CN115948574B

CN115948574B - Three-generation sequencing-based individual identification system, kit and application thereof

Info

Publication number: CN115948574B
Application number: CN202211700477.1A
Authority: CN
Inventors: 王广云; 罗渊; 梅竹松; 严江伟; 刘晋玎; 张家榕; 杨婷婷; 叶巧; 房龙梅
Original assignee: Air Force Specialty Medical Center of PLA
Current assignee: Air Force Specialty Medical Center of PLA
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2023-11-10
Anticipated expiration: 2042-12-28
Also published as: CN115948574A

Abstract

The application provides an individual identification system based on three-generation sequencing, a kit and application thereof, and relates to the field of biotechnology, wherein the individual identification system comprises a primer sequence for amplifying 6 STR loci, and the corresponding primer sequences are shown in SEQ ID NO.1 to SEQ ID NO. 12; the 6 STR loci comprise D3S1358, CSF1PO, D6S1043, D7S820, D12S391 and D13S317 loci; the application also provides a kit for individual identification, which comprises the primer sequence, a DNA template, a long fragment amplification buffer solution, a long fragment amplification polymerase and deionized water, can be used for mixed complex sample typing, individual identification and genetic relationship identification, is applicable to a nanometer sequencer, has portability, low requirement on sequencing environment and accurate sequencing result.

Description

Three-generation sequencing-based individual identification system, kit and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to an individual identification system based on third-generation sequencing, a kit and application thereof.

Background

Short tandem repeats (short tandem repeat, STR), also known as microsatellite DNA, refer to the presence of a class of repeats of 2bp-6bp in length in genomic DNA, which is a human DNA fingerprint, widely distributed in the genome, wherein it is enriched in A-T base pairs. STRs are widely found in the human and mammalian genome and are highly polymorphic, they typically consist of 2-6 bases in a core sequence, the core sequences are tandem repeats, and length polymorphisms result from variations in the number of core sequence repeats. The number of repeats of a repeat sequence at a particular location on a chromosome is fixed for a particular individual, while the number of repeats at the same location may vary for different individuals, which constitutes a polymorphism of these repeat sequences in the population. Since the number of such repeated sequences in human and mammalian genomes is very large, by detecting such polymorphisms, it is possible to clearly distinguish individuals from each other, and to determine the relationship between parents and parents, and a method of distinguishing different individuals is called "individual identification", and is also called "identity identification".

Human genetics is an important basis for forensic material science. Human genetics and variation make the genetic information of each individual similar to that of its parent and offspring, but each individual has its own genetic characteristics. Forensic physical evidence is to research and apply the genetic rules to individual identification and right-of-authority identification practice, and provide accurate and effective scientific evidence for case forensic and judgment. Current individual identification techniques mainly include: a generation of STR detection technology and a detection technology based on Next generation sequencing (Next-generation sequencing technology, NGS). In the first generation STR detection technology, STR is a polymorphic locus in a human genome, a core sequence is formed by 2-6 base pairs, the STR loci are in tandem repeated arrangement, the length of each STR locus is generally between 100 and 300bp, the STR length difference among individuals forms polymorphism, the Mendelian's genetic law is followed in gene transfer, and the STR locus has been widely applied to forensic individual identification and paternity test, and the detection kit is generally about 18 STR loci in the market at present; the next generation sequencing technology, also known as high throughput sequencing (High throughput sequencing), allows for the simultaneous sequencing of millions of DNA molecules, which enables a careful overview of the analysis of the transcriptome and genome of a species, a milestone in the development of sequencing technology. However, the first-generation STR detection technology and the infrastructure based on the next-generation sequencing are sensitive to optical components, and have strict storage and use environment conditions, so that the optical components are extremely difficult to carry and use at will. Chinese patent CN108517363B discloses a second-generation sequencing-based individual identification system, a kit and application thereof, wherein the system can simultaneously obtain second-generation or third-generation genetic marker information on autosomes, X chromosomes and Y chromosomes by only one-time amplification, reduces operation steps, shortens experimental time, is suitable for DNA typing detection of people in different regions of different countries worldwide, but the sequencing needs a high-throughput sequencer, has no portability, and cannot solve the problem of immediately identifying individuals by collecting samples on site. At present, the application of on-site individual identification is not realized in the field of forensic physics and science, and an STR composite amplification system suitable for nano-pore on-site sequencing is not available.

Therefore, a method and a system suitable for on-site sequencing, which can be suitable for on-site sequencing of a nanopore, and have accurate results and convenient operation are needed.

