CN110499373B

CN110499373B - High-throughput STR typing system and kit for identifying complex genetic relationship

Info

Publication number: CN110499373B
Application number: CN201910883270.4A
Authority: CN
Inventors: 严江伟; 范庆炜; 张更谦; 张君; 程凤; 王萌春; 李万婷; 石林玉; 张晓梦; 张家榕
Original assignee: Shanxi Medical University
Current assignee: Shanxi Medical University
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2020-06-05
Anticipated expiration: 2039-09-18
Also published as: CN110499373A

Abstract

The invention discloses a high-throughput STR typing system and a typing kit for identifying complex genetic relationship, and relates to the technical field of nucleic acid in-vitro detection. The STR typing system comprises PCR primers for amplifying 60, 118 or 179 linked autosomal STR loci, and the kit comprises a PCR primer combination, an Index adaptor sequence, an IGT-EM707polymerase mixture, an amplification buffer enhancer NB, a YF buffer B, a library construction reagent and the like. The STR typing system and the kit thereof provided by the invention can realize single-tube amplification of 60, 118 or 179 linked autosomal STR loci, have good balance, high sensitivity, good specificity and accurate typing result, and can be used for identifying complex genetic relationships of different genetic relationships at the same level.

Description

High-throughput STR typing system and kit for identifying complex genetic relationship

Technical Field

The invention relates to the technical field of forensic detection, in particular to a high-throughput STR typing system and a typing kit for identifying complex genetic relationship.

Background

Short Tandem Repeat (STR) is a special sequence formed by tandem connection of 2-6bp repeat units, the STR locus has large quantity and wide distribution, accounts for about 3 percent of the whole genome, has high polymorphism, and the polymorphism mainly derives from the difference of the repeat times of a core sequence among individuals, and the difference follows Mendelian genetic rules in the genetic process. Therefore, STR amplification detection techniques are widely used in forensic individual identification, genetic identification and population genetics research. The most commonly used STR typing techniques are fluorescence-labeled multiplex amplification combined with Capillary Electrophoresis (CE) and the second generation sequencing technique.

The identification of complex genetic relationship is one of the technical problems which are urgently needed to be solved in the field of judicial identification at present, and the identification is mainly used in multiple fields of lawsuits, inheritance, remains in large-scale disasters, revenues of remains, traffic accidents, immigration cases, relativity identification and the like in judicial practice, and the cases tend to increase in recent years. The success of such case identification is often dependent on the social and bloody relationships of several families, and is a great challenge for forensic geneticists and a significant impact on the personal interests of the general public.

In the complex genetic relationship identification, the identification requirements of different genetic relationships in the same class of the same class between a half-sib and a tertiary nephew, between the half-sib and a grandchild or between the tertiary nephew and a grandchild are met, but the Identity by term (IBD) value and the coordinate coefficient (theta) value derived through Mendel's genetic law in the class of the third class of relationships are consistent, so that the three relationships cannot be distinguished by applying the conventional independent genetic marker and calculation method. It is suggested that although the identification of such relationships can aid in the determination of such problems through age information and other DNA information, such as mitochondrial DNA and sex chromosomal DNA, the linked autosomal genetic markers can better address the above problems in the general case of the same level of different relationships. In theoretical research, Thompson deduces different genetic relationship coefficients among grandfather, half-sibling and tertiary-nephew in 1998 by using recombination rate of linkage genetic law, and with the application of computer technology in the field of forensic physical evidence, Egeland makes a theoretical calculation model by using a certain number of linkage genetic markers according to the different genetic relationship coefficients obtained by Thompson in 2008.

Current state of the art for genetic relationship identification, for example: chinese patent CN104818323B discloses a genotyping detection kit for 20 STR loci of human chromosomes 13, 18 and 21, which can realize single-tube amplification of 20 STR loci; chinese patent CN106906292A discloses a 22 short tandem repeat sequence composite amplification method and a kit thereof, wherein the kit can be used for amplifying 22 STR loci and 1 individual locus. The majority of the chain STR researches are X-STR and Y-STR genetic markers, and the genetic markers can be applied only by special genetic relationship; autosomal STRs that are independently inherited also do not distinguish between complex relationships.

The invention aims at the characteristics of the chain autosomal STR, and designs an STR typing system for multiple PCR targeted capture sequencing, wherein the STR typing system can amplify all STR genetic markers at one time, the adopted primer combination sequence has good balance, all loci in a group can be ensured to be detected, the typing and sequence information of each STR can be obtained through second-generation sequencing and subsequent data analysis, and the STR typing system can be used for identifying the complex genetic relationship of different genetic relationships at the same level.

Disclosure of Invention

The invention aims to provide an STR typing system for identifying complex genetic relationship and a kit thereof. The STR typing system and the kit thereof can realize single-tube amplification of 179 linked autosomal STR loci, have good balance, high sensitivity, good specificity and accurate typing result, and can be used for identifying complex genetic relationships of different genetic relationships at the same level.

On the other hand, in the research process, it is found that the 179 STR genomes can be divided into three groups independent from each other, and the 6 STR locus combinations formed by the three groups of STR loci alone or in pairwise cooperation can achieve the same, similar or equivalent technical effects as the typing system formed by the 179 STR loci.

Based on the above:

the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 1 for amplifying 60 linked autosomal STR loci;

the corresponding physical positions, chromosome partitions and genetic distances of the 60 linked autosomal STR loci and the reference genome Hg38 are as follows:

the PCR primer combination 1 comprises a forward primer group and a reverse primer group; the forward primer group is as follows:

the reverse primer group is as follows:

secondly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 2 for amplifying 58 linked autosomal STR loci;

the 58 linked autosomal STR loci, the corresponding physical locations on the reference genome Hg38, the chromosomal partitions and the genetic distances are:

the PCR primer combination 2 comprises a forward primer group and a reverse primer group; the forward primer group is as follows:

the reverse primer group is as follows:

thirdly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 3 for amplifying 61 linked autosomal STR loci;

the 61 linked autosomal STR loci, the corresponding physical locations on the reference genome Hg38, the chromosomal partitions and the genetic distances are:

the PCR primer combination 3 comprises a forward primer group and a reverse primer group; the forward primer group is as follows:

the reverse primer group is as follows:

fourthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 4 for amplifying 118 linked autosomal STR loci; the 118 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-118 in the description of the invention; the PCR primer combination 4 is a forward primer combination and a reverse primer combination which are used for amplifying STR loci with STR locus sequence numbers of 1-118 in the description of the invention, and specifically consists of

primer combinations

1 and 2.

Fifthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 5 for amplifying 121 linked autosomal STR loci; the 121 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-60 and 119-179 in the description of the invention; the PCR primer combination 5 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 1-60 and 119-179 in the description of the invention, and specifically comprises

primer combinations

1 and 3.

Sixthly, the invention provides an STR typing system for identifying complex genetic relationship, which comprises a PCR primer combination 6 for amplifying 119 linked autosomal STR loci; the 119 linked autosomal STR loci are STR loci with STR locus sequence numbers of 61-179 in the description of the invention; the PCR primer combination 6 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 61-179 in the description of the invention, and specifically comprises

primer combinations

2 and 3.

