A one-step library-based multiplex SNP combined microhaplotype amplification system, construction method, product and application

Abstract

The application belongs to the technical field of forensic medicine, and particularly relates to a multiple SNP combined microhaplotype amplification system and construction method, product and application based on one-step library construction. The application is based on RC-PCR technology, and constructs a one-step library construction composite amplification system. The amplification system comprises RC-Primer with a sequence as shown in SEQ ID No. 1-SEQ ID No. 210, I5-Primer with a sequence as shown in SEQ ID No. 211-SEQ ID No. 214, and I7-Primer with a sequence as shown in SEQ ID No. 215-SEQ ID No. 222. The method provided by the application realizes one-time PCR reaction, simultaneously completes targeted amplification and library construction, greatly shortens experimental time and simplifies experimental steps, and the advantage is more significant when a large number of samples are simultaneously detected.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of forensic science, and particularly relates to a one-step library construction-based amplification system and construction method for multiple SNPs combined with microhaplotypes, products and applications. BACKGROUND

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, has been widely used in forensic genetics due to its high throughput, fast sequencing speed, high accuracy and low cost. Based on the principle of next-generation sequencing technology, a sequencing primer binding sequence for initiating the sequencing reaction, a tag sequence for distinguishing samples, and an anchoring sequence that can be connected to a sequencing chip are added to both ends of the DNA before the DNA sample is sequenced. This step is called library construction. This step is different from conventional first-generation sequencing, and the operation is complicated, the experimental process is long, and the experimental personnel have high requirements.

[0003] The commonly used library construction method at present is TA cloning method, the main steps of which include: targeted site amplification, end repair, A tail addition, T-tailed linker ligation, and magnetic bead purification or magnetic bead sorting between steps. This method is currently relatively mature, but it has many steps, a long experimental process, and high requirements for experimental personnel, especially when a large number of samples are subjected to library construction, which is time-consuming and labor-intensive.

[0004] With the development of library construction technology, the current targeted sequencing commonly used library construction method is two-step library construction, the main method of which is: the amplification primers at both ends of the first step targeted amplification are designed to add specific sequences that can be complementary to the second step amplification primers, and after the first step PCR is completed, the target product with complementary second step amplification primers is obtained, and then the sequencing adapter is connected through the second step PCR reaction, thereby obtaining a library that can be used for sequencing. Each amplification needs to be purified by magnetic beads. The total experimental time from amplification to magnetic bead purification is about 5.5-7.5 hours (the total time of different amplification systems is slightly different). And the PCR product is contaminated due to multiple tube transfers.

[0005] Therefore, it is urgent to develop a method that is simple to operate, shorter in time, and higher in detection efficiency and accuracy. SUMMARY

[0006] The application is based on a one-step library construction method designed by using a reverse complement PCR (RC-PCR) technology, which simplifies experimental steps, and only needs one PCR and one magnetic bead purification to complete library construction, and the PCR time is only 1 hour and 42 minutes. The experimental time is greatly shortened, the operation steps are reduced, the pollution risk is reduced, and the efficiency and accuracy of judicial identification are improved.

[0007] The technical scheme of the application is:

[0008] In one aspect, the application provides a one-step library construction-based multiplex SNP combined microhaplotype amplification system, which comprises a primer combination for simultaneously amplifying 80 high MAF value SNP sites, 1 Y-Indel and 24 microhaplotype sites.