Disclosure of Invention

Aiming at the problems existing in the prior art, the application provides an individual identification system based on three-generation sequencing, a kit and application thereof, wherein the individual identification system comprises primer sequences for amplifying 6 STR loci D3S1358, CSF1PO, D6S1043, D7S820, D12S391 and D13S317, and can be suitable for nanopore on-site sequencing for mixed complex sample typing, individual identification and genetic relationship identification.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

the application provides an individual recognition system based on three-generation sequencing, which comprises a primer sequence for amplifying 6 STR loci, wherein the corresponding primer sequences are shown in SEQ ID NO.1 to SEQ ID NO. 12; the 6 STR sites comprise D3S1358, CSF1PO, D6S1043, D7S820, D12S391 and D13S317.

The size of 6 STR sites is between 1.5 and 2kb, and the specific information is shown in Table 1:

TABLE 1STR information summary table

Site(s)	Chromosome of the human body	Motif repeat range	Motif structure
				D3S1358	3	8–22	TCTA[TCTG] _n [TCTA] _n
CSF1PO	5	5–17	[AGAT] _n
				D6S1043	6	8–26	[AGAT] _n
D7S820	7	5–21.1	[GATA] _n
				D12S391	12	13–28	[AGAT] _n [GAT] _n [AGAT] _n [AGAC] _n [AGAT] _n
D13S317	13	5–17	[TATC] _n [AATC] _n

The application also provides an individual identification method, which adopts the individual identification system to carry out individual identification.

Preferably, the method of individual identification comprises the steps of:

s1, extracting and amplifying sample nucleic acid to obtain 6 STR loci;

s2, third generation library construction;

s3, nanometer sequencing is carried out on a machine;

s4, analyzing sequencing data, and carrying out individual identification.

Preferably, the extraction method in step S1 includes Chelex100 method, magnetic bead extraction method or organic extraction method.

Preferably, the amplified system of step S1 comprises: 11. Mu.L of long fragment amplification buffer, 0.5. Mu.L of long fragment amplification polymerase, 5. Mu. L, DNA of primer mixture and 13.5. Mu.L of template; the amplification procedure includes: (1) 95 ℃ for 3min; (2) 28-30 cycles, each cycle at 98 ℃ for 10s and 60 ℃ for 8min; (3) 72 ℃ for 10min; (4) 4 ℃, and preserving.

Specific amplification procedures are shown in Table 4, and specific amplification reagents are shown in Table 5.

Preferably, the step of third generation library construction in step S2 comprises: DNA targeted amplification target fragment end repair: connecting barcode; mixing the samples; and connecting an adapter.

After the mixed sample is obtained, if the volume exceeds 65 mu L, the mixed library is purified and concentrated by using AMPure XP magnetic beads with the volume being 2.5 times that of the mixed library, and finally 67 mu L of nuclease-free water is used for eluting the mixed library, and 65 mu L of the mixed library is sucked out for the connection of a next adapter.

Preferably, the calculation mode of the input amount of the end repair of the target fragment of the DNA targeted amplification is as follows: according to the concentration (ng/. Mu.L) of the sample to be detected measured by the Qubit quantitative analyzer, the sample volume (mu.L) required for end repair is (200 fmol multiplied by 660 multiplied by the average length (bp) of the target fragment of targeted composite amplification, and the concentration (ng/. Mu.L) of the sample to be detected is (10) ⁶ 。

Wherein, concentration conversion formula: fmol/. Mu.L = sample concentration to be detected (ng/. Mu.L)/(660%)/average length of target complex amplified fragment of interest ×10 ⁶ 。

Preferably, the step of mixing samples includes mixing together each sample of connected barcode; the volume calculation formula of each sample mixture connected with the barcode is as follows: mixed volume (mul) =200×660×targeting composite amplified fragment of interest average length (bp)/(sample to be detected Qubit quantitative instrument quantitative concentration (ng/. Mu.l)/(10) ⁶ The number of samples to be tested.

Preferably, the identification criteria of the individual identification in step S4 are: the identification efficiency of the coincidence of two random genotypes in the population is not lower than 1:10000; specifically, all 6 STR sites are required, if the probability of agreement of any two genotypes at the ith site is P _i Then the product pi _i <0.0001。

Specifically, the identification of individuals standard uses medical parameters: personal identification rate; the personal recognition rate is the combined probability of randomly likely finding two individuals of identical genotype in what population.

The application also provides an individual identification kit, which comprises the primer sequence; the individual identification kit also comprises a DNA template, long PCR buffer, long PCR polymerase and deionized water.

The application also provides application of the individual identification kit in mixed complex sample typing, individual identification and genetic relationship identification.