Seventhly, the invention provides an STR typing system for identifying complex genetic relationship, wherein the STR typing system comprises a PCR primer combination 7 for amplifying 179 linkage autosomal STR loci; the 179 linked autosomal STR loci are STR loci with STR locus sequence numbers of 1-179 in the description of the invention; the PCR primer combination 7 is a forward primer combination and a reverse primer combination for amplifying STR loci with STR locus sequence numbers of 1-179 in the description of the invention, and specifically comprises

primer combinations

1, 2 and 3.

In order to more intuitively embody the technical scheme of the invention, the key points of the first to seventh technical schemes are summarized as follows:

eighth, the present invention provides a kit for identification of complex relationships, which comprises the PCR primer set 1, 2, 3, 4, 5, 6 or 7 described in the present specification.

In the primer combination, the concentrations of the positive primer and the negative primer are both 0.1 mu M.

Further, the kit also comprises an Index adaptor sequence and DNA polymerase;

further preferably, the Index linker sequences comprise IGT-I5Index and IGT-I7Index, and the concentration of the working solution is 10. mu.M.

The Index linker sequence is a universal linker sequence for the illiminia sequencing platform and is used for discrimination between samples.

Preferably, the kit further comprises reagents for preparing genomic DNA into a library for sequencing.

The invention also provides a method for identifying complex genetic relationship, which comprises the following steps:

(1) extracting human genome DNA, and quantifying the concentration of the genome DNA to be 1-20 ng/mu L;

(2) multiplex PCR library construction:

A. obtaining a purified multiplex PCR product through a first round of multiplex PCR reaction and a first round of magnetic bead purification;

B. performing a second round of multiplex PCR reaction by taking the purified multiplex PCR product as a template, inserting an Index adaptor sequence, and purifying by a second round of magnetic beads to obtain a multiplex PCR library;

C. carrying out quantitative and quality detection on the obtained multiple PCR library;

(3) and (3) sequencing and analyzing data of the multiple PCR library obtained in the step (2) to obtain STR typing results and forensic parameters corresponding to each individual.

Further preferably, the concentration of genomic DNA is 10-20 ng/. mu.L.

The STR typing system and the kit are suitable for all samples containing DNA, including but not limited to blood, seminal plasma, hair, bones, skin, solid tissues and the like.

Compared with the prior art, the invention has the advantages that:

(1) the STR typing system and the kit thereof for identifying complex genetic relationship provided by the invention can simultaneously detect 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR loci at one time, and the mutual interference between each pair of primers can be increased along with the increase of the number of the detected STR loci in the process of establishing a multiple PCR amplification system.

(2) In the prior art, neither autosomal STR inherited independently nor STR genetic markers linked to sex chromosomes can identify complex relationships such as the same-level different relationships between a half-sib and a tertiary (girl) nephew, a half sib and a grande (milk) grande or a tertiary nephe and a grande. The STR typing system and the kit thereof provided by the invention aim at the characteristics of the chain autosomal STR, can be used for identifying the complex genetic relationship, and have accurate identification result.

(3) The STR typing system and the kit thereof provided by the invention adopt two rounds of PCR reactions to construct the library, have the advantages of short library construction period, high comparison rate, good capture efficiency, good repeatability, simple and convenient operation and the like, have high sensitivity of detection results, good specificity and accurate typing results, and can effectively amplify 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR loci at one time.

It should be clear that, based on the disclosure of the present invention of all 179 STR loci described and their corresponding amplified forward and reverse primers, one skilled in the art can reasonably expect, based on the disclosure of the present invention: the technical scheme of the invention, which is formed by any collocation combination with genetic testing efficiency quantity in 179 STR loci recorded in the invention or the collocation combination of corresponding forward and reverse primers, can obtain the technical effect which is equal to or similar to the technical effect of the invention. The amount of any genetic test potency may specifically be any integer between 3 and 179. Therefore, the technical solutions obtained by the embodiments described in this paragraph are all within the technical extension, the protection scope, and the infringement scope of the present invention.

Drawings

FIG. 1 is a diagram of quality control peaks of a library prepared using the primer combinations disclosed in example 1.

FIG. 2 is a diagram of quality control peaks of a library prepared using the primer combinations disclosed in example 2.

FIG. 3 is a peak inspection diagram of a library prepared using the primer combinations disclosed in example 3.

FIG. 4 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 4.

FIG. 5 is a peak inspection diagram of a library prepared using the primer combinations disclosed in example 5.

FIG. 6 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 6.

FIG. 7 is a peak inspection chart of a library prepared by using the primer combination disclosed in example 7.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1: STR typing system for complex genetic relationship identification and kit thereof

1.1 autosomal linked STR locus constitution and characteristics:

(1) the STR typing system in this example includes the following 60 STR loci:

D1S2131, D1S3721, D1S2130, D1S1600, D1S1653, D1S1660, D1S3732, D2S1364, D2S2734, D2S1396, D2S428, D2S435, D2S1387, D2S1792, D2S1399, D2S2959, D3S2431, D3S4547, D4S1643, D4S2408, D4S3326, D4S2368, D5S2845, D5S1473, D5S813, D5S1716, D5S1459, D5S1487, D5S1466, D5S2496, D5S2501, D6S1019, D6S2417, D6S2412, D6S1284, D7S 1280, D7S 376S 821 0, D2S 2328S 1469, D2S 2501, D2S 1019, S14611, S1469, D2S 14611, S1469, D2S 20, S14611, S1469, D2S 1469, D5S 14613, D2S 20, D2S 1469, D2S 20S 1469, D2S 20, D2S 1469, D2S;

it has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

3) each group comprises at least two STRs and the genetic distance of the STRs is less than 3 cM;

4) each STR has the name DXSXX.

(2) Primer set 1 used to simultaneously amplify the 60 STR loci in this example was:

table 1: forward primer set of primer combination 1

Table 2: reverse primer set of primer set 1

(3) The working concentration of the forward primer in this example was 0.1. mu.M; the working concentration of the reverse primer was 0.1. mu.M.

1.2 a kit for identification of complex relationships:

comprises the PCR primer combination 1, Index adaptor sequence and IGT-EM707polymerase mixture.

The Index linker sequences include IGT-I5Index and IGT-I7Index, and the concentrations of the working solution are 10 μ M.

The Index linker sequence is: the I7 end linker sequence of IGT-I7index Aijiekang, with working concentration of 10 μ M; one contains 96, linker sequence information as follows

The I5 end linker sequence of IGT-I5Index Aijiekang, working concentration 10 μ M; one contains 4, linker sequence information as follows

Note: linker sequences are those commonly used by the illumina sequencing platform for discrimination between samples.

Example 2: STR typing system for complex genetic relationship identification and kit thereof

2.1 autosomal linked STR loci constitution and Properties:

(1) the STR typing system in this example contains the following 58 STR loci:

D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、D14S301、D14S583、D15S816、D15S1514、D18S872、D18S972、D18S548、D20S1145、D20S477；

it has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer set 2 used to simultaneously amplify the 58 STR loci in this example was:

table 3: forward primer set of primer combination 2

Table 4: reverse primer set of primer combination 2

2.2 a kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer set used was primer set 2.