[0009] rs1253805, rs565645, rsl 1208133, rs681968, rsl 171043, rs880329, rs7515856, rs891700 on chromosome 1, rs876724, rs950661, rsl2622958, rs6754768, rsl385172, rs6705667, rs7597826, rs6714809, rs6436688 on chromosome 2, rs6793085, rs7649122, rs9310740, rs57579284 on chromosome 3, rs2295499, rs3096579, rs2048961, rs10034991, rs7668855, rsl605463, rs6535598, rs10034372 on chromosome 4, rs10866562, rs252369, rs10472761, rs272735, rs61467430, rs264748, rsl3182883 on chromosome 5, rs3799000, rs2747740, rs214955, rs316032, rs9366013 on chromosome 6, rs2686920 on chromosome 7, rs10092491, rs7815202 on chromosome 8, rs10758556, rs7046317, rsl0821241, rsl463729 on chromosome 9, rsl418407, rsl0826551, rs7088769, rsl 1146518 on chromosome 10, rsl319309 on chromosome 11, rs2269355, rs6581429, rs959897 on chromosome 12, rs9510177, rs6563522, rsl2585235, rsl602204, rs354439 on chromosome 13, rsl958395, rsl454361, rs7157655, rsl956467, rsl202668, rsl 1846252, rs7151788 on chromosome 14, rs8041340 on chromosome 15, rs8050385, rs9928480, rs8043925 on chromosome 16, rs9951171, rs28690792, rs3991209 on chromosome 18, rs576261 on chromosome 19, rs6041563 on chromosome 20, rs2070650 on chromosome 21, rs361844, rsl028528 on chromosome 22;

[0010] The one Y-Indel consists of rs34733631;

[0011] The 24 microhaplotype sites consist of F1-012, F3-019, F6-026, F7-0210, F9-033, F12-045, F16-057, F17-058, F18-061, F22-071, F23-072, F24-074, F26-086, F27-092, F28-101, F29-107, F30-114, F31-117, F32-122, F33-124, F34-131, F36-164, F38-175, F39-184;

[0012] The primer combination comprises RC-Primer, I5-Primer and I7-Primer, and the design method is as follows:

[0013] S1, designing a conventional primer according to a target sequence;

[0014] S2, designing a reverse complementary primer RC-Primer according to the conventional primer;

[0015] S3, designing I5 / I7 end adapter primers I5-Primer and I7-Primer;

[0016] The RC-Primer comprises a reverse complementary sequence of a complementary region of the I5-Primer and the I7-Primer;

[0017] The 3' end of the RC-Primer is blocked with TAGGTGT, and the last GT base is thio-modified, and the last base T is phosphorylated;

[0018] Specifically, the sequence of the RC-Primer is shown in SEQ ID No. 1-SEQ ID No. 210;

[0019] The sequence of the I5-Primer is shown in SEQ ID No. 211-SEQ ID No. 214;

[0020] The sequence of the I7-Primer is shown in SEQ ID No. 215-SEQ ID No. 222.

[0021] Specifically, the addition amount of the RC-Primer is 0.25 μM, and the addition amount of the I5-Primer and the I7-Primer is 10 μM.

[0022] Specifically, the one-step library construction amplification system further comprises one or more of a buffer, dNTPs, and a high-fidelity enzyme.

[0023] In another aspect, the present application provides a library construction method based on multiple SNPs and microhaplotypes, which comprises performing PCR amplification on a sample using the aforementioned amplification system.

[0024] Specifically, the program of the PCR amplification is as shown in the following table:

[0025]

[0026] In the program of the PCR amplification, 68-60℃ represents a decrease of 0.5℃ after 68℃ in the first cycle.

[0027] Specifically, the sample comprises one or more of blood, blood stains, buccal cells, semen, semen stains, bone, hair, saliva, saliva stains, sweat, amniotic fluid containing fetal cells, or urogenital tract exfoliated cells.

[0028] In another aspect, the present application provides a kit comprising the aforementioned amplification system.

[0029] Specifically, the kit further comprises a library purification kit.

[0030] Preferably, the library purification kit comprises one or more of a purification column or magnetic beads, a binding buffer, a washing buffer, and an elution buffer.

[0031] In another aspect, the present application provides the aforementioned amplification system or library construction method or kit for use in forensic rapid individual identification.

[0032] The present application has the following beneficial effects:

[0033] (1) The library construction typing result based on the one-step library construction method of the present application has good repeatability.