Compared with the prior art, the application has the following beneficial effects:

(1) The application is an STR long amplified fragment composite system, the long fragment amplicon is suitable for a nanopore connection library-building kit, and high-efficiency specific amplification can be realized;

(2) The amplification system can be used for on-site individual identification, has extremely high portability and low requirement on a sequencing environment, and is suitable for on-site sequencing;

(3) At least 6 target amplified STR target fragments of 1.5-2kb can be obtained, and the sequencing result is accurate.

Drawings

FIG. 1 is a graph showing the effect of 6 STR locus homogeneity.

Detailed Description

The following description of the present application is provided by way of specific examples to facilitate understanding and grasping of the technical solution of the present application, but the present application is not limited thereto, and the described examples are only some, but not all, examples of the present application.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

Reagent consumable: DNA samples (50 ng template DNA per sample); PCR (enzyme, primer, buffer); 3.NEBNext Ultra II End repair/dA-tapering Module; ligation sequencencing Kit (SQK-LSK 109); 5.Flow Cell Priming Kit (EXP-FLP 002); 6.MinION Flow Cell (R9.4.1); 7.Agencourt AMPure XP beads (AMPure XP beads are used after being taken out of the refrigerator in advance and returned to room temperature); qubit ^TM 1X dsDNA HS Assay Kit; a qubit quantitative tube, absolute ethyl alcohol, a 1.5mL centrifuge tube and a 0.2mL PCR centrifuge tube; NEB Blunt/TA Ligase Master Mix;11.NEBNext Quick Ligation Module;12.Native Barcoding Expansion 1-12 (EXP-NBD 104), native Barcoding Expansion-24 (EXP-NBD 114), native Barcoding Expansion 96 (EXP-NBD 196), note that: when the number of the detected samples is less than or equal to 12, the EXP-NBD104, the EXP-NBD114 or the EXP-NBD196 is singly used; when the number of samples is more than or equal to 13 and less than or equal to 24, the EXP-NBD104 and the EXP-NBD114 are used in combination or the EXP-NBD196 is used singly; when the number of samples is more than or equal to 25 and less than or equal to 96, EXP-NBD196 is singly used

Instrument: qubit quantitative instrument and Minion MK1B sequencer

Example 1 screening for effective sites that can be identified confidence by nanopore sequencing

Screening conditions: STR sites common to individual identification; primers for specific amplification products of 1.5-2kb can be designed; the core repeat motif is one that does not comprise a single base tandem repeat; site-accumulating individual recognition can meet judicial identification specifications. STR sites shown in Table 1 were selected as D3S1358, CSF1PO, D6S1043, D7S820, D12S391, D13S317, respectively.

The final STR detection system constructed contains 6 STR loci altogether, the individual recognition (DP) of each locus is shown in the following table, and the cumulative individual recognition (CDP) of the system is 1-1.328×10 ^-7 。

TABLE 2 personal identification (CDP) statistics for STR loci

The forward and reverse primer sequence information for amplifying 6 STR sites is shown in Table 3

TABLE 3 primer information

Example 2 multiplex PCR amplification technique based on third Generation sequencing

Amplification was performed according to the amplification procedure of Table 4 and the amplification reagents of Table 5.

TABLE 4 multiplex PCR amplification procedure

TABLE 5 multiplex PCR amplification reagents

Reagent name	Volume (mu L)	Final concentration
			Long PCR buffer	11	1X
Long PCR polymerase	0.5
			Primer mixture(μM)	5	16μM/μL
DNA template	13.5	50ng
			Total	30	-

Test case identification of unrelated individuals

1.DNA targeted amplification capturing target fragment

A. Reagent preparation:

a) Thawing the reagents of table 5 at room temperature to ensure that the reagents are fully dissolved, and placing all the reagents on an ice box after the reagents are dissolved:

b) Shaking and mixing the reagent and centrifuging to ensure that the reagent is fully and uniformly mixed.

c) The tube is centrifuged before being opened for the first time, and the tube cover and the reagent on the tube wall are centrifuged to the bottom of the test tube. Calculating the input amount of a sample to be detected:

B. calculating the input amount of a sample to be detected:

50ng of template DNA was required for each sample, and a sufficient amount (XuL) of the original DNA sample was diluted to 3.8 ng/. Mu.L according to the concentration C1 (ng/. Mu.L) of the sample to be examined measured by a Qubit 4 quantitative analyzer. (X (μl) =50/c1+2)

The quantitative system is as follows:

table 6 Standard substance System

TABLE 7 sample system

Reagent(s)	Volume (mu L)
		Qubit ^TM 1X dsDNA HS Working Solution	199
DNA	1

And C, preparing a PCR reaction premix:

TABLE 8 premix liquid system for PCR reactions

Remarks: when a plurality of samples are processed simultaneously, more than 2 reaction mixed liquid systems are needed to be matched, so that the loss of the reaction mixed liquid caused by the sample adding operation is ensured to be complemented. The mixture is blown and sucked by a pipette and centrifuged.