Example 3: STR typing system for complex genetic relationship identification and kit thereof

3.1 autosomal linked STR locus constitution and Properties:

(1) the STR typing system in this example comprises the following 61 STR loci:

D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、D14S302、D14S749、D16S767、D16S3393、D18S537、D18S875、D18S1367、D20S1152、D20S206、D20S607、D20S1146；

it has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer set 3 used to simultaneously amplify the 61 STR loci in this example was:

table 5: forward primer set of primer combination 3

Table 6: reverse primer set of primer combination 3

3.2A kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer set used was primer set 3.

Example 4: STR typing system for complex genetic relationship identification and kit thereof

4.1 autosomal linked STR loci constitution and Properties:

(1) the STR typing system in this embodiment includes 118 STR loci, specifically including 60 STR loci described in embodiment 1 and 58 STR loci described in embodiment 2.

It has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer set 4 for simultaneously amplifying the 118 STR loci in this example is a set of primer sets in examples 1 and 2.

4.2 a kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer set used was primer set 4.

Example 5: STR typing system for complex genetic relationship identification and kit thereof

5.1 autosomal linked STR loci constitution and Properties:

(1) the STR typing system in this embodiment includes 121 STR loci, specifically including 60 STR loci described in embodiment 1 and 61 STR loci described in embodiment 3.

It has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer set 5 for simultaneously amplifying 121 STR loci in this example is a set of primer sets in examples 1 and 3.

5.2A kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer set used was primer set 5.

Example 6: STR typing system for complex genetic relationship identification and kit thereof

6.1 autosomal linked STR loci constitution and Properties:

(1) the STR typing system in this embodiment includes 119 STR loci, specifically including 58 STR loci described in embodiment 2 and 61 STR loci described in embodiment 3.

It has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer combination 6 for simultaneously amplifying the 119 STR loci in this example is a combination of the primer combinations in examples 3 and 2.

6.2A kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer combination used is primer combination 6.

Example 7: STR typing system for identifying complex genetic relationship

7.1 autosomal linked STR locus constitution and Properties:

(1) the STR typing system in this embodiment includes 179 STR loci, specifically including 60 STR loci described in embodiment 1, 58 STR loci described in embodiment 2, and 61 STR loci described in embodiment 3.

It has the following measurable characteristics:

1) the core region repeat unit is tetranucleotide;

2) the heterozygosity of each STR is more than 0.6;

4) each STR has the name DXSXX.

(2) Primer set 7 for simultaneously amplifying the 179 STR loci in this example is a set of primer sets in example 1, example 3, and example 2.

7.2A kit for identification of complex relationships:

the only difference from example 1 is that the PCR primer set used was primer set 7.

Examples of the experiments

In the following experimental examples, the reagents used were all available from the legal commercial sources as follows:

tissue and blood DNA extraction kit: purchased from Beijing Tiangen Biochemical technology Ltd; the goods number is: DP 304-03;

QIAamp DNA investigetor Kit (50): purchased from qiagen, germany; the goods number is: 5650;

enhancer buffer NB (1N): a PCR reaction enhancer purchased under the name NB of agitta conggins; the goods number is: MT017035

IGT-EM707polymerase mix: a DNA polymerase cocktail available from Aijiekang under the designation EM 707; the goods number is: MT 017035;

YF Buffer B: magnetic bead rinse buffer purchased from agutazone under the name YF; the goods number is: MT 017035;

the above reagent is Beijing Aikitikang (iGeneTech)^TM) The customized composition has the following goods number: MT017035

Primer or Index sequence: hodgkin Eitykang Co., Ltd (iGeneTech)^TM) Performing customized synthesis;

1. detecting a sample: 108 unrelated individual samples from the Peking Han nationality, including 48 blood samples and 60 FTA blood cards.

2. Detecting an object: the typing system and the kit described in embodiments 1 to 7 are respectively adopted to detect a detection sample according to a detection flow.

3. And (3) detection flow:

(1) the whole genome DNA was extracted from the blood sample and FTA blood card using tissue, blood DNA extraction Kit and QIAampDNAinvestigator Kit (50), respectively. The concentration of the genomic DNA was measured by using a nucleic acid quantitative analyzer at 1, 5, 10 and 20 ng/. mu.L.

(2) Multiplex PCR library construction:

library construction was performed using 1, 5, 10, 20 ng/. mu.L of genomic DNA as described above, with library numbers F01, F02, F03 and F04, respectively.

A. First round multiplex PCR reaction: preparing reaction mixed solution according to the table 7, wherein each tube is 25 mu L, and performing multiple PCR reaction according to the reaction conditions of the table 8 to obtain multiple PCR products; wherein the primer combinations referred to in the different detection assays refer to the primer combinations of examples 1-7, respectively;

table 7: first round multiple PCR reaction system

Reagent	Volume (μ L)
		Double distilled water	4
Enhancer buffer NB(1N)	7
		Primer combination	8
Genomic DNA	1
		IGT-EM707polymerase mixture	5

Table 8: conditions for the first round of multiplex PCR reactions

First round magnetic bead purification:

adding 23 μ L of AMPure XP magnetic beads balanced at room temperature into 25 μ L of multiplex PCR products, sucking and beating the mixture for several times by using a pipettor, after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3min, completely removing the supernatant, taking the PCR tube off the magnetic frame, adding 40 μ L of YF buffer B (magnetic bead rinsing buffer), sucking and beating the mixture for several times by using a pipettor, incubating for 5min at room temperature, removing the supernatant, adding 180 μ L of 80% ethanol solution, standing for 30s, completely removing the supernatant, standing for 3min at room temperature to completely volatilize the residual ethanol, adding 24 μ L of nucleic-free water or 1 × TE buffer (pH 8.0), sucking and beating and re-suspending the mixture by using a pipettor gently to avoid generating bubbles, standing for 2min at room temperature, placing the PCR tube on the magnetic frame again and standing for 3min, and (4) sucking the supernatant by using a liquid transfer machine, and transferring the supernatant into a new PCR tube, wherein the supernatant in the tube is the purified multiplex PCR product.

B. Second round multiplex PCR reaction: b, taking the purified multiplex PCR product obtained in the step A as a template, performing a second round of multiplex PCR reaction, inserting an Index adaptor sequence, wherein the system of the second round of multiplex PCR reaction is shown in Table 9, and the reaction conditions are shown in Table 10;

table 9: second round multiplex PCR reaction system

Reagent	Volume (μ L)
		Purified multiplex PCR product	18
IGT-I5Index(10μM)	1
		IGT-I7Index(10μM)	1
IGT-EM707polymerase mixture	5

Table 10: second round of multiplex PCR reaction conditions

And (3) second round of magnetic bead purification:

the specific operation is the same as the first round of magnetic bead purification, and the supernatant transferred to a new PCR tube in the last step is a prepared multiplex PCR library.

C. And (3) carrying out quantitative and quality detection on the obtained multiplex PCR library:

taking 1 mu L of the multiple PCR library obtained in the step B, measuring the library concentration by using a nucleic acid quantifier, and recording the library concentration;

and (4) taking 1 mu L of the multiple PCR library obtained in the step (B), and detecting the length and the purity of the library fragment by using a full-automatic nucleic acid protein analysis system, wherein the distribution interval of the target fragment of the normal library is 300bp-450bp, and the main peak is about 339 bp.