[0034] (2) The library construction based on the one-step library construction method of the present application has certain anti-humic acid, EDTA, and indigo inhibition capacity.

[0035] (3) The amplification system established based on the one-step library construction method of the present application has good species specificity. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a schematic diagram of the one-step library construction core principle.

[0037] Figure 2 It is a comparison result diagram of the 3' end blocking method. DETAILED DESCRIPTION

[0038] The application will be further clarified by the following examples. The examples are only a part of the application and are not used to limit the application. The experimental methods used in the following examples are conventional experiments. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0039] Example 1 One-step library construction method

[0040] The core principle of the one-step library construction method provided by the application is shown in Figure 1 . The one-step library construction target amplification system first needs to design a conventional PCR amplification primer as shown in Figure 1 ①. Then, according to the conventional PCR amplification primer, design the RC-Primer at Figure 1 ②. The RC-Primer contains the reverse complementary sequence of the amplification primer, and the reverse complementary sequence of the complementary region of the I5-Primer / I7-Primer at Figure 1 ③, and a blocking sequence to stop the amplification reaction.

[0041] Put the RC-Primer and the I5-Primer / I7-Primer into the PCR reaction system. At the beginning of the PCR reaction, the RC-Primer can combine with the I5-Primer / I7-Primer to generate the Final-Primer at Figure 1 ④. The Final-Primer contains the sequencing anchor sequence, barcode, sequencing primer, and target primer. Finally, the Final-Primer binds to the DNA template to obtain the sequencing library, and the library construction is completed.

[0042] The specific content is as follows:

[0043] (1) RC-Primer 3' end blocking method: use the non-human genomic DNA sequence "TAGGTGT" for blocking, and perform thio modification between the last "GT" bases, and perform phosphorylation modification on the last base "T". The complete blocking sequence is: "TAGGTG*T-P".

[0044] (2) One-step library construction method PCR reaction system: the total system is 30 μL, the recommended starting amount of DNA is 3.5-20 ng, the enzyme is produced by Yixing Company, and the item number is Cat:10154ES03. The reaction system is shown in Table 1:

[0045] Table 1

[0046] Component Volume (μL) ddH2O 7-X 2x Hieff Canace 15 I5-Primer (10 μM) 2 I7-Primer (10 μM) 2 RC-Primer MIX (0.25 μM) 3 gDNA X

[0047] Note: X in the table represents the input volume of gDNA.

[0048] (3) PCR reaction program for one-step library construction method is shown in Table 2:

[0049] Table 2

[0050]

[0051]

[0052] Note: 68-60°C represents a decrease of 0.5°C for each cycle after 68°C in the first cycle.

[0053] (4) Magnetic bead purification

[0054] a. Take Beckman Agencourt AM Pure XP magnetic beads (Beckman Coulter, USA) out of the 4°C refrigerator and equilibrate at room temperature for 30 minutes.

[0055] b. Transfer 30 μL of PCR product into a 1.5 mL EP tube, and add 1.8 times the volume of 54 μL Beckman magnetic beads, mix by pipetting or vortexing, and incubate at room temperature for 5 minutes.

[0056] c. After incubation, place the EP tube on the magnetic stand for 3 minutes until the solution is clear.

[0057] d. Remove the supernatant completely, add 180 μL of 70% ethanol solution to the EP tube, rotate the EP tube 180°, stand for 10 seconds, and rotate the EP tube 180° again.

[0058] e. Remove the supernatant completely, add 190 μL of 70% ethanol solution to the EP tube, rotate the EP tube 180°, stand for 10 seconds, and rotate the EP tube 180° again.

[0059] f. Cover the tube cap, centrifuge briefly, centrifuge the remaining ethanol to the bottom of the tube, place the EP tube on the magnetic stand, and carefully use a 10 μL pipette to remove the residual ethanol at the bottom, taking care not to suck up the magnetic beads.

[0060] g. Keep the EP tube on the magnetic stand, and stand at room temperature for 3-5 minutes until the magnetic beads are completely dried, and the residual ethanol is completely volatilized.