D.PCR reaction system

TABLE 9PCR reaction System

Reagent(s)	Volume (mu L)
		50ng DNA(3.8ng/μL)	13.5
PCR reaction premix	16.5
		Total amount of reaction system	30

The liquid on the tube wall and the tube cover are mixed into the tube by blowing and sucking the mixture uniformly by a liquid-transferring gun, ensuring that the reaction system is fully mixed and then slightly centrifuged.

E. The reaction system was amplified using a PCR instrument according to the following procedure set up:

TABLE 10PCR amplification procedure

Purification of PCR products (30. Mu.L sample+30. Mu.L AMPure XP magnetic beads, magnetic bead amount: 30. Mu.L sample count+5. Mu.L)

1) After thoroughly shaking and mixing the AMPure XP magnetic beads, a new 1.5mL centrifuge tube is taken and the sample name is marked, and 30 mu L of AMPure XP magnetic beads are added into each centrifuge tube.

2) All the products of each sample after the reaction are respectively transferred into a 1.5mL centrifuge tube with the corresponding number, and the tube is flicked to be uniformly mixed.

3) Incubate for 5 minutes at room temperature. And during the process, the tube is flicked for a plurality of times, so that the DNA amplification product is ensured to be fully combined with the magnetic beads. During which a 70% fresh ethanol solution was prepared. Volume required for 70% new formulation of ethanol solution = 400 μl x number of samples +500 μl.

4) And (3) standing the sample on a magnetic frame, when the magnetic beads are adsorbed on one side, keeping the test tube on the magnetic frame after the liquid is clarified, sucking out the clarified liquid by using a pipette, and discarding the clarified liquid.

5) The tube was kept on a magnetic rack and the beads were washed with 200 μl of freshly prepared 70% ethanol without blowing off the beads. The centrifuge tube was rotated 180 degrees clockwise every 30 seconds, left to stand after 4 rotations, and when the beads were adsorbed on one side, the liquid was clarified, and the ethanol was aspirated with a pipette and discarded.

6) Repeating step 5) once.

7) The tube was centrifuged and returned to the magnetic rack, and a 10. Mu.L pipette was selected to aspirate the remaining alcohol. The centrifuge tube cover is opened to dry the alcohol for about 1 minute, and the magnetic beads are prevented from drying and cracking. And (3) injection: the specific alcohol airing time is determined according to the temperature and humidity of the operating environment. 8) The centrifuge tube was removed from the magnetic rack, 30. Mu.L nuclease-free water was added and mixed well, incubated at room temperature for 2 minutes, and the end repair product was eluted.

9) The centrifuge tube was placed back on the magnet rack waiting for the beads to be adsorbed to one side until the eluate was clear and colorless, and the process was waiting for at least 1 minute. Mu.l of the eluate was aspirated and retained in a clean 1.5mL centrifuge tube for third generation library construction, 1. Mu.l was taken for Qubit quantification and the centrifuge tube containing the magnetic beads was discarded.

2. Third generation library construction

DNA targeted amplification of fragment end repair

A. Preparation of reagents

a) Thawing the reagent at room temperature, and placing all reagents on ice after the reagent is dissolved:

b) Flick or turn the reagent tube upside down to ensure the reagent is fully mixed.

c) The tube is centrifuged before being opened for the first time, and the tube cover and the reagent on the tube wall are centrifuged to the bottom of the test tube.

d) The Ultra II End-prep buffer may have some white precipitate. When the reagent returns to room temperature, the pellet is naturally dissolved by sucking it several times with a pipette. And vibrating the Ultra II End-prep buffer reagent tube to ensure that the reagents are fully and uniformly mixed.

e) Ultra II End-Prep Enzyme Mix prohibits forced concussion.

B. And calculating the input amount of the sample to be detected for DNA end repair.

Based on the sample concentration to be examined (ng/. Mu.L) measured by the Qubit quantitative apparatus, the sample volume required for end repair was calculated, 200fmol DNA per sample was required: sample volume required for end repair (μl) =200 fmol×660×target complex amplification fragment mean length of interest (bp)/(μl) 10 sample concentration to be detected (ng/μl) ⁶ 。

(concentration conversion formula: fmol/. Mu.L = sample concentration to be tested (ng/. Mu.L)/(660%)/targeting)Average length of multiplex amplified target fragment X10 ⁶ )

C. Sample preparation

The 200fmol amplified product was transferred to a 0.2mL PCR centrifuge tube, the volume was adjusted to 48. Mu.L with nuclease free water, thoroughly mixed by flick tube and briefly centrifuged in a microcentrifuge.