(3) Performing second-generation sequencing on the multiple PCR library obtained in the step (2), performing quality control on a fastq file obtained by sequencing by using FASTQC software, filtering data by using Trimmomatic software, trimming a sequence lower than Q30, removing a sequence with a sequencing fragment lower than 100bp, typing the STR on the fastq file with STRaitRazor3.0 after quality control, and finally obtaining an STR typing file corresponding to each individual.

4. And (3) detection results:

(1) the typing system and the kit disclosed in the embodiments 1 to 7 can accurately realize typing and genetic relationship identification for 108 samples.

(2) The quality control peak patterns of the libraries prepared by using the primer combinations disclosed in examples 1 to 7 are shown in FIGS. 1 to 7: the results show that the 7 STR typing systems and the kits thereof provided by the invention can simultaneously amplify the 60, 58, 61, 118, 121, 119 or 179 STR loci at one time, have good balance, good primer specificity and high sensitivity, and can effectively detect gDNA with the concentration as low as 1 ng/. mu.L.

(3) Effective typing information can be obtained for 108 samples by using the typing system and the kit disclosed in the embodiments 1 to 7, and in order to reasonably simplify the application document, typing information of 3 samples is listed:

analysis software: software STRAIT Razor3.0

Interpretation of the tables: the lower blank represents that the site of the sample is homozygote, and the two types represent heterozygote; the number of reads of the sample after each typing represents the coverage of the typing corresponding to the site obtained by using the configuration file of the STRait Razor 3.0.

The typing results information of STR loci in example 1 in 3 samples are as follows:

the typing results information of STR loci in example 2 in 3 samples are as follows:

typing results information of STR loci in example 3 in 3 samples is as follows:

example 4 the typing results of the above 3 samples are equivalent to the combination of the results of example 1 and example 2; example 5 the typing results of the above 3 samples are equivalent to the combination of the results of example 1 and example 3; example 6 the typing results of the above 3 samples are equivalent to the combination of the results of example 2 and example 3; example 7 the typing results of the above 3 samples are equivalent to the combination of the results of example 1, example 2 and example 3; to avoid redundancy, the tables are not repeated here.

(4) And typing efficiency:

the forensic parameters of the 60, 58, 61, 118, 121, 119, 179 STR sites contained in the typing systems and kits disclosed in examples 1-7, respectively, are as follows:

an analysis tool: STRAF 1.0.5 (STR Analysis for Forensecs);

analysis data sources: typing result information of the 108 Beijing Han nationality irrelevant individual samples;

interpretation of the tables: locus denotes the name of the STR Locus; n represents the number of alleles, and the total number of 108 Beijing Han nationality unrelated individuals exist, so that each STR genetic marker has 216 alleles; nall represents the total number of genotypes typed at each locus, the range of typing is 5-19; GD (Genetic diversity), ranging from 0.4911 to 0.9013; PIC (polymorphic Information Content) with a range of 0.4368-0.8880; PM (match probability), which ranges from 0.0297-0.5111; PD (power of diagnosis, personal identification probability), its range is 0.4889-0.9703.

(5) Library concentration:

the RFU (fluorescence signal intensity) values of the gDNA initial amounts were slightly lower at 1ng and 5ng, but both were normal main peaks, and when the gDNA initial amounts were 10ng and 20ng, not only the library was mainly normal main peaks but also the RFU values were better.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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aaaccccgtc tgtacaaaaa tatt 24