[0061] h. Add 24 μL of DNase / RNase-Free Water (Thermo Scientific, USA), remove the EP tube from the magnetic stand, mix by pipetting or vortexing, and stand at room temperature for 5 minutes.

[0062] i. After centrifugation, place the EP tube on the magnetic stand for 2 minutes until the solution is clear.

[0063] j. Pipette 19 μL of supernatant with a pipette into a new EP tube, which is the library sample after magnetic bead purification, and label it.

[0064] (5) Comparison of 3' end blocking methods

[0065] According to the reaction system, reaction primer and reaction procedure of Example 1, the 3' end blocking method of RC-primer was replaced, and the effects of various 3' end blocking methods on the method were compared. The blocking methods are shown in Table 3:

[0066] Table 3

[0067]

[0068]

[0069] Note: "*" indicates thio modification; "-P" indicates phosphorylation modification.

[0070] The results are shown in Figure 2 (Desired fragments are between 300-400bp) It can be seen that the 3' end blocking method used in the application is most beneficial to the one-step library construction method established by the application.

[0071] Example 2 Construction of forensic SNP combined microhaplotype multiplex system based on one-step library construction method

[0072] Based on the method of Example 1, a one-step library construction method multiplex SNP+microhaplotype MH composite amplification system was constructed for forensic daily case detection. The multiplex system contains 80 high MAF value SNP sites, 1 Y-Indel and 24 microhaplotype sites, and the individual identification rate of the system reaches 1-2.0265×10 -39 , which provides a new method for forensic next-generation sequencing rapid individual identification. This study strictly follows the ethical guidelines of the Helsinki Declaration. Blood samples of 74 volunteers were collected by intravenous blood sampling and stored in EDTA anticoagulant tubes.

[0073] 2.1 SNP site selection results

[0074] Based on the information of autosomal SNP sites in 1000Genomes Project and Genome Aggregation Database, the SNP sites were screened by the following conditions:

[0075] ①Screen out SNP sites with MAF>0.4 in global population;

[0076] ②Select SNP sites with MAF>0.4 in East Asian population at the same time;

[0077] ③ Add illumina Verogen ForenSeq DNA Signature Prep Kit, Thermo Fisher Precision ID Identity Panel kit, autosomal SNP sites of forensic SNP detection system of Huada Gene Co., Ltd.;

[0078] ④ Delete SNP sites with a distance of 1 Mb (to ensure linkage equilibrium between sites);

[0079] ⑤ According to the different lengths of each chromosome, allocate the number of SNP sites on each chromosome (to avoid the situation that sites are concentrated on a few chromosomes);

[0080] ⑥ Delete sites with other mutations upstream and downstream, which cause primer design difficulties.

[0081] The selection results are shown in Table 4:

[0082] Table 4

[0083]

[0084]

[0085]

[0086] Note: Ref is the wild type typing; Allele% is the allele frequency; Alt is the mutant typing; MAF is the minimum allele frequency; del is the deletion.

[0087] 2.2 Selection results of microhaplotype sites

[0088] This study strictly followed the ethical guidelines of the Helsinki Declaration. Blood samples from 95 volunteers were collected by intravenous blood sampling and stored in EDTA anticoagulant tubes.

[0089] To make up for the defects of SNP sites in mixed typing, this study further screened MH sites and included them in the multiplex amplification system based on the one-step library construction method, with the following screening conditions:

[0090] ① Select fragments within the range of 230 bp, containing 2 or more (as many as possible) MAF>0.01 SNP sequences as microhaplotype sites;

[0091] ② Delete sites with mutations upstream and downstream, which cause primer design difficulties;

[0092] ③In order to ensure linkage equilibrium, the sites with distance less than 1 Mb were deleted (the MH sites with distance less than 1 Mb from the final screened SNP sites were also not selected).