D. Preparing a premix for the end repair reaction:

TABLE 11 premixed liquid System for end repair reactions

Reagent(s)	Volume (mu L)	Multiple samples (mu L)
			Ultra II End-prep reaction buffer	3.5μL	Mu L of 3.5 x (number of samples+2)
Ultra II End-prep enzyme mix	3μL	Mu L (number of samples +2)

Remarks: when a plurality of samples are processed simultaneously, more than 2 reaction mixed liquid systems are needed to be matched, so that the loss of the reaction mixed liquid caused by the sample adding operation is ensured to be complemented. And blowing and sucking by using a pipetting gun, and fully and uniformly mixing each reaction system.

E. Preparation of terminal repair system

TABLE 12 terminal repair system

Reagent(s)	Add volume (μL)
		200fmol sample amplification product to be detected	48μL
Premix for end repair reaction	6.5μL
		Total amount of reaction system	54.5μL

And (3) blowing and sucking by using a pipetting gun to fully mix, ensuring that the reaction system is fully mixed, and slightly centrifuging to mix the liquid on the pipe wall and the pipe cover into the pipe.

F. Using a PCR instrument, the reaction system was incubated, and the hot lid temperature set according to the following procedure: the reaction system is set at 80 ℃: 55. Mu.L.

TABLE 13PCR procedure

Temperature (. Degree. C.)	Time (min)
		20℃	5min
65℃	5min
		4℃	∞

G. Purification of samples after end repair (magnetic bead amount: 60. Mu.L. Sample count+5. Mu.L)

a) After thoroughly shaking and mixing the AMPure XP magnetic beads, a new 1.5mL centrifuge tube is taken and the sample name is marked, and 60 mu L of AMPure XP magnetic beads are added into each centrifuge tube.

b) All the products of each sample after the reaction are respectively transferred into a 1.5mL centrifuge tube with the corresponding number, and the tube is flicked to be uniformly mixed.

c) Incubate for 5 minutes at room temperature. And during the process, the tube is flicked for a plurality of times, so that the DNA amplification product is ensured to be fully combined with the magnetic beads. During which a 70% fresh ethanol solution was prepared. Volume required for 70% new formulation of ethanol solution = 400 μl x number of samples +500 μl.

d) And (3) standing the sample on a magnetic frame, when the magnetic beads are adsorbed on one side, keeping the test tube on the magnetic frame after the liquid is clarified, sucking out the clarified liquid by using a pipette, and discarding the clarified liquid.

e) The tube was kept on a magnetic rack and the beads were washed with 200 μl of freshly prepared 70% ethanol without blowing off the beads. The centrifuge tube was rotated 180 degrees clockwise every 30 seconds, left to stand after 4 rotations, and when the beads were adsorbed on one side, the liquid was clarified, and the ethanol was aspirated with a pipette and discarded.

f) Repeating step e) once.

g) The tube was centrifuged and returned to the magnetic rack, and a 10. Mu.L pipette was selected to aspirate the remaining alcohol. The centrifuge tube cover is opened to dry the alcohol for about 1 minute, and the magnetic beads are prevented from drying and cracking. Note that the specific alcohol airing time is determined according to the temperature and humidity of the operating environment.

h) The centrifuge tube was removed from the magnetic rack, 25. Mu.L of nuclease-free water was added and mixed well, incubated for 2 minutes at room temperature, and the end repair product was eluted.

i) The centrifuge tube was placed back on the magnet rack waiting for the beads to be adsorbed to one side until the eluate was clear and colorless, and the process was waiting for at least 1 minute. Aspirate and hold 22.5 μl of eluate into a clean 1.5mL centrifuge tube for the next barcode ligation. Discard centrifuge tube containing magnetic beads.

2. Connecting barcode

A. Preparation of reagents

a) Each sample corresponds to one barcode individually, and a unique number barcode is selected from the 24 barcodes for each sample for this chip sequencing.

b) Thawing the reagent at room temperature, and placing all reagents on ice after the reagent is dissolved.

c) The reagent tube cover is required to be centrifuged before being opened for the first time, and the reagent on the tube cover and the tube wall is centrifuged to the bottom of the test tube.

d) And (3) fully and uniformly mixing all the reagents one by one in a blowing and sucking mode of a pipetting gun.

