

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

[0095] ④ Place the centrifuge tube or 96-well plate on the magnetic stand, 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 remaining in the tube, centrifuge briefly and use a 20μL pipette tip to remove 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 stand 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 excessively and causing loss of PCR products.

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

[0100] 4. Sequencing tag connection

[0101] ① In this step, the following reagents are prepared in the kit of the present invention: reaction buffer premixed with PCR amplification enzyme, sequencing tag combination primer pairs (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 tag combination primer pair, 2 μL; the purified product from the previous step, 13 μL.

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

[0104] 5. Library purification

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

[0106] ① Remove the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex thoroughly before use. Pipette 35 μL of purified magnetic beads and 25 μL of water into a new tube. Transfer the PCR product with the sequencing tags attached to the new tube containing the purified magnetic beads and water. Mix thoroughly by pipetting 8-10 times quickly, avoiding bubbles.

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

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

[0109] ④ Place the centrifuge tube or 96-well plate on the magnetic stand, 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 remaining in the tube, centrifuge briefly and use a 20μL pipette tip to remove 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 beads from drying out excessively 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 a 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 the GeneMind FASTASeq 300 gene sequencer were used to obtain all the typing of the 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 and their corresponding sequence structures of the positive standard 9948 were 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 sex 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, and DYS551.

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 hypervariable regions of mitochondrial mtDNA are: HVS-I, HVS-II, and HVS-III; 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: 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: 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 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 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、SEQ ID NO:179~180;DYS533、SEQ ID NO:181~182;DYS576、SEQ ID NO:183~184;DYS643、SEQ ID 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 IDNO:201~202;DYS444、SEQ ID NO:203~204;DYS447、SEQ ID NO:205~206;DYS557、SEQ IDNO:207~208;DYS596、SEQ ID NO:209~210;DYS510、SEQ ID NO:211~212;DYS622、SEQ IDNO: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、SEQID NO:225~226;DYS593、SEQ ID NO:227~228;DYS531、SEQ ID NO:229~230;DYS459a / b、SEQID NO:231~232;DYS508、SEQ ID NO:233~234;DYS388、SEQ ID NO:235~236;DYS617、SEQ IDNO:237~238;DYS645、SEQ ID NO:239~240;DYS630、SEQ ID NO:241~242;DYF404S1a / b、SEQID NO:243~244;DYS626、SEQ ID NO:245~246;DYS612、SEQ ID NO:247~248;DYS434、SEQ IDNO:249~250;DYS722、SEQ ID NO:251~252;DYS454、SEQ ID NO:253~254;DYS450、SEQ IDNO:255~256;DYS453、SEQ ID NO:257~258;DYS435、SEQ ID NO:259~260;DYS476、SEQ IDNO:261~262;DYS485、SEQ ID NO:263~264;DYS513、SEQ ID NO:265~266;DYS590、SEQ IDNO:267~268;DYS455、SEQ ID NO:269~270;DYS565、SEQ ID NO:271~272;DYS568、SEQ IDNO:273~274;DYS556、SEQ ID NO:275~276;DYS641、SEQ ID NO:277~278;DYS578、SEQ IDNO:279~280;DYS638、SEQ ID NO:281~282;DYS511、SEQ ID NO:283~284;DYS472、SEQ IDNO:285~286;DYS502、SEQ ID NO:287~288;DYS530、SEQ ID NO:289~290;DYS538、SEQ IDNO:291~292;DYS541、SEQ ID NO:293~294;DYS571、SEQ ID NO:295~296;DYS572、SEQ IDNO:297~298;DYS573、SEQ ID NO:299~300;DYS585、SEQ ID NO:301~302;DYS613、SEQ IDNO:303~304;DYS640、SEQ ID NO:305~306;DYS512、SEQ ID NO:307~308;DYS616、SEQ IDNO:309~310;DYS644、SEQ ID NO:311~312;31-328; 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; DXS7423, SEQ ID NOs: 337-338; DXS6809, SEQ ID NOs: 338-339; DXS10159, SEQ ID NOs: 340-341; DXS10134, SEQ ID NOs: 342-343; DXS7424, SEQ ID NOs: 344-345. IDNO: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 NOs: 387-388; two sex-indicating locus-specific amplification primers: Amelogenin, SEQ ID NOs: 389-390; SRY, SEQ ID NOs: 391-392; three mitochondrial mtDNA hypervariable region-specific amplification primers: HVS-I, SEQ ID NOs: 393-394; HVS-II, SEQ ID NOs: 395-396; HVS-III, SEQ ID NOs: 397-398; 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.

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

3. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 2, characterized in that: The library purification kit includes purification magnetic beads, anhydrous ethanol, water and TE Buffer.

4. The forensic genetic marker detection kit based on high-throughput sequencing according to claim 2, 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 their nucleotide sequences are shown in SEQ ID Nos. 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 their nucleotide sequences are shown in SEQ ID Nos. 407-418.

5. The method for using the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 2 to 4, characterized in that: The method includes two rounds of amplification and two rounds of purification, and the specific steps are: S1. First round of amplification: Target enrichment, using specific amplification primers to amplify the sample and enrich the target sequence; S2. First round of purification: Target purification: Use a 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 ligation, using PCR to connect the sequencing tags to the purified amplification products to form a complete library; S4, second round of purification: Use a library purification kit to purify the library in S3 to remove excess sequencing index primers, reaction buffer, and template to obtain a homogenized library.

6. Use of the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 1 to 4 in forensic identification of individuals in a population.

7. Use of the forensic genetic marker detection kit based on high-throughput sequencing according to any one of claims 1 to 4 in kinship identification.

Images