<210>5

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>5

ctaaagaccc caccaacaaa atttt 25

<210>6

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>6

acacataaac tagttatata tttgtgggtt atat 34

<210>7

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>7

actcctgctt accactattc tggct 25

<210>8

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>8

gtatgtactc ctgagaatct gattctcag 29

<210>9

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>9

aaattttaaa tcttctgaag gaaaagtgtt ctgatc 36

<210>10

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>10

taacttgaga ccataagttc acaaatggcc tg 32

<210>11

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>11

ctacttatgt tgaatggtca ttaagtcaaa gttt 34

<210>12

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>12

cacaagaaca ctgactaata cactatacca ca 32

<210>13

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>13

actgctgtca acacattgta tct 23

<210>14

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>14

aatagtcact gctatactaa aatgaataaa ttggt 35

<210>15

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>15

ctgctctcaa ggtgaatcta aactac 26

<210>16

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>16

agttcttttg ctctggtagt gaaattg 27

<210>17

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>17

attctgctcg cactgtagtc caa 23

<210>18

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>18

cctgtctcca gagctttgtt tt 22

<210>19

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>19

cttcatcatg tgagtcaatt ctgcaga 27

<210>20

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>20

actatgattc atttccatag ggtaag 26

<210>21

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>21

ttcctccgtg taatgctata tctagcat 28

<210>22

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>22

tcatgaaggg attttaggaa ctgatacgat g 31

<210>23

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>23

atctcacttc caaaggaaat ctttgt 26

<210>24

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>24

agaattaagt ccatttacgt tcaatgttat 30

<210>25

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>25

attagtcagg gttctccaaa gagactg 27

<210>26

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>26

gtacttgtat cacaacacat aacaacat 28

<210>27

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>27

aactaattct gtggttcctg taatatgatt 30

<210>28

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>28

gttctttaga aaaacacaac taatcagatg 30

<210>29

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>29

aggcacctag gtttgttctg aaggt 25

<210>30

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>30

aagtctttta cccattcttt ttatttgaat tgttg 35

<210>31

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>31

gaggaaataa agagggcctt tccctt 26

<210>32

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>32

gatggattga tttaaaggga attatatata gatata 36

<210>33

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>33

catcatgggg ttatgaagct ttggcct 27

<210>34

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>34

ctaggtaaat tctacattat ccctttggtt t 31

<210>35

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>35

aattttctga gttagccatg aaagattctc 30

<210>36

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>36

aggatatggt ttggaagaaa caagattt 28

<210>37

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>37

atagagcgag accccctctc 20

<210>38

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>38

tcaggaacac tcaactgctc tataagc 27

<210>39

<211>37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>39

gtagatttta tatatataca cacacgctat atatatt 37

<210>40

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>40

gagagattga aattaatata tataaatgaa ttata 35

<210>41

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>41

cattgtccat ttttctacag taccattagt cttt 34

<210>42

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>42

ttctctttct cattacacct atgtgaaacc a 31

<210>43

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>43

ggaggatcat ttgagaattt gagaccag 28

<210>44

<211>37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>44

attgggtaat tatctcactc atttttatta gtttttg 37

<210>45

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>45

cagagaaaca gaacttgtag gatatatag 29

<210>46

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>46

gattagaaca ttgtattatt caaggctttc 30

<210>47

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>47

gttcaggcct caatatatac ctgtattt 28

<210>48

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>48

atatctgaga cttgtagtag aaggccttga g 31

<210>49

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>49

tgttatctca agggtactta gaatctttat g 31

<210>50

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>50

ccagtcattc atttaaatag ataaagataa atgaaa 36

<210>51

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>51

acccatctaa cgcctatctg tattt 25

<210>52

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>52

caattcccct actgcctaga cacctt 26

<210>53

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>53

tcccagcact cacaatcttg tgag 24

<210>54

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>54

ggacttgtaa ggctccacaa ttg 23

<210>55

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>55

attggcagac tcacttacaa ataaaacatt c 31

<210>56

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>56

agaacacaag atcacatctg ttggcca 27

<210>57

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>57

ttccttgtgt acttgttact ggtaattttt 30

<210>58

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>58

tcctaacttt tgaggcaatt tgttacaga 29

<210>59

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>59

gaaggtagag aaatactgag gaaaaagc 28

<210>60

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>60

tagacagaca gatagacaga tcttaac 27

<210>61

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>61

cacctataac caatatagaa aatgaggcag g 31

<210>62

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>62

ttccgttctc tggagaaccc 20

<210>63

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>63

tctatctcca gttggctctg tttctct 27

<210>64

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>64

tcctagtagc ctcttcctac aggctt 26

<210>65

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>65

tactaatagt gaactacctg caaaagaaat taagaa 36

<210>66

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>66

ctgtgttctc ttttctccat ttggtta 27

<210>67

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>67

ttacaacatt tctgtatctc tggggtagg 29

<210>68

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>68

tgggtaactt gtgtgtcact gag 23

<210>69

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>69

tgtctgtctg agtttggaaa tgttta 26

<210>70

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>70

attgagggtg cagtaagcta tgatca 26

<210>71

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>71

ttccaaggcc aggtacagtg gct 23

<210>72

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>72

tttactatga agaagcagct cacacaat 28

<210>73

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>73

ttagtcaata aaagcctttc catgaaca 28

<210>74

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>74

tcactgcctt cctcatatcc ta 22

<210>75

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>75

aacattatat agaatattat acaaaaatga gcaaga 36

<210>76

<211>21

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>76

tccctgagct gccacaatcc t 21

<210>77

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>77

aactttaaac aggtcattta agctctctgg gc 32

<210>78

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>78

agggcagaaa ccactgttcc 20

<210>79

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>79

tctctggaga acatggacta aaacagcc 28

<210>80

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>80

gatgaattga agttgaagtt tattgttaat ttagt 35

<210>81

<211>18

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>81

caagggtcag ggatgcca 18

<210>82

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>82

ctctgtatag ctattgttct aagtgttg 28

<210>83

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>83

aatggctgta ttaatagtgg gaaagaca 28

<210>84

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>84

tttcttcttt ctgtgaaggt tattttctcg g 31

<210>85

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>85

ggatttatta ggtaaattgg ttcacgtgat 30

<210>86

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>86

atgtatgtct gtctgtctat ctatttgtct ca 32

<210>87

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>87

gaagtaaatt ctatttatct gctcccttaa atatg 35

<210>88

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>88

tatgataagg aattagctca cactgttatg gag 33

<210>89

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>89

tattttccag ctttattgag gtattattgg taa 33

<210>90

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>90

gtttgttaca taggtttaca acatgccat 29

<210>91

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>91

ctaccttttc tatcctcaac cccac 25

<210>92

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>92

agatgagaat gaggcactgg ag 22

<210>93

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>93

catccactca tctaaaataa aatctcatga aa 32

<210>94

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>94

cccttgctac ctttgaaaag actt 24

<210>95

<211>19

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>95

tcccacacca cctgccttt 19

<210>96

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>96

atatgcatac tcaccaaagt cattcaattc a 31

<210>97

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>97

caccagcaat aataaaactt agcaaccta 29

<210>98

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>98

tatccatcca tccacccgtt tttgt 25

<210>99

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>99

ggttcaggtt ctgtggaaaa ccct 24

<210>100

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>100

aggggaagag tagatacaaa ggaa 24

<210>101

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>101

aaatgtgtcc atgtttttca gtattacttt t 31

<210>102

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>102

gtttaaattt gcttctggat attggcac 28

<210>103

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>103

ttctctgaaa ggtactgtca agaaaat 27

<210>104

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>104

cagtgtttac tattagaggt gttttgggtc 30

<210>105

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>105

ctcaagcagt tattttggct gacata 26

<210>106

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>106

ttcaatacta catcatttac aagctagaga ac 32

<210>107

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>107

ataaggaaaa tgacacccca gccc 24

<210>108

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>108

actgtcttgt tgaagttggc agtaggg 27

<210>109

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>109

ttttgaatgt gtgtaactaa gggaagaatc t 31

<210>110

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>110

agagaaagag agacagacag aca 23

<210>111

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>111

caagctctct gaatatgttt tgaaaataat gtat 34

<210>112

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>112

tgtgaatatc gatgaatgtc aataaaagat tgt 33

<210>113

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>113

tgttcatatt atctctttta ttttgagttc tttgc 35

<210>114

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>114

ttctctggaa aactctaata aaagtatcaa aga 33

<210>115

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>115

aatgttcctt agtcccacct ttctaaga 28

<210>116

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>116

atgagtaagt aggcagtgtg catgtg 26

<210>117

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>117

gcatccttag ggacagggtt gg 22

<210>118

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>118

ttttgttttt ggtaccaggt acatt 25

<210>119

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>119

atcatccttt cctctctttc tagtgcaa 28

<210>120

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>120

gctgatggaa cctgcttgcc 20

<210>121

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>121

cctctctttt tcaatctcta gatagataaa tgtta 35

<210>122

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>122

aaattcatca gtgctatgtg gaataaaaaa 30

<210>123

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>123

agaaacaagt agtcaaagga gcctttta 28

<210>124

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>124

atagtcttta atagtctagt ctgttttgga taac 34

<210>125

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>125

tagggataag ttaggacaaa taaaaaatg 29

<210>126

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>126