B. Sample and barcode corresponding list:

the samples Sample01-24 correspond to the Barcode numbers Barcode01-24.

C.Barcode connection system

The reagents were added to a 1.5mL centrifuge tube of 22.5. Mu.L purified eluate product from step 1 in the following order:

TABLE 14Barcode connection System

Reagent(s)	Add volume (μL)
		Completed end repair sample	22.5μL
Barcode	2.5μL
		Blunt/TA Ligase Master Mix	25μL
Total amount of reaction system	50μL

And (3) blowing and sucking a fully mixed Barcode connecting system by using a pipetting gun, slightly centrifuging, and centrifuging the tube cover and the solution on the tube wall to the bottom of the test tube.

D. Incubation of Barcode ligation system: the tube was allowed to stand for 10 minutes at room temperature.

E. Purification of the ligated barcode samples (magnetic bead amount: 50. Mu.L. Sample count+5. Mu.L)

a) After fully vibrating, blowing and sucking and uniformly mixing AMPure XP magnetic beads, 50 mu L of AMPure XP magnetic beads are added into each 1.5mL centrifuge tube containing a completed connection barcode reaction system, and the centrifuge tube is flicked to be uniformly mixed.

b) Incubate for 5 minutes at room temperature. And during the process, the tube is flicked for a plurality of times, so that the DNA amplification product is ensured to be fully combined with the magnetic beads. During which a 70% fresh ethanol solution was prepared. Volume required for 70% new formulation of ethanol solution = 400 μl x number of samples +500 μl.

c) And (3) standing the sample on a magnetic frame, when the magnetic beads are adsorbed on one side, keeping the test tube on the magnetic frame after the liquid is clarified, sucking out the clarified liquid by using a pipette, and discarding the clarified liquid.

d) The tube was kept on a magnetic rack and the beads were washed with 200 μl of freshly prepared 70% ethanol without blowing off the beads. The centrifuge tube was rotated 180 degrees clockwise every 30 seconds, left to stand after 4 rotations, and when the beads were adsorbed on one side, the liquid was clarified, and the ethanol was aspirated with a pipette and discarded.

e) Repeating step d) once.

f) The tube was centrifuged and returned to the magnetic rack, and a 10. Mu.L pipette was selected to aspirate the remaining alcohol. The centrifuge tube cover is opened to dry the alcohol for about 1 minute, and the magnetic beads are prevented from drying and cracking. Note that the specific alcohol airing time is determined according to the temperature and humidity of the operating environment.

g) The centrifuge tube was removed from the magnetic rack, 26. Mu.L of nuclease-free water was added and mixed well, incubated for 2 minutes at room temperature, and the end repair product was eluted.

h) The centrifuge tube was placed back on the magnet rack waiting for the beads to be adsorbed to one side until the eluate was clear and colorless, and the process was waiting for at least 1 minute. Aspirate and hold 24 μl of eluate into a clean 1.5mL centrifuge tube for the next sample mix. Discard centrifuge tube containing magnetic beads.

3. Mixing samples

A. Quantification of the ligated barcode samples. 1. Mu.L of each sample after the connection of the barcode in step 2 and purification and elution was quantified using a Qubit quantifier.

B. The mixing volume is required for each connected barcode sample when calculating the mixing sample. According to the quantitative result of the Qubit quantitative instrument, the mixing volume (mu L) =200x660×targeted composite amplification target fragment average length (bp) and the quantitative concentration (ng/mu L) of the sample to be detected are required to be mixed into each sample when the samples are mixed ⁶ The number of samples to be tested.

C. Taking a clean 1.5mL centrifuge tube, according to the calculation result in the step B, sequentially mixing all samples connected with the barcode in the step (2) and purified and eluted into the centrifuge tube according to the calculated volume to finally obtain a mixed library with the final concentration of 200 fmol; the total volume (μl) of the samples after mixing was recorded simultaneously.

D. If the volume of the mixed library obtained in C is less than 65. Mu.L, the mixed library is supplemented with nuclease-free water to 65. Mu.L.

4. Connection adapter

A. Preparation of reagents

a) Thawing the reagent at room temperature, and placing all reagents on ice after the reagent is dissolved.

b) The reagent tube cover is required to be centrifuged before being opened for the first time, and the reagent on the tube cover and the tube wall is centrifuged to the bottom of the test tube.

c) And (3) fully and uniformly mixing all the reagents one by one in a blowing and sucking mode of a pipetting gun.

d) NEBNext Quick Ligation Reaction Buffer may have a small amount of sediment, and after the temperature is restored to room temperature, the sediment is blown and sucked by a pipetting gun for a plurality of times, and the sediment is dissolved, so that the reagent is ensured to be completely mixed.

e) Avoid violent shock of Quick T4 DNA ligation.

