SNP (Single Nucleotide Polymorphism) site combination for identifying complex genetic relationship by utilizing nanopore sequencing technology, kit and application of SNP site combination and kit

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site combination for identifying a complex genetic relationship by utilizing a nanopore sequencing technology, a kit and application of the SNP site combination and the kit, and relates to the technical field of biology. The SNP locus combination comprises 5198 autosomal SNP loci, the 5198 autosomal SNP loci are detected based on a nanopore sequencing technology, genetic relationship identification can be achieved, identification of a parent-child relationship, a full-sib relationship and a secondary genetic relationship can be achieved, and the identification result is more accurate and more efficient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a SNP site combination, a kit and an application thereof for complex kinship identification using nanopore sequencing technology. Background Art

[0002] Kinship analysis is one of the most important tasks in forensic practice. It can be used to identify kinship in inheritance disputes, search for missing persons, identify victims in disasters, and even provide important information for the identification of key figures in some important events.

[0003] At present, the main forensic DNA detection technologies include DNA fingerprint analysis technology and fluorescent labeling multi-locus STR composite amplification detection technology. DNA fingerprint analysis technology belongs to restriction fragment length polymorphism markers (RPLPS). It uses restriction endonucleases that can recognize and cut specific DNA sequences to cut human DNA into DNA fragments of different lengths. After electrophoresis separation, the enzyme-cut fragments are transferred to nylon membranes or nitrocellulose membranes using Souhem blotting. Specific probes labeled with fluorescent or radioactive isotopes are used for hybridization to detect DNA fragments of different lengths, that is, to obtain the DNA map of the restriction endonuclease and specific probe. The limitations of this method are that the method is complicated, the cycle is long, and the experimental conditions are high. Fluorescent labeling multi-locus STR composite amplification detection technology is currently the most widely used identification technology. It first attaches fluorescent luminescent groups to the amplification primers. When the DNA fragments with luminescent groups pass through the excitation light irradiation area, they emit fluorescence of the corresponding wavelength. After the fluorescence light harmonic information is collected by the photoelectric detector, it is stored and processed by the electronic system and computer. By comparing the appearance time of the sample spectral signal with the signal of the standard reference material (the standard reference material is a series of DNA fragments of different sizes but known length), the length of the corresponding DNA fragment is calculated to obtain the test result. Then, according to the spectral range of fluorescence, the test result refers to the full allele mixture of the amplified site, and the allele name of the detected DNA fragment can be known. Recording it is the DNA typing of the sample at this site. There are many STR polymorphic loci, and the fragment length is generally less than 400bp, which is easy to amplify and suitable for the inspection of trace samples; the allele size is relatively close, and the preferential amplification of smaller alleles is not obvious: the difference in the length of the STR loci at different sites is small, which is convenient for composite amplification and can improve the detection efficiency. In addition, the STR locus analysis method is simple, the typing is accurate, the typing procedure is clear and standardized, and the typing result graphics are simple, which is convenient for the standardization and automation of DNA typing, and convenient for computer storage and network retrieval of typing data.

[0004] Nanopore sequencers have received widespread attention in recent years. As an emerging high-throughput detection technology, nanopore sequencers have the advantages of high throughput, long read length, easy portability and operation, real-time data analysis, and can be used without high capital investment or complex information technology infrastructure. Chinese patent CN115976014 A provides a method for detecting genetic markers with good forensic application value in the human genome. The detection method can be performed through a nanopore sequencing platform. Nanopore sequencing has the same advantages as mixed sample sequencing, no need to establish reference standards, and can obtain specific sequence and mutation information other than STR length to help improve detection resolution. It also has the advantages of unlimited sequencing fragment length, low equipment cost, compactness and portability, and convenient application in grassroots laboratories and field environments.