gtcaattcct tgttataaaa ttatatatac atatat 36

<210>127

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>127

tactatcaag aaaacaagaa tatttcagaa gaata 35

<210>128

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>128

aaaattgagc tactgatctt aactacaaag aac 33

<210>129

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>129

ggcgtgtatc tgtagtccta gttac 25

<210>130

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>130

aataagataa gagtgtctgg ctcatagaaa 30

<210>131

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>131

ctaatgcacc caacattcta ac 22

<210>132

<211>37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>132

aaccattgat atataggaat catgtgaatt atatctc 37

<210>133

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>133

agtggattca tgcagttcaa atccatgct 29

<210>134

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>134

cacaggatca gagatgcaaa ta 22

<210>135

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>135

ctgcctgaac aaagtaatga aagtgg 26

<210>136

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>136

aattctttgc aagttctagg aagagttaaa a 31

<210>137

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>137

ataatgagat gaaagaaaga gagaaagaga ga 32

<210>138

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>138

accacatgag ccaattctgt ataat 25

<210>139

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>139

ggaaaagaaa tcagtatgtc aaagagatat ct 32

<210>140

<211>21

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>140

agccccagga ggattatatt t 21

<210>141

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>141

tggcaaaact ctgttagcta ttta 24

<210>142

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>142

ttaaaatacc tcaatatgcc acttcataaa cgtat 35

<210>143

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>143

aggctgacag tttaccatgt agac 24

<210>144

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>144

agccagaata tcatgagcag agaactgg 28

<210>145

<211>21

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>145

catctttgct tggcacactt c 21

<210>146

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>146

cattaaaaac attatgaata aaaactgaaa aattc 35

<210>147

<211>37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>147

ctctttaaaa ttttattgta tctcaggtta tcttttt 37

<210>148

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>148

aaagaacaga atcaattgta tatgtatata gatat 35

<210>149

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>149

atgtgaaagg cattgtatta gtgttct 27

<210>150

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>150

gtctttttaa ataataataa taatacccat gataa 35

<210>151

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>151

gtatgttatg agctgagtac attctagg 28

<210>152

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>152

tggataaaag gcagctgtag ctg 23

<210>153

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>153

atggggcctt ggaaagtata ttagt 25

<210>154

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>154

agattcaaca gtggcaaact tttt 24

<210>155

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>155

gatatggaat tgacctaagt atccattaat gga 33

<210>156

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>156

attgcaatat tcctgaccta aagaaaacat aa 32

<210>157

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>157

tgctaaataa cttacacaag ctatcttaag tt 32

<210>158

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>158

tctgcaactc tctgaacata tcttcaa 27

<210>159

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>159

atctcacacc tccctctctt cc 22

<210>160

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>160

accatatagt caggttgtgt tttcataatt tg 32

<210>161

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>161

gccagcctgc cttacaggtt 20

<210>162

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>162

aagaggggaa tcaaattcaa ccaat 25

<210>163

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>163

caagccataa tctcacctca gctt 24

<210>164

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>164

ttcagtccat tttccaccag agaa 24

<210>165

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>165

ttcagtaatt ctgctgtctc tcccatta 28

<210>166

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>166

atttcagagt gaacagtccc acagt 25

<210>167

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>167

aagaaaaaca tctcccttct gttttta 27

<210>168

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>168

caggcctata tacacaccta tat 23

<210>169

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>169

taccatataa gatgctcaat agatagaaat 30

<210>170

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>170

aatggtatac ataaattccc atttctgaaa a 31

<210>171

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>171

ttttgagtag gcttgcaatc tcttacttt 29

<210>172

<211>18

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>172

cgagtcaggc gagttttt 18

<210>173

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>173

gtacaaaatt taggaaaaat taagcaaaac cctac 35

<210>174

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>174

atgtgaaatt tggctttacg ctaaatt 27

<210>175

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>175

ttttgggtat ctagcctgcc aagag 25

<210>176

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>176

acattatgtc tacctatatt tatctctatt tcttt 35

<210>177

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>177

tcaccaatta ttaacgtttt gctcaatttg 30

<210>178

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>178

actctctgcc cattctgaac tttaca 26

<210>179

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>179

agtacatact cacacgcaca cagac 25

<210>180

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>180

ttttgtaggt ggcaacaggc cat 23

<210>181

<211>36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>181

aggataatag tacaaatgat aatgatgatg atgctc 36

<210>182

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>182

agtagatagg ggaagcctgt ta 22

<210>183

<211>37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>183

gtactaaaat tattaaaaat cttattcaat gagctaa 37

<210>184

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>184

ctctggagaa ctctaatgca gttgc 25

<210>185

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>185

gggaataaaa agcaaaatat tcaagataat gacta 35

<210>186

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>186

cctgcttagg aatccaaata agaaac 26

<210>187

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>187

atagtcctgc tttcctattt gtactgttca 30

<210>188

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>188

ccatgctcat tatttgtaag ttgtaagaaa a 31

<210>189

<211>32

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>189

gattaatcat aaacatttgg gaaggagagt ga 32

<210>190

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>190

ggtatgtgtt cttgagcctc ctga 24

<210>191

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>191

tgtacatcct ctgcatccgt ttttt 25

<210>192

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>192

atcctcctaa gagaccattt atgaaacaat g 31

<210>193

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>193

acctaatgat tggatatgga ggtgag 26

<210>194

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>194

taacaactct aggaaaagaa ttacaggggg t 31

<210>195

<211>29

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>195

gacttcacca aactgatatt tcgaagtct 29

<210>196

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>196

gtttacttac aatattccct tctaggtaac 30

<210>197

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>197

ataaagctgg agaacatgtc atgtg 25

<210>198

<211>18

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>198

ctgtctgcgc tggccttt 18

<210>199

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>199

gcagggtgat agaaaggtag aaaagg 26

<210>200

<211>28

<212>DNA

<213> Artificial sequence (artificailseq)

<400>200

aaaaacatac taatcaacca aggcaatt 28

<210>201

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>201

ccaactatct gctatctgga aaacc 25

<210>202

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>202

atacaaacgc tcctctgact gccac 25

<210>203

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>203

ccaacaaaag gactcaccca tca 23

<210>204

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>204

ccaacaaaag gactcaccca tca 23

<210>205

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>205

gggaattggc tcaatcacag agac 24

<210>206

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>206

tataaagaat tggcttacat aattatggat gctga 35

<210>207

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>207

gagtgccaat atttgaggac agaag 25

<210>208

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>208

tattctgtct gacatcaagc tactac 26

<210>209

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>209

gatgatacag tgaggttggg gtatgc 26

<210>210

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>210

tctcacccca tgttcttccc acc 23

<210>211

<211>20

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>211

gaagcccagg gaagacaata 20

<210>212

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>212

aactatttca atcattggca gatattatga ctt 33

<210>213

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>213

tgaaatcatg tcttttgcag caaca 25

<210>214

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>214

ggtgtccttg ctcattcctg ggc 23

<210>215

<211>24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>215

ccatgttatc ttctgggatg caaa 24

<210>216

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>216

cattcctctt ttgatgaaca tttaggttct t 31

<210>217

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>217

ttaaaaattc ctcaaactgg ctgggtgt 28

<210>218

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>218

tcttcaatga gtccgtagtc ttaagaat 28

<210>219

<211>27

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>219

tatagtgtca tctgtttcag cctgaga 27

<210>220

<211>19

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>220

tcacccagct ggagtgcag 19

<210>221

<211>23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>221

gtttccctaa gttgcccaga ctg 23

<210>222

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>222

ttatactaag cttctcgagg gttggagg 28

<210>223

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>223

gctattttta tcacggatgt tacatttcat 30

<210>224

<211>34

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>224

cacagtgtta tatttacaca aacctagatt ggtc 34

<210>225

<211>28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>225

tttagttctg actctgtcac ctaggctg 28

<210>226

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>226

gggttgcttt taaacctttg tttaa 25

<210>227

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>227

gtcctggtgt catgctttta aagat 25

<210>228

<211>33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>228

ttgataattt tacaaagatt ctctgcccta cag 33

<210>229

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>229

cttatctctc tctacttgtt ttgtagttac 30

<210>230

<211>35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>230

gtttctattt ttcaggcata aataaattta ctaag 35

<210>231

<211>21

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>231

gcagtgacgc acctaacact c 21

<210>232

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>232

cacatctaaa catgcataca cacataaacg a 31

<210>233

<211>31

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>233

ggagaaccct aaaacaaaat aaaaatctat t 31

<210>234

<211>25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>234

tttctggaga accctgacta ataca 25

<210>235

<211>26

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>235

tgcacacata acctttgatc tgcaga 26

<210>236

<211>30

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>236

atcttggtga ataaaataaa gccaggaaag 30

Claims

1. A kit for identifying complex genetic relationship is characterized in that the kit comprises reagents for 60 linked autosomes, wherein the reagents comprise a PCR primer combination 1 for amplifying 60 linked autosomes STR loci;

the 60 linked autosomal STR loci are D1S2131, D1S3721, D1S2130, D1S1600, D1S1653, D1S1660, D1S3732, D2S1364, D2S2734, D2S1396, D2S428, D2S435, D2S1387, D2S1792, D2S1399, D2S2959, D3S2431, D3S4547, D4S1643, D4S2408, D4S3326, D4S2368, D5S2845, D5S1473, D5S813, D5S1716, D5S1459, D5S1487, D5S1466, D5S2496, D5S2501, D6S1019, D6S2417, D6S2412, D6S 821, D6S 1487, D5S1466, D2S 1466, D5S2496, D5S 2501S 1468, D2S 1469, D2S 14611, D2S 1468, D2S 14611, D2S 1469, D2S 1468, D2S 14611, D2S 24, D2S 1469, D2S 24, D2S 1469, D2S 24, D2S 9, D2S 1469, D2S 24, D;

the PCR primer combination 1 comprises a forward primer and a reverse primer; the forward primers are as follows:

STR site sequence number Forward primer name Primer sequence (5 '-3') 1 D1S2131_F SEQ ID NO:1 2 D1S3721_F SEQ ID NO:2 3 D1S2130_F SEQ ID NO:3 4 D1S1600_F SEQ ID NO:4 5 D1S1653_F SEQ ID NO:5 6 D1S1660_F SEQ ID NO:6 7 D1S3732_F SEQ ID NO:7 8 D2S1364_F SEQ ID NO:8 9 D2S2734_F SEQ ID NO:9 10 D2S1396_F SEQ ID NO:10 11 D2S428_F SEQ ID NO:11 12 D2S435_F SEQ ID NO:12 13 D2S1387_F SEQ ID NO:13 14 D2S1792_F SEQ ID NO:14 15 D2S1399_F SEQ ID NO:15 16 D2S2959_F SEQ ID NO:16 17 D3S2431_F SEQ ID NO:17 18 D3S4547_F SEQ ID NO:18 19 D4S1643_F SEQ ID NO:19 20 D4S2408_F SEQ ID NO:20 21 D4S3326_F SEQ ID NO:21 22 D4S2368_F SEQ ID NO:22 23 D5S2845_F SEQ ID NO:23 24 D5S1473_F SEQ ID NO:24 25 D5S813_F SEQ ID NO:25 26 D5S1716_F SEQ ID NO:26 27 D5S1459_F SEQ ID NO:27 28 D5S1487_F SEQ ID NO:28 29 D5S1466_F SEQ ID NO:29 30 D5S2496_F SEQ ID NO:30 31 D5S2501_F SEQ ID NO:31 32 D6S1019_F SEQ ID NO:32 33 D6S2417_F SEQ ID NO:33 34 D6S2412_F SEQ ID NO:34 35 D6S1284_F SEQ ID NO:35 36 D7S1820_F SEQ ID NO:36 37 D7S3050_F SEQ ID NO:37 38 D7S821_F SEQ ID NO:38 39 D8S594_F SEQ ID NO:39 40 D8S1468_F SEQ ID NO:40 41 D8S2322_F SEQ ID NO:41 42 D8S569_F SEQ ID NO:42 43 D8S1475_F SEQ ID NO:43 44 D9S746_F SEQ ID NO:44 45 D9S319_F SEQ ID NO:45 46 D9S2149_F SEQ ID NO:46 47 D11S2010_F SEQ ID NO:47 48 D11S1392_F SEQ ID NO:48 49 D12S376_F SEQ ID NO:49 50 D12S1052_F SEQ ID NO:50 51 D13S317_F SEQ ID NO:51 52 D13S790_F SEQ ID NO:52 53 D14S1432_F SEQ ID NO:53 54 D14S1428_F SEQ ID NO:54 55 D15S644_F SEQ ID NO:55 56 D15S1507_F SEQ ID NO:56 57 D16S752_F SEQ ID NO:57 58 D16S485_F SEQ ID NO:58 59 D20S481_F SEQ ID NO:59 60 D20S1151_F SEQ ID NO:60

；

The reverse primers are as follows:

STR site sequence number Reverse primer name Primer sequence (5 '-3') 1 D1S2131_R SEQ ID NO:61 2 D1S3721_R SEQ ID NO:62 3 D1S2130_R SEQ ID NO:63 4 D1S1600_R SEQ ID NO:64 5 D1S1653_R SEQ ID NO:65 6 D1S1660_R SEQ ID NO:66 7 D1S3732_R SEQ ID NO:67 8 D2S1364_R SEQ ID NO:68 9 D2S2734_R SEQ ID NO:69 10 D2S1396_R SEQ ID NO:70 11 D2S428_R SEQ ID NO:71 12 D2S435_R SEQ ID NO:72 13 D2S1387_R SEQ ID NO:73 14 D2S1792_R SEQ ID NO:74 15 D2S1399_R SEQ ID NO:75 16 D2S2959_R SEQ ID NO:76 17 D3S2431_R SEQ ID NO:77 18 D3S4547_R SEQ ID NO:78 19 D4S1643_R SEQ ID NO:79 20 D4S2408_R SEQ ID NO:80 21 D4S3326_R SEQ ID NO:81 22 D4S2368_R SEQ ID NO:82 23 D5S2845_R SEQ ID NO:83 24 D5S1473_R SEQ ID NO:84 25 D5S813_R SEQ ID NO:85 26 D5S1716_R SEQ ID NO:86 27 D5S1459_R SEQ ID NO:87 28 D5S1487_R SEQ ID NO:88 29 D5S1466_R SEQ ID NO:89 30 D5S2496_R SEQ ID NO:90 31 D5S2501_R SEQ ID NO:91 32 D6S1019_R SEQ ID NO:92 33 D6S2417_R SEQ ID NO:93 34 D6S2412_R SEQ ID NO:94 35 D6S1284_R SEQ ID NO:95 36 D7S1820_R SEQ ID NO:96 37 D7S3050_R SEQ ID NO:97 38 D7S821_R SEQ ID NO:98 39 D8S594_R SEQ ID NO:99 40 D8S1468_R SEQ ID NO:100 41 D8S2322_R SEQ ID NO:101 42 D8S569_R SEQ ID NO:102 43 D8S1475_R SEQ ID NO:103 44 D9S746_R SEQ ID NO:104 45 D9S319_R SEQ ID NO:105 46 D9S2149_R SEQ ID NO:106 47 D11S2010_R SEQ ID NO:107 48 D11S1392_R SEQ ID NO:108 49 D12S376_R SEQ ID NO:109 50 D12S1052_R SEQ ID NO:110 51 D13S317_R SEQ ID NO:111 52 D13S790_R SEQ ID NO:112 53 D14S1432_R SEQ ID NO:113 54 D14S1428_R SEQ ID NO:114 55 D15S644_R SEQ ID NO:115 56 D15S1507_R SEQ ID NO:116 57 D16S752_R SEQ ID NO:117 58 D16S485_R SEQ ID NO:118 59 D20S481_R SEQ ID NO:119 60 D20S1151_R SEQ ID NO:120

；

The working concentration of the forward primer and the reverse primer in the PCR primer is 0.1 mu M;

the kit further comprises an Index linker sequence and a DNA polymerase; the Index linker sequences include IGT-I5Index and IGT-I7Index, both at 10. mu.M working concentration.