Adapter connection system

The reagents were added to a 1.5mL centrifuge tube containing a final purified eluted volume of 65. Mu.L of the mixed library in step 3) in the following order:

table 15 adapter connection system

Reagent(s)	Add volume (μL)
		200fmol hybrid library	65μL
Adapter Mix II(AMII)	5μL
		NEBNext Quick Ligation Reaction Buffer	20μL
Quick T4 DNA Ligase	10μL
		Total amount of reaction system	100μL

And (3) blowing and sucking by using a pipetting gun, fully mixing the adapter connecting system, slightly centrifuging, and centrifuging the tube cover and the solution on the tube wall to the bottom of the test tube.

C. Incubation of the adapter ligation system: the tube was allowed to stand for 10 minutes at room temperature.

D. Purification of the ligated adapter Mixed library (magnetic bead usage: 50. Mu.L)

a) After fully vibrating, blowing and sucking the mixed AMPure XP magnetic beads, adding 50 mu L of AMPure XP magnetic beads into a 1.5mL centrifuge tube connected with an adapter reaction system, and flicking the centrifuge tube to mix uniformly.

b) Incubate for 5 minutes at room temperature. And during the process, the tube is flicked for a plurality of times, so that the DNA amplification product is ensured to be fully combined with the magnetic beads.

d) The tube was held on a magnetic rack and the beads were washed with 200 mu L Short Fragment Buffer (SFB) without blowing off the beads. The centrifuge tube was rotated 180 degrees clockwise every 30 seconds, left to stand after 4 rotations, and when the magnetic beads were adsorbed on one side, after the liquid was clarified, the clarified liquid was aspirated with a pipette and discarded.

e) Repeating step d) once.

f) The tube was centrifuged and the tube was returned to the magnet rack and a 10. Mu.L pipette was selected to aspirate the remaining clarified liquid. And opening the cover of the centrifugal tube to dry the magnetic beads for about 1 minute, and avoiding the magnetic beads from drying and cracking. Note that the specific bead airing time is determined according to the temperature and humidity of the operating environment.

g) The centrifuge tube was removed from the magnetic rack, 17. Mu.L of the addition Buffer (EB) was added and mixed well, incubated at room temperature for 10 minutes, and the end repair product was eluted.

h) The centrifuge tube was placed back on the magnet rack waiting for the beads to be adsorbed to one side until the eluate was clear and colorless, and the process was waiting for at least 1 minute. Aspirate and hold 15 μl of the eluted pooled library into a clean 1.5mL centrifuge tube. Discard centrifuge tube containing magnetic beads. E. The adapter-connected mixed library was quantified and diluted. The final library mixture after the adapter was ligated and eluted was purified by 1. Mu.L in step (4) and quantified using a Qubit quantifier.

F. The volume of the 100fmol mixed library was calculated from the quantitative results. Volume of 100fmol mixed library (μl) =100 fmol×660×target complex amplified fragment of interest average length (bp)/(μl) 10 sample concentration to be tested (ng/μl) ⁶ 。

G. According to the calculation result, 100fmol of the mixed library is sucked into a clean 1.5mL centrifuge tube, and the mixture library is complemented to 12 mu L by an Elutation Buffer (EB) for preparing the sequencing final library on the machine.

3. Nanometer sequencing upper machine

Preparation before chip loading, preparation of final library and sequencing on chip loading

A. Preparation of reagents

a) Thawing Sequencing Buffer (SQB), loading Beads (LB), flush teathers (FLT) and a tube of Flush Buffer (FB) at room temperature, after dissolution all reagents were placed on ice.

B. Chip starting premix preparation: mu.L of Flush teather (FLT) was pipetted directly into a tube of Flush Buffer (FB) and thoroughly mixed at room temperature by pipetting.

C. And opening the cover of the MinION equipment, inserting the chip into a clamping groove on the right side of the instrument, and forcibly pressing down the chip to ensure stable connection between the temperature and the current contact position.

a) Inserting the chip into a clamping groove on the right side of the instrument; b) Forcefully pressing down the chip

D. Sliding the priming port cover clockwise opens the priming port.

E. Check if there are small bubbles under the priing port lid. A small volume was sucked back using a pipette to remove all bubbles (20-30 μl):

a) The 1000. Mu.L range pipette was adjusted to 200. Mu.L.

b) The gun head is completely inserted into the priming port, so that the gun head is tightly embedded with the priming port.

c) The pipette range adjustment knob is turned counterclockwise until the dial displays 220-230 uL, or until a small amount of liquid is seen to enter the pipette tip.