[0093] The selection results are shown in Table 5 and Table 6:

[0094] Table 5

[0095]

[0096]

[0097] Note: Ae is the effective allele number, the larger the Ae value, the better the polymorphism of the MH site; DP is the individual identification ability, the higher the DP value, the higher the individual identification efficiency of the MH site, and the maximum value is 1.

[0098] Table 6

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Note: POS is the position of the SNP site in the reference genome hg38; MH is the abbreviation of microhaplotype; Rs is the SNP site rs number; SNP is the abbreviation of single nucleotide polymorphism; Ref is the wild type typing; Alt is the mutant typing; Data from is the data source database; gnomAD is Genome Aggregation Database; ALFA is Allele Frequency Aggregator; 1KG is 1000 Genomes Project; 1KG 30x is 1000 Genomes Project 30x.

[0105] 2.3 Requirements for designing conventional amplification primers

[0106] ① The length of the primer is between 16-35 bp;

[0107] ② The length of the amplified fragment is between 81-116 bp;

[0108] ③ The Tm value of the primers of each locus is as consistent as possible;

[0109] ④ Avoid forming dimers and hairpin structures between primers;

[0110] 5. Avoid consecutive identical bases in the primer; 6. The amplification product from the target region should be single.

[0111] 2.4 One-step library construction primer design results

[0112] 2.4.1 RC-Primer design results

[0113] The results are shown in Table 7:

[0114] Table 7

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] Note: "*" indicates thio modification; "-P" indicates phosphorylation modification.

[0124] The results are shown in Table 8:

[0125] Table 8

[0126]

[0127] Note: The underlined part is Barcode.

[0128] Example 3 Reproducibility verification results of forensic SNP combined microhaplotype multiplex composite system based on one-step library construction method

[0129] According to the reaction system, reaction primers and reaction procedures in Example 1, 3.5 ng of 9947A DNA standard and one example of Chinese northern Han male DNA were repeatedly input into the composite system one-step library construction, and the second generation sequencing was performed on the illumina platform Novaseq X plus sequencer. The 3 times of sequencing gender site typing was accurate, and the typing accuracy of each site in 3 times of repetition was 100%, and the results are shown in the following table, which shows that the library construction typing results based on one-step library construction method have good repeatability. This study strictly follows the ethical guidelines of the "Helsinki Declaration". The blood sample of a male volunteer was collected by intravenous blood sampling and stored in an EDTA anticoagulant tube.

[0130] The results are shown in Tables 9, 10 and 11:

[0131] Table 9

[0132]

[0133]

[0134]

[0135] Table 10

[0136]

[0137]

[0138] Table 11

[0139]

[0140]

[0141] The results of Tables 9, 10 and 11 show that according to the experimental results, the system can maintain good result repeatability under the condition of 3.5 ng of DNA input.

[0142] Example 4 Anti-inhibitor results of forensic SNP combined microhaplotype multiplex composite system based on one-step library construction method

[0143] According to the reaction system, reaction primer and reaction procedure of embodiment 3, humic acid, EDTA and indigo were added to the PCR amplification system to reach the final concentration of 25 μmol and 50 μmol, and 10 ng of DNA of a Chinese northern Han female was added to the humic acid inhibition system. 10 ng of DNA of a Chinese northern Han male was added to the EDTA and indigo inhibition system. And the Illumina platform Novaseq X plus sequencer was used for second-generation sequencing. The results showed that the typing accuracy reached 100% under the addition of different concentrations of inhibitors, indicating that the library construction based on one-step library construction method has certain anti-humic acid, EDTA and indigo inhibition ability. This study strictly followed the ethical guidelines of the Helsinki Declaration. The blood samples of 2 volunteers were collected by intravenous blood sampling and stored in EDTA anticoagulant tubes.

[0144] The results are shown in Tables 12, 13 and 14:

[0145] Table 12

[0146]

[0147]

[0148]

[0149] Table 13

[0150]

[0151]

[0152] Table 14

[0153]

[0154]

[0155] The results of Tables 12, 13 and 14 show that accurate analysis results can be obtained under the conditions of humic acid, EDTA and indigo at 25 μmol / L and 50 μmol / L.