[0005] SNP is a genomic variation at a single base position in DNA, which manifests as a DNA sequence polymorphism with a low mutation rate. The 1000 Genomes Project (1KGP) completed a comprehensive description of common human genetic variation by sequencing the whole genome of different individuals from multiple populations, and discovered 84.7 million SNPs in the human genome. Although the polymorphism of bi-allelic loci is poor, the identification ability of a large number of combined use of SNPs is significantly increased, which has great advantages for the analysis of complex kinship. At present, there is still a lack of effective methods for kinship identification using SNP loci in combination with nanopore sequencers. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a SNP site combination, a kit and its application for complex kinship identification using nanopore sequencing technology, which realizes accurate sequencing and typing of 5198 SNP sites using nanopore sequencing technology, and realizes accurate identification of parent-child relationships, full sibling relationships, and secondary kinship using 5198 SNP sites.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a SNP site combination, comprising SNP sites with the following rs numbers:

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[0050] Preferably, the SNP site combination also includes at least one of the position information and allele frequency of the 5198 SNP sites.

[0051] Further preferably, the SNP site combination also includes the position information and allele frequencies of the 5198 SNP sites.

[0052] The allele frequency can be obtained according to the rs number by conventional methods in the art, such as querying a gene database.

[0053] In another aspect, the present invention provides a detection kit comprising the above-mentioned SNP site combination.

[0054] In another aspect, the present invention provides applications of the above-mentioned SNP site combination or detection kit, wherein the applications include at least one of kinship identification, human individual identification, forensic DNA database construction, suspect family investigation, and degraded sample identification.

[0055] Preferably, the application is kinship identification.

[0056] Finally, the present invention provides a method for identifying kinship based on nanopore sequencing technology, comprising the following steps:

[0057] S1, construction of nanopore library;

[0058] S2, connect to Adaptor;

[0059] S3. Nano sequencing to compare the above SNP site combinations.

[0060] Preferably, the step S1 comprises the following steps:

[0061] (1) configuring the sample to be tested into an end-repair system;

[0062] (2) Incubate and purify the end repair system to obtain a nanopore library.

[0063] Further preferably, in step (1), the end repair system comprises a sample to be tested and an end repair reaction premix. Furthermore, in terms of volume, the end repair system comprises 45-55 parts of 150-250 fmol of sample to be tested and 5-8 parts of the end repair reaction premix. Most preferably, the end repair system comprises 48 parts of 200 fmol of sample to be tested and 6.5 parts of the end repair reaction premix.

[0064] Further preferably, the end repair reaction premix comprises a reaction buffer and an enzyme reagent. Furthermore, in terms of volume, the end repair reaction premix comprises 3-5 parts of reaction buffer and 2-4 parts of enzyme reagent. Most preferably, the end repair reaction premix comprises 3.5 parts of reaction buffer and 3 parts of enzyme reagent.

[0065] More preferably, the incubation is: incubation at 15-25°C for 3-8 min, then incubation at 60-70°C for 3-8 min, and then storage at 4°C. Most preferably, the incubation is: incubation at 20°C for 5 min, then incubation at 65°C for 5 min, and then storage at 4°C.

[0066] Further preferably, the purification is as follows: the incubated end repair system is combined with magnetic beads, placed on a magnetic stand, and the clarified liquid is discarded after the liquid is clarified; the magnetic beads are washed with 60-80% ethanol, and the ethanol is discarded after the liquid is clarified, and the process is repeated 1-3 times, the ethanol is removed, and nuclease-free water is added to mix well, and the mixture is incubated at 20-30°C for 1-5 minutes to elute the end repair product, and the eluate is clear and colorless, and then the nanopore library is obtained by aspirating the nanopore library.

[0067] Furthermore, nuclease-free water was added and mixed thoroughly, and then incubated at room temperature for 3 min.

[0068] Preferably, step S2 comprises the following steps:

[0069] (1) configuring the nanopore library into an adaptor connection system, incubating, and purifying to obtain a connected mixed library 1;

[0070] (2) Calculate the volume of the ligated mixed library corresponding to 100 fmol and adjust the concentration to obtain ligated mixed library 2.

[0071] Preferably, the Adaptor ligation system comprises a nanopore library, a Native Adapter, NEBNext Quick Ligation Reaction Buffer and NEBNext Quick T4 DNA Ligase. Further preferably, in terms of volume, the Adaptor ligation system comprises 60-70 portions of 150-250 fmol of the sample to be tested, 3-8 portions of Native Adapter, 15-25 portions of NEBNext Quick Ligation Reaction Buffer and 5-15 portions of NEBNext Quick T4 DNA Ligase. Most preferably, in terms of volume, the Adaptor ligation system comprises 65 portions of 200 fmol of the sample to be tested, 5 portions of Native Adapter, 20 portions of NEBNext Quick Ligation Reaction Buffer and 10 portions of NEBNext Quick T4 DNA Ligase.