F. 800. Mu.L of the priming premix was added to the chip via the prime port, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, the sequencing library was prepared as follows.

G. The Loading Beads (LB) were thoroughly mixed by a pipette. The Loading Beads (LB) tube contained suspended Beads. These beads settle quickly and must be mixed as soon as they are used.

H. Library preparation

TABLE 16 library System

Reagent(s)	Add volume (μL)
		Mixed library	12μL
Sequencing Buffer(SQB)	37.5μL
		Loading Beads(LB)	25.5μL
Total amount of reaction system	75μL

Note that the library was added to the chip immediately after the addition of Sequencing Buffer (SQB) and Loading Beads (LB) because the fuel in the buffer would begin to be consumed by the adapter.

I. Completing the chip start

a) The SpotON sample well lid is gently lifted to open the SpotON sample well.

b) 200. Mu.L of priming premix was added to the chip via a priming port (not a Spoton sample well) to avoid the introduction of air bubbles. Note that the library was added to the chip as soon as possible after this step.

J. Before the library is loaded, the prepared library is gently blown and sucked by a pipette.

K. Through the Spoton sample loading well, 75. Mu.L of library was added dropwise to the chip, ensuring that each drop of library entered the well and the next drop was added.

And L, lightly covering the Spoton sample hole cover, ensuring that a cover plug is inserted into the Spoton hole, closing the print port cover and the MinION device cover. Sequencing was started.

The sequencing results are shown in Table 17: the MiSeq FGx second generation sequencing result is used as a standard result to evaluate the third generation sequencing result.

Based on the second generation sequencing result, the result proves that the typing results of 6 sites are consistent on both sequencing platforms through comparing the third generation sequencing result, and the correct typing result can be obtained in the third generation sequencing. The 6 sites are indicated to be suitable for individual identification based on the third-generation sequencing platform, and the resolution of irrelevant individuals can be accurately realized.

TABLE 17 second and third generation sequencing results

Finally, it should be noted that the above description is only for illustrating the technical solution of the present application, and not for limiting the scope of the present application, and that the simple modification and equivalent substitution of the technical solution of the present application can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present application.

Claims

1. A method of individual identification, characterized by: performing individual identification by adopting an individual identification system based on third-generation sequencing; the primer comprises primers for amplifying 6 STR loci, and the corresponding primer sequences are shown in SEQ ID NO.1 to SEQ ID NO. 12; the 6 STR loci comprise D3S1358, CSF1PO, D6S1043, D7S820, D12S391 and D13S317 loci;

the method comprises the following steps:

s1, extracting and amplifying sample nucleic acid to obtain 6 STR loci;

s2, third generation library construction;

s3, nanometer sequencing is carried out on a machine;

s4, analyzing sequencing data, and identifying individuals;

the extraction method in the step S1 comprises a Chelex100 method, a magnetic bead extraction method or an organic extraction method;

the amplified system of step S1 comprises: 11 mu L of long-fragment amplification buffer solution, 0.5 mu L of long-fragment amplification polymerase, and 13.5 mu L of primer mixture 5 mu L, DNA template; the amplification procedure includes: (1) 95 ℃ for 3min; (2) 28-30 cycles, each cycle at 98 ℃ for 10s and 60 ℃ for 8min; (3) 72 ℃ for 10min; (4) preserving at 4 ℃;

the third generation library construction step in step S2 comprises the following steps: DNA targeted amplification target fragment end repair: connecting barcode; mixing the samples; connecting an adapter;

the step of mixing samples includes mixing together each sample of connected barcode; the volume calculation formula of each sample mixture connected with the barcode is as follows: mixed volume=200x660×average length of target fragment of target composite amplification/quantitative concentration of sample to be detected Qubit quantitator/10 ⁶ The number of samples to be detected; the unit of the mixed volume is [ mu ] L; the unit of the average length of the fragment is bp; the unit of the quantitative concentration is ng/MuL;

the identification criteria of the individual identification in step S4 are: the identification efficiency of the coincidence of two random genotypes in the population is not lower than 1:10000; all 6 STR sites are required, if the probability of agreement of any two genotypes at the ith site is Pi, then the product Pi <0.0001.

2. An individual identification kit, characterized in that: comprising the primer set forth in claim 1; the individual identification kit also comprises a DNA template, a long fragment amplification buffer solution, a long fragment amplification polymerase and deionized water.

3. The use of the individual identification kit of claim 2 in mixed complex sample typing, individual identification, and genetic relationship identification.