[0156] Example 5 Species-specific detection results of the forensic SNP combined microhaplotype multiplex system based on one-step library construction method

[0157] According to the reaction system, reaction primer and reaction procedure of embodiment 1, 10 ng of DNA of chicken, cow, pig, mouse, rabbit and sheep was added for one-step library construction of the complex system, and the Illumina platform Novaseq X plus sequencer was used for second-generation sequencing. The results are shown in Table 15:

[0158] Table 15

[0159] Chicken Cow Sheep Pig Mouse Rabbit Number of SNP sites detected 0 1 2 0 2 0 Number of microhaplotype (MH) sites detected 1 1 1 1 0 0

[0160] As shown in Table 15, the amplification system based on the one-step library construction method of the application has good species specificity, and non-human DNA cannot successfully construct a library and be sequenced.

[0161] The above detailed description is a specific description of one of the feasible embodiments of the application, which is not used to limit the scope of the application. It should be pointed out that any equivalent implementation or change made without departing from the application should be included in the scope of the technical solutions of the application. Therefore, the protection scope of the application should be subject to the appended claims.

Claims

1. A one-step library-based multiplex SNP combined microhaplotype amplification system, characterized in that, The one-step library construction amplification system comprises a primer combination for simultaneously amplifying 80 high MAF value SNP sites, 1 Y-Indel and 24 microhaplotype sites; rs1253805, rs565645, rsl 1208133, rs681968, rsl 171043, rs880329, rs7515856, rs891700 on chromosome 1, rs876724, rs950661, rsl2622958, rs6754768, rsl385172, rs6705667, rs7597826, rs6714809, rs6436688 on chromosome 2, rs6793085, rs7649122, rs9310740, rs57579284 on chromosome 3, rs2295499, rs3096579, rs2048961, rs10034991, rs7668855, rsl605463, rs6535598, rs10034372 on chromosome 4, rsl0866562, rs252369, rs10472761, rs272735, rs61467430, rs264748, rsl3182883 on chromosome 5, rs3799000, rs2747740, rs214955, rs316032, rs9366013 on chromosome 6, rs2686920 on chromosome 7, rs10092491, rs7815202 on chromosome 8, rs10758556, rs7046317, rsl0821241, rs1463729 on chromosome 9, rsl418407, rsl0826551, rs7088769, rsl 1146518 on chromosome 10, rsl319309 on chromosome 11, rs2269355, rs6581429, rs959897 on chromosome 12, rs9510177, rs6563522, rsl2585235, rsl602204, rs354439 on chromosome 13, rsl958395, rsl454361, rs7157655, rsl956467, rsl202668, rsl 1846252, rs7151788 on chromosome 14, rs8041340 on chromosome 15, rs8050385, rs9928480, rs8043925 on chromosome 16, rs9951171, rs28690792, rs3991209 on chromosome 18, rs576261 on chromosome 19, rs6041563 on chromosome 20, rs2070650 on chromosome 21, rs361844, rsl028528 on chromosome 22; rs1253805, rs565645, rsl 1208133, rs681968, rsl 171043, rs880329, rs7515856, rs891700 on chromosome 1, rs876724, rs950661, rsl2622958, rs6754768, rsl385172, rs6705667, rs7597826, rs6714809, rs6436688 on chromosome 2, rs6793085, rs7649122, rs9310740, rs57579284 on chromosome 3, rs2295499, rs3096579, rs2048961, rs10034991, rs7668855, rsl605463, rs6535598, rs10034372 on chromosome 4, rsl0866562, rs252369, rs10472761, rs272735, rs61467430, rs264748, rsl3182883 on chromosome 5, rs3799000, rs2747740, rs214955, rs316032, rs9366013 on chromosome 6, rs2686920 on chromosome 7, rs10092491, rs7815202 on chromosome 8, rs10758556, rs7046317, rsl0821241, rs1463729 on chromosome 9, rsl418407, rsl0826551, rs7088769, rsl 1146518 on chromosome 10, rsl319309 on chromosome 11, rs2269355, rs6581429, rs959897 on chromosome 12, rs9510177, rs6563522, rsl2585235, rsl602204, rs354439 on chromosome 13, rsl958395, rsl454361, rs7157655, rsl956467, rsl202668, rsl 1846252, rs7151788 on chromosome 14, rs8041340 on chromosome 15, rs8050385, rs9928480, rs8043925 on chromosome 16, rs9951171, rs28690792, rs3991209 on chromosome 18, rs576261 on chromosome 19, rs6041563 on chromosome 20, rs2070650 on chromosome 21, rs361844, rsl028528 on chromosome 22; The 1 Y-Indel consists of rs34733631; The 24 microhaplotype sites consist of F1-012, F3-019, F6-026, F7-0210, F9-033, F12-045, F16-057, F17-058, F18-061, F22-071, F23-072, F24-074, F26-086, F27-092, F28-101, F29-107, F30-114, F31-117, F32-122, F33-124, F34-131, F36-164, F38-175, F39-184, and the SNPs of the 24 microhaplotype sites consist of the following: Wherein, POS is the position of the SNP site in the reference genome hg38; MH is the abbreviation of microhaplotype; Rs is the SNP site rs number; SNP is the abbreviation of single nucleotide polymorphism; Ref is the wild type typing; Alt is the mutant typing; Data from is the data source database; gnomAD is Genome Aggregation Database; ALFA is Allele Frequency Aggregator; 1KG is 1000 Genomes Project; 1KG 30x is 1000 Genomes Project 30x; The primer combination comprises RC-Primer, I5-Primer and I7-Primer, and the design method is as follows: S1, designing a conventional primer according to a target sequence; S2, designing a reverse complementary primer RC-Primer according to the conventional primer; S3, designing I5 / I7 end adapter primers I5-Primer and I7-Primer; The RC-Primer comprises a reverse complementary sequence of the complementary region of the I5-Primer and the I7-Primer; The 3' end of the RC-Primer is blocked with TAGGTGT, and the last GT base is thio-modified, and the last base T is phosphorylated; The sequence of the RC-Primer is shown in SEQ ID No. 1-SEQ ID No. 210; The sequence of the I5-Primer is shown in SEQ ID No. 211-SEQ ID No. 214; The sequence of the I7-Primer is shown in SEQ ID No. 215-SEQ ID No.