[0072] More preferably, the incubation is: incubation at 20-30° C. for 5-15 min. Most preferably, incubation is: incubation at room temperature for 10 min.

[0073] Further preferably, the purification is as follows: the ligated mixed library 1 is combined with magnetic beads, placed on a magnetic stand, and the clarified liquid is discarded after the liquid is clarified; the magnetic beads are washed with Short Fragment Buffer, and the clarified liquid is discarded after the liquid is clarified, and the process is repeated 1-3 times to remove all the clarified liquid, add Elution Buffer and mix thoroughly, incubate at 20-30° C. for 5-15 min, elute the end repair product, and wait until the eluate is clear and colorless, aspirate it, and obtain the ligated mixed library 2.

[0074] Furthermore, after adding Elution Buffer and mixing thoroughly, incubate at room temperature for 10 min.

[0075] Further preferably, the step S3 comprises the following steps:

[0076] (1) preparing a starting premix solution;

[0077] (2) preparing a sequencing library;

[0078] (3) Add the starting premix and sequencing library to the chip for sequencing and comparison.

[0079] More preferably, in step (1), the start-up premix solution includes Flush Tether and Flush Buffer. Further, in terms of volume fraction, the start-up premix solution includes 20-40 parts of Flush Tether and 1100-1200 parts of Flush Buffer. Most preferably, in terms of volume fraction, the start-up premix solution includes 30 parts of Flush Tether and 1170 parts of Flush Buffer.

[0080] More preferably, in step (2), the sequencing library comprises 2 connected mixed libraries, Sequencing Buffer and Library Beads. Further, in terms of volume fractions, the priming premix comprises 10-15 parts of connected mixed libraries, 35-40 parts of Sequencing Buffer and 20-30 parts of Library Beads. Most preferably, in terms of volume fractions, the priming premix comprises 12 parts of connected mixed libraries, 37.5 parts of Sequencing Buffer and 25.5 parts of Library Beads.

[0081] Preferably, in step (3), the chip is at least one of R9 and R10, and more preferably is an R10 chip.

[0082] Preferably, in step (3), the sequencing time is 24-70 hours, more preferably 48 hours.

[0083] The beneficial effects of the present invention are:

[0084] The present invention is based on nanopore sequencing technology and detects 5198 autosomal SNP sites to realize kinship identification, and can realize the identification of parent-child relationship, full sibling relationship, and secondary kinship, and the identification results are more accurate and efficient. DETAILED DESCRIPTION

[0085] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0086] In the following examples, allele frequencies are derived from the southern Chinese population data and northern Chinese population data from the 1000 Genomes dataset.

[0087] The ITO calculation method refers to "Lu Huiling, Yang Qingen. Using the ITO method to calculate the chance of blood relationship between two individuals. Chinese Journal of Forensic Medicine. 2002, 17(3): 188-191".

[0088] The standard reference for likelihood ratio calculation is "Technical Specifications for Forensic Identification" (SF / Z JD0105003-2015).

[0089] In the following examples, the specific information and sources of the primers and reagents used are as follows:

[0090]

[0091] Example 1

[0092] S1. Construction of nanopore library

[0093] (1) Sample end repair

[0094] Transfer 200 fmol of the DNA sample to be tested into a 0.2 mL PCR centrifuge tube, adjust the volume to 48 μL with nuclease-free water, mix thoroughly by flicking the tube, and centrifuge briefly in a microcentrifuge.

[0095] Prepare the end repair reaction premix according to the following table:

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[0098] Note: When processing multiple samples at the same time, prepare 2 more reaction mixture systems to ensure that the loss of reaction mixture caused by sample addition is compensated. Use a pipette to mix each reaction system thoroughly.

[0099] Prepare the end repair system according to the following table:

[0100] Reagents Addition volume (μL) 200fmol sample to be tested 48μL End Repair Reaction Master Mix 6.5μL Total amount of reaction system 54.5μL

[0101] Use a pipette to mix thoroughly by pipetting. Ensure that the reaction system is fully mixed and then centrifuge slightly to mix the liquid on the tube wall and tube cap into the tube.