2. The kit of claim 1, further comprising a PCR primer combination 2 for amplifying the 58 linked autosomal STR loci;

the 58 linked autosomal STR loci are D1S3736, D1S1665, D1S2127, D1S2138, D1S1642, D1S518, D1S1604, D1S3729, D1S3727, D2S1336, D2S1779, D2S1771, D2S437, D2S2970, D2S2944, D2S1327, D3S2402, D3S1766, D4S2411, D4S3351, D4S243, D4S2426, D4S2373, D5S1490, D5S2856, D5S2495, D5S2855, D5S1722, D5S1725, D6S1048, D6S1275, D7S796, D7S1799, D8S 174, D2320, D8S 2320, D745S 1249, D4S 1249S 1725, D2S 1725S 13611, D2S 13611, D11411, D3S 10614, D3S 11411, D3S 10611, D3S 11411;

the PCR primer combination 2 comprises a forward primer and a reverse primer;

the forward primers are as follows:

STR site sequence number Forward primer name Primer sequence (5 '-3') 61 D1S3736_F SEQ ID NO:121 62 D1S1665_F SEQ ID NO:122 63 D1S2127_F SEQ ID NO:123 64 D1S2138_F SEQ ID NO:124 65 D1S1642_F SEQ ID NO:125 66 D1S518_F SEQ ID NO:126 67 D1S1604_F SEQ ID NO:127 68 D1S3729_F SEQ ID NO:128 69 D1S3727_F SEQ ID NO:129 70 D2S1336_F SEQ ID NO:130 71 D2S1779_F SEQ ID NO:131 72 D2S1771_F SEQ ID NO:132 73 D2S437_F SEQ ID NO:133 74 D2S2970_F SEQ ID NO:134 75 D2S2944_F SEQ ID NO:135 76 D2S1327_F SEQ ID NO:136 77 D3S2402_F SEQ ID NO:137 78 D3S1766_F SEQ ID NO:138 79 D4S2411_F SEQ ID NO:139 80 D4S3351_F SEQ ID NO:140 81 D4S243_F SEQ ID NO:141 82 D4S2426_F SEQ ID NO:142 83 D4S2373_F SEQ ID NO:143 84 D5S1490_F SEQ ID NO:144 85 D5S2856_F SEQ ID NO:145 86 D5S2495_F SEQ ID NO:146 87 D5S2855_F SEQ ID NO:147 88 D5S1722_F SEQ ID NO:148 89 D5S1725_F SEQ ID NO:149 90 D6S1048_F SEQ ID NO:150 91 D6S1275_F SEQ ID NO:151 92 D7S796_F SEQ ID NO:152 93 D7S1799_F SEQ ID NO:153 94 D8S2324_F SEQ ID NO:154 95 D8S2330_F SEQ ID NO:155 96 D8S1144_F SEQ ID NO:156 97 D9S745_F SEQ ID NO:157 98 D9S301_F SEQ ID NO:158 99 D9S1124_F SEQ ID NO:159 100 D10S1246_F SEQ ID NO:160 101 D10S2485_F SEQ ID NO:161 102 D11S1983_F SEQ ID NO:162 103 D11S2363_F SEQ ID NO:163 104 D11S1368_F SEQ ID NO:164 105 D12S393_F SEQ ID NO:165 106 D12S1063_F SEQ ID NO:166 107 D13S1807_F SEQ ID NO:167 108 D13S1492_F SEQ ID NO:168 109 D14S738_F SEQ ID NO:169 110 D14S301_F SEQ ID NO:170 111 D14S583_F SEQ ID NO:171 112 D15S816_F SEQ ID NO:172 113 D15S1514_F SEQ ID NO:173 114 D18S872_F SEQ ID NO:174 115 D18S972_F SEQ ID NO:175 116 D18S548_F SEQ ID NO:176 117 D20S1145_F SEQ ID NO:177 118 D20S477_F SEQ ID NO:178

；

The reverse primers are as follows:

STR site sequence number Reverse primer name Primer sequence (5 '-3') 61 D1S3736_R SEQ ID NO:179 62 D1S1665_R SEQ ID NO:180 63 D1S2127_R SEQ ID NO:181 64 D1S2138_R SEQ ID NO:182 65 D1S1642_R SEQ ID NO:183 66 D1S518_R SEQ ID NO:184 67 D1S1604_R SEQ ID NO:185 68 D1S3729_R SEQ ID NO:186 69 D1S3727_R SEQ ID NO:187 70 D2S1336_R SEQ ID NO:188 71 D2S1779_R SEQ ID NO:189 72 D2S1771_R SEQ ID NO:190 73 D2S437_R SEQ ID NO:191 74 D2S2970_R SEQ ID NO:192 75 D2S2944_R SEQ ID NO:193 76 D2S1327_R SEQ ID NO:194 77 D3S2402_R SEQ ID NO:195 78 D3S1766_R SEQ ID NO:196 79 D4S2411_R SEQ ID NO:197 80 D4S3351_R SEQ ID NO:198 81 D4S243_R SEQ ID NO:199 82 D4S2426_R SEQ ID NO:200 83 D4S2373_R SEQ ID NO:201 84 D5S1490_R SEQ ID NO:202 85 D5S2856_R SEQ ID NO:203 86 D5S2495_R SEQ ID NO:204 87 D5S2855_R SEQ ID NO:205 88 D5S1722_R SEQ ID NO:206 89 D5S1725_R SEQ ID NO:207 90 D6S1048_R SEQ ID NO:208 91 D6S1275_R SEQ ID NO:209 92 D7S796_R SEQ ID NO:210 93 D7S1799_R SEQ ID NO:211 94 D8S2324_R SEQ ID NO:212 95 D8S2330_R SEQ ID NO:213 96 D8S1144_R SEQ ID NO:214 97 D9S745_R SEQ ID NO:215 98 D9S301_R SEQ ID NO:216 99 D9S1124_R SEQ ID NO:217 100 D10S1246_R SEQ ID NO:218 101 D10S2485_R SEQ ID NO:219 102 D11S1983_R SEQ ID NO:220 103 D11S2363_R SEQ ID NO:221 104 D11S1368_R SEQ ID NO:222 105 D12S393_R SEQ ID NO:223 106 D12S1063_R SEQ ID NO:224 107 D13S1807_R SEQ ID NO:225 108 D13S1492_R SEQ ID NO:226 109 D14S738_R SEQ ID NO:227 110 D14S301_R SEQ ID NO:228 111 D14S583_R SEQ ID NO:229 112 D15S816_R SEQ ID NO:230 113 D15S1514_R SEQ ID NO:231 114 D18S872_R SEQ ID NO:232 115 D18S972_R SEQ ID NO:233 116 D18S548_R SEQ ID NO:234 117 D20S1145_R SEQ ID NO:235 118 D20S477_R SEQ ID NO:236

The working concentration of the forward primer and the reverse primer in the PCR primer is 0.1 mu M.

3. The kit of claim 1 or 2, wherein the kit further comprises reagents for preparing genomic DNA into a library for sequencing.