222.

2. The amplification system of claim 1, wherein The addition amount of the RC-Primer is 0.25 μM, and the addition amount of the I5-Primer and the I7-Primer is 10 μM.

3. The amplification system according to claim 1 or 2, characterized by, The one-step library construction amplification system further comprises one or more of a buffer, dNTPs and a high-fidelity enzyme.

4. A library construction method based on multiple SNPs and microhaplotypes, characterized by, The library construction method comprises performing PCR amplification on a sample by using the amplification system according to any one of claims 1-3.

5. The library building method according to claim 4, wherein The program of the PCR amplification is as follows: 68-60°C in the PCR amplification procedure means a decrease of 0.5°C per cycle after 68°C in the first cycle.

6. The library building method according to claim 4 or 5, characterized by, The sample comprises one or more of blood, bloodstain, buccal cell, semen, seminal stain, bone, hair, saliva, saliva stain, sweat, amniotic fluid containing fetal cells, or urogenital tract exfoliated cells.

7. A kit characterized in that, The kit comprises the amplification system of any one of claims 1-3.

8. The kit of claim 7, wherein The kit further comprises a library purification kit.

9. Use of the amplification system of any one of claims 1-3 or the library building method of any one of claims 4-6 or the kit of any one of claims 7-8 for forensic rapid individual identification.

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