[0102] (2) Incubation and purification

[0103] Use a PCR instrument and incubate the reaction system according to the following program settings:

[0104] Temperature(℃) Time (min) 20℃ 5min 65℃ 5min 4℃ save

[0105] The temperature of the hot cover was set to 80°C and the reaction volume was set to 55 μL.

[0106] Purify the end repair system after incubation according to the following steps (magnetic bead dosage: 60 μL × number of samples + 5 μL):

[0107] A. After thoroughly mixing the AMPure XP beads, take new 1.5 mL centrifuge tubes and label them with the sample names. Add 60 μL of AMPure XP beads to each centrifuge tube.

[0108] B. Transfer all products of each sample after the reaction to the corresponding 1.5 mL centrifuge tubes and flick the tubes to mix evenly.

[0109] C. Incubate at room temperature for 5 minutes.

[0110] During this period, flick the tube several times to ensure that the DNA amplification product is fully bound to the magnetic beads.

[0111] During this time, prepare 70% fresh ethanol solution. The volume required for 70% fresh ethanol solution = 400 μL × number of samples + 500 μL.

[0112] D. Place the sample on the magnetic rack. When the magnetic beads are adsorbed to one side and the liquid is clarified, keep the test tube on the magnetic rack, use a pipette to suck out the clarified liquid and discard it.

[0113] E. Keep the test tube on the magnetic rack and wash the magnetic beads with 200 μL of freshly prepared 70% ethanol. Do not blow the magnetic beads away. Rotate the centrifuge tube 180 degrees clockwise every 30 seconds. After rotating 4 times, let the centrifuge tube stand. When the magnetic beads are adsorbed to one side and the liquid is clear, use a pipette to suck out the ethanol and discard it.

[0114] F. Repeat step E once.

[0115] G. Centrifuge the tube, place it back on the magnetic rack, and use a 10μL pipette to remove the remaining alcohol. Open the tube cap and let the alcohol dry for about 1 minute. Be careful not to let the magnetic beads dry out.

[0116] Note: The specific alcohol drying time is determined by the temperature and humidity of the operating environment.

[0117] H. Remove the centrifuge tube from the magnetic stand, add 25 μL of nuclease-free water and mix thoroughly, incubate at room temperature for 2 minutes, and elute the end-repair product.

[0118] I. Place the centrifuge tube back on the magnetic stand and wait for the magnetic beads to be adsorbed to one side until the eluate is clear and colorless. This process should take at least 1 minute. Aspirate and retain 22.5 μl of the eluate into a clean 1.5 mL centrifuge tube to obtain the nanopore library for the next barcode connection. Discard the centrifuge tube containing the magnetic beads.

[0119] S2. Connect to Adaptor

[0120] Reagent preparation: Nanopore library prepared in step (1), NEBNext Quick T4 DNA Ligase, NEBNextQuick Ligation Reaction Buffer, Native Adapter, Short Fragment Buffer, Elution Buffer.

[0121] A. Thaw reagents at room temperature and place all reagents on ice after they have dissolved.

[0122] B. Before opening the reagent tube cap for the first time, be sure to centrifuge to centrifuge the reagent on the tube cap and tube wall to the bottom of the test tube.

[0123] C. Mix all reagents thoroughly one by one by pipetting.

[0124] D.NEBNext Quick Ligation Reaction Buffer may have a small amount of precipitation. After returning to room temperature, use a pipette to mix several times. The precipitation will dissolve. Make sure the reagents are completely mixed.

[0125] E. Avoid violent shaking of Quick T4 DNALigase.

[0126] (1) The nanopore library is configured into an adaptor connection system, incubated, and purified to obtain a connected mixed library 1:

[0127] Add the reagents to the 1.5 mL centrifuge tube containing the final purified eluted volume of 65 μL nanopore library in the following order:

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[0129]

[0130] Use a pipette to mix the Adaptor connection system thoroughly, centrifuge slightly, and centrifuge the solution on the tube cap and tube wall to the bottom of the test tube.

[0131] Incubation: Incubate the tubes at room temperature for 10 minutes.

[0132] Purification (magnetic beads dosage: 50 μL):

[0133] A. After thoroughly mixing the AMPure XP beads by pipetting, add 50 μL of AMPure XP beads to the 1.5 mL centrifuge tube connected to the adaptor reaction system and flick the tube to mix evenly.

[0134] B. Incubate at room temperature for 5 minutes. During this time, flick the tube several times to ensure that the DNA amplification product is fully bound to the magnetic beads.

[0135] C. Place the sample on the magnetic rack. When the magnetic beads are adsorbed to one side and the liquid is clarified, keep the test tube on the magnetic rack, use a pipette to aspirate the clarified liquid and discard it.

[0136] D. Keep the test tube on the magnetic rack and wash the magnetic beads with 200μL Short Fragment Buffer. Do not blow the magnetic beads away. Rotate the centrifuge tube 180 degrees clockwise every 30 seconds. After rotating 4 times, let the centrifuge tube stand. When the magnetic beads are adsorbed to one side and the liquid is clear, use a pipette to suck out the clear liquid and discard it.

[0137] E. Repeat step D once.

[0138] F. Centrifuge the test tube, put the centrifuge tube back on the magnetic rack, and use a 10μL pipette to aspirate the remaining clear liquid. Open the centrifuge tube cover and let the magnetic beads dry for about 1 minute. Be careful not to let the beads dry out.

[0139] Note: The specific drying time of the magnetic beads is determined by the temperature and humidity of the operating environment.

[0140] G. Remove the centrifuge tube from the magnetic stand, add 17 μL Elution Buffer and mix thoroughly, incubate at room temperature for 10 minutes to elute the end repair product.

[0141] H. Place the centrifuge tube back on the magnetic stand and wait for the magnetic beads to be adsorbed to one side until the elution solution is clear and colorless. This process should last at least 1 minute. Aspirate and retain 15 μL of the eluted mixed library into a clean 1.5 mL centrifuge tube to obtain the connected mixed library 1. Discard the centrifuge tube containing the magnetic beads.

[0142] (2) Quantification and dilution of ligated mixed library 1: Take 1 μL of ligated mixed library 1 and quantify using a Qubit quantifier. Calculate the volume of 100 fmol of the mixed library based on the quantification result.

[0143] The volume of 100 fmol mixed library (μL) = 100 fmol × 660 × average length of target complex amplification target fragment (bp) ÷ concentration of sample to be tested (ng / μL) ÷ 106.

[0144] According to the calculation results, 100 fmol of the ligated mixed library 1 was pipetted into a clean 1.5 mL centrifuge tube and filled to 12 μL with Elution Buffer to obtain the ligated mixed library 2.

[0145] S3, Nano-sequencing

[0146] Preparation before chip loading, final library preparation and sequencing

[0147] Reagent preparation: Library Beads, Sequencing Buffer, Flush Tether, Flush Buffer

[0148] A. Thaw Sequencing Buffer, Library Beads, Flush Tether, and a tube of Flush Buffer at room temperature. After thawing, place all reagents on ice.

[0149] B. Centrifuge before opening the reagent tube cap for the first time to centrifuge the reagent on the tube cap and tube wall to the bottom of the test tube.

[0150] C. Mix all reagents thoroughly one by one by pipetting.

[0151] (1) Prepare chip startup premix:

[0152] Pipette 30 μL Flush Tether and add directly into the tube containing 1170 μL Flush Buffer, and mix thoroughly by pipetting at room temperature.

[0153] Open the MinION device cover and insert the chip into the slot on the right side of the instrument. Press down firmly on the chip to ensure that the temperature and current contacts are connected stably.

[0154] Slide the priming port cover clockwise to open the priming port.

[0155] Check for small bubbles under the priming port cap. Remove any bubbles by aspirating a small volume using a pipette (20-30 μL):

[0156] A. Adjust the 1000μL range pipette to 200μL.

[0157] B. Insert the gun tip completely into the priming port so that the gun tip fits tightly into the priming port.

[0158] C. Turn the pipette volume adjustment knob counterclockwise until the dial reads 220-230uL, or until a small amount of liquid is seen entering the pipette tip.

[0159] Add 800 μL of priming solution to the chip through the priming port, avoiding the introduction of bubbles. Wait for 5 minutes.

[0160] (2) Preparation of sequencing library:

[0161] Mix the Library Beads thoroughly by pipetting.

[0162] Prepare sequencing libraries according to the following table:

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[0165] Note: Load the library onto the chip immediately after adding the Sequencing Buffer and Library Beads, as the fuel in the buffer will begin to be consumed by the adapter.

[0166] (3) Complete chip startup

[0167] A. Gently lift the SpotON sample well cover to open the SpotON sample well.

[0168] B. Add 200 μL of the priming solution into the chip through the priming port (not the SpotON sample injection port), avoiding the introduction of bubbles.

[0169] Note: Add the library to the chip as soon as possible after this step.

[0170] C. Add 75 μL of sequencing library to the chip through the SpotON sample well, making sure that every drop of library enters the well before adding the next drop of sequencing library. Before adding the sequencing library, gently pipette and mix the prepared sequencing library.

[0171] D. Gently close the SpotON sample well cover, making sure the cover plug is inserted into the SpotON well, close the primingport cover, and the MinION device cover. You are ready to start sequencing.

[0172] E. Conversion between sequencing electrical signals and base signals:

[0173] The R10 chip was used and the sequencing time was 48 hours. The ONT official base recognition software Guppy (v7.1.4, high-precision recognition model) was used to perform base recognition on the data from the machine.

[0174] The information of 5198 sites in the sequencing results was compared, and the comparison information of 5198 sites is as follows:

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[0295] In addition, according to the rs number, the ref (reference allele), ref_freq (reference allele frequency), alt (alternative allele), and alt_freq (alternative allele frequency) information were queried in the 1000Genomes Projects Phase 3 database and compared simultaneously.

[0296] A total of 24 samples (276 pairs) were used in this test, and the actual relationship is shown in the table below.

[0297] Family Full siblings Level 2 1-7 1-3 1-9 1-8 1-6 1-10 2-9 3-6 1-11 3-10 7-8 1-12 6-15 1-15 7-11 2-13 8-12 3-7 9-13 3-8 10-14 3-9 15-16 3-14 3-15 4-15 5-15 6-7 6-8 6-9 6-10 6-16 7-12 8-11

[0298] The actual test results are shown in the following table:

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[0301] The results show that the likelihood values ​​of the tested sample pairs are much greater than 1, supporting the parent-child relationship between the tested sample pairs.

[0302] Sample pair Likelihood ratio (full siblings) 1-3 421.954302 1-6 427.127961 7-8 340.5947778 3-6 381.1569213

[0303] The results show that the likelihood values ​​of the tested sample pairs are much greater than 1, supporting the full-sibling relationship between the tested sample pairs.

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[0306] The results show that the likelihood values ​​of the tested sample pairs are much greater than 1, supporting the secondary kinship relationship between the tested sample pairs.

[0307] The likelihood ratios between the above 276 sample pairs were calculated by the ITO algorithm. Based on the 5198 SNP site combinations in the present invention, 10 parent-child sample pairs, 4 full sibling sample pairs, and 20 secondary kinship sample pairs were accurately identified among the 276 pairs of kinship.

[0308] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP site combination, characterized in that: Included are SNP sites with the following rs numbers:

2. The SNP site combination according to claim 1, characterized in that: It also includes at least one of the position information and allele frequency of the 5198 SNP sites.

3. A detection kit, characterized in that: Comprising the SNP site combination described in claim 1 or 2.

4. Use of the SNP site combination according to claim 1 or 2 or the detection kit according to claim 3, characterized in that: The applications include at least one of kinship identification, human individual identification, forensic DNA database construction, suspect family investigation, and degraded specimen identification.

5. The use according to claim 4, characterized in that: The application is kinship identification.

6. A method for identifying kinship based on nanopore sequencing technology, characterized in that: The following steps are involved: S1, construction of nanopore library; S2, connect to Adaptor; S3. Nanosequencing, comparing the SNP site combination described in claim 1 or 2.

7. The method for identifying kinship according to claim 6, characterized in that: The step S1 comprises the following steps: (1) configuring the sample to be tested into an end-repair system; (2) Incubating and purifying the end repair system to obtain a nanopore library, Preferably, in step (1), the end repair system comprises a sample to be tested and an end repair reaction premix. Furthermore, in terms of volume, the end repair system comprises 45-55 parts of 150-250 fmol of the sample to be tested and 5-8 parts of the end repair reaction premix. Most preferably, the end repair system comprises 48 parts of 200 fmol of the sample to be tested and 6.5 parts of the end repair reaction premix. Further preferably, the end repair reaction premix comprises a reaction buffer and an enzyme reagent. Further, in terms of volume, the end repair reaction premix comprises 3-5 parts of a reaction buffer and 2-4 parts of an enzyme reagent. Most preferably, the end repair reaction premix comprises 3.5 parts of a reaction buffer and 3 parts of an enzyme reagent. Preferably, the incubation is: incubation at 15-25°C for 3-8 min, then incubation at 60-70°C for 3-8 min, and then incubation at 4°C; most preferably: incubation at 20°C for 5 min, then incubation at 65°C for 5 min, and then incubation at 4°C; Preferably, the purification is as follows: the incubated end repair system is combined with magnetic beads, placed on a magnetic stand, and the clarified liquid is discarded after the liquid is clarified; the magnetic beads are washed with 60-80% ethanol, and the ethanol is discarded after the liquid is clarified, and the process is repeated 1-3 times, the ethanol is removed, nuclease-free water is added and mixed thoroughly, and the mixture is incubated at 20-30°C for 1-5 minutes to elute the end repair product, and the eluate is clear and colorless, and then the nanopore library is obtained by aspirating the solution.

8. The method for identifying kinship according to claim 6, characterized in that: The step S2 comprises the following steps: (1) configuring the nanopore library into an adaptor connection system, incubating, and purifying to obtain a connected mixed library 1; (2) Calculate the volume of the ligated mixed library corresponding to 100 fmol and adjust the concentration to obtain ligated mixed library 2. Preferably, the Adaptor ligation system comprises a nanopore library, a Native Adapter, NEBNext Quick Ligation Reaction Buffer and NEBNext Quick T4 DNA Ligase; further preferably, in terms of volume, the Adaptor ligation system comprises 60-70 portions of 150-250 fmol of a sample to be tested, 3-8 portions of a Native Adapter, 15-25 portions of a NEBNext Quick Ligation Reaction Buffer and 5-15 portions of a NEBNext Quick T4 DNA Ligase; most preferably, in terms of volume, the Adaptor ligation system comprises 65 portions of a 200 fmol sample to be tested, 5 portions of a Native Adapter, 20 portions of a NEBNext Quick Ligation Reaction Buffer and 10 portions of a NEBNext Quick T4 DNA Ligase; Preferably, the incubation is: incubating at 20-30°C for 5-15 minutes; most preferably: incubating at room temperature for 10 minutes; Preferably, the purification is as follows: the ligated mixed library 1 is combined with magnetic beads, placed on a magnetic stand, and the clarified liquid is discarded after the liquid is clarified; the magnetic beads are washed with Short Fragment Buffer, and the clarified liquid is discarded after the liquid is clarified, and the process is repeated 1-3 times to remove all the clarified liquid, add Elution Buffer and mix thoroughly, incubate at 20-30° C. for 5-15 min, elute the end repair product, and aspirate the eluate until it is clear and colorless to obtain the ligated mixed library 2.

9. The method for identifying kinship according to claim 6, characterized in that: The step S3 comprises the following steps: (1) preparing a starting premix solution; (2) preparing a sequencing library; (3) Add the starting premix and sequencing library to the chip, sequence and compare. Preferably, in step (1), the start-up premix solution includes Flush Tether and Flush Buffer; further, in terms of volume fraction, the start-up premix solution includes 20-40 parts of Flush Tether and 1100-1200 parts of Flush Buffer; most preferably, in terms of volume fraction, the start-up premix solution includes 30 parts of Flush Tether and 1170 parts of Flush Buffer; Preferably, in step (2), the sequencing library comprises a connected mixed library 2, Sequencing Buffer and Library Beads; further, in terms of volume fraction, the priming premix comprises 10-15 parts of the connected mixed library, 35-40 parts of Sequencing Buffer and 20-30 parts of Library Beads; most preferably, in terms of volume fraction, the priming premix comprises 12 parts of the connected mixed library, 37.5 parts of Sequencing Buffer and 25.5 parts of Library Beads.

10. The method for identifying kinship according to claim 9, characterized in that: In step (3), the chip is at least one of R9 and R10, preferably R10 chip; the sequencing time is 24-70h, preferably 48h.

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