CN114574595B - Application of human chromosome InDel gene locus, primer group, product thereof and individual identification method of test material - Google Patents
Application of human chromosome InDel gene locus, primer group, product thereof and individual identification method of test material Download PDFInfo
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Abstract
The invention provides an application of a human chromosome InDel gene locus, a primer group, a product thereof and an individual identification method of a test material, and relates to the technical field of biology. The invention provides application of 66InDel gene loci of human chromosomes in individual identification or paternity identification. The inventor autonomously screens 66InDel loci from a dbSNP database, evaluates the group genetics parameters of 66-plex InDels (66 InDel loci) in 251 Chinese Han population and analyzes the forensic application value. The results show that: the 66InDel gene loci have good genetic polymorphism in Chinese Han population, and can be independently applied to forensic individual identification and paternity identification research.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an application of a human chromosome InDel gene locus, a primer group, a product thereof and an individual identification method of a test material.
Background
Short Tandem Repeat (STR) has been widely used in the field of individual identification and paternity test due to its high sensitivity and strong identification ability. However, the STR mutation rate is relatively high, about 10 -3 There may be misjudgment in the paternity test; and the fragment is longer (100-400 bp), and the detection success rate in degraded test materials is lower. Insertion/deletion polymorphisms (InDel) as novel genetic markers, widely distributed in the human genome; the mutation rate is significantly lower than STR, about 10 -8 (ii) a Amplicons are shorter (<200 bp) is more suitable for detecting highly degraded samples; the two-state structure is easier to type and detect.
At present, some researches design InDel heredity aiming at different countries and different groupsThe marker multiplex amplification system provides basic data for individual identification and paternity identification by evaluating population genetics related parameters of the loci. The InDel commercial typing kit widely applied at home and abroad mainly comprises
DIPplex kit (QIAGEN, germany) and AGCU InDel 50kit (german biotechnology limited, wunst).
The DIPplex kit takes the gene frequency of the German population as a main screening basis, and has lower identification efficiency on the Chinese population. The cumulative individual recognition rate (TDP) of 30 InDel in Beijing Han nationality population is 0.999 999 999 999 999 999 985, the TDP in Guizhou Gelao and Miao nationality population is 0.999 999 999 999 999 999 999 999 999 999 999 999 999 34 and 0.999 999 996 respectively, but the TDP in Spanish population is up to 0.999 999 999 999 999 999. The AGCU InDel 50kit is a commercialized typing kit developed in China, and comprises 47 autosomal InDel loci, 2Y chromosome InDel loci and 1 individual identification locus (Amelogenin). The cumulative individual identification (TDP) and cumulative non-paternity exclusion probability (CPE) for 47 indels in the chinese population were 0.999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 and 0.999 7, respectively. Although 47 InDel identification potencies are greater than TDP (0.999 999 999 999 999 999 999 999 98) of the conventional STR system, and can meet individual identification requirements, CPEtrio<0.999 9, paternity testing efficacy is low and needs to be further improved.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
A first object of the present invention is to provide an application of the human chromosomal InDel locus to solve at least one of the above problems.
The second objective of the invention is to provide a primer group for amplifying the InDel locus.
It is a third object of the present invention to provide a reagent.
The fourth object of the present invention is to provide a kit.
The fifth purpose of the invention is to provide an application of the primer group, the reagent or the kit in individual identification or paternity test.
A sixth object of the present invention is to provide a method for identifying an individual of a material to be tested.
In a first aspect, the invention provides the use of a human chromosomal InDel locus in any of the following a) -b):
a) Individual identification;
b) Performing paternity test;
the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs 146109109109101, rs151142037, rs59258861, rs4053254, rs145041883, rs 12156094, rs 7430767, rs 36363676043, rs34812419, rs144650377, rs 338383838328, rs 914009127, rs 2737701, rs 444593, rs56110100, rs 6656375635799756974, rs 34785635563579975633794, rs 343556355633799756335634798, rs 335634563456345794, rs 3356345794, rs 335634563456345794, rs 33563456345633563356335633563356335633563356339753, rs 34563456345634563456345634975634975634975634975634975634569, rs 335634563457401, rs.
As a further technical solution, the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs4053254, rs145041883, rs56012194, rs3074767, rs 36043, rs 34241819, rs144650377, rs 92338328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs34564973, rs144581522, rs10656522, rs10623704, rs58303500, rs71102499, rs 230787807, rs 56053535353535353760, rs 955557, rs59306401, rs 530139962, rs10544053, rs35615387, rs71843136, rs34339565, rs 61315 and rs11471707.
In a second aspect, the invention provides a primer group for amplifying the InDel locus, wherein the sequence of the primer group is shown as SEQ ID No. 1-SEQ ID No. 144.
In a third aspect, the present invention provides a reagent comprising the above primer set.
In a fourth aspect, the present invention provides a kit comprising the above primer set or the above reagent.
As a further technical scheme, the kit also comprises a reaction buffer solution and a hot start Taq enzyme.
In a fifth aspect, the present invention provides the use of a primer set, reagent or kit for individual identification or paternity testing.
In a sixth aspect, the present invention provides a method for identifying individuals of a material to be tested, comprising the steps of:
a. extracting DNA of a material to be detected;
b. obtaining the typing result of the DNA InDel gene locus;
c. performing individual identification according to the genotype of the locus;
the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs4053254, rs145041883, rs56012194, rs3074767, rs 36043, rs 34241819, rs144650377, rs 92338328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs34564973, rs144581522, rs10656522, rs10623704, rs58303500, rs71102499, rs 230787807, rs 56053535353535353760, rs 955557, rs59306401, rs 530139962, rs10544053, rs35615387, rs71843136, rs34339565, rs 61315 and rs11471707.
As a further technical scheme, the step b further comprises a step of amplifying the InDel locus by using a primer group.
As a further technical scheme, the sequences of the primer group are shown in SEQ ID NO. 1-SEQ ID NO. 144.
Compared with the prior art, the invention has the following beneficial effects:
the inventor independently screens 66InDel loci from a dbSNP database, evaluates population genetics parameters of 66-plex InDels (66 InDel loci) in 251 Chinese Han population, and analyzes the forensic application value of the parameters. The research result shows that: through the correction of the Bonferroni method, 66InDel loci all accord with Hardy-Weinberg equilibrium law (P) in Chinese Han population>0.000 758), in linkage equilibrium between the loci. The average observed heterozygosity (Ho) for each locus was 0.482, the average expected heterozygosity (He) was 0.483, the average individual recognition (DP) was 0.612, the average Polymorphism Information Content (PIC) was 0.365, and the cumulative individual recognition (TDP) was 0.999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 428. Diad cumulative non-paternal exclusion probabilities (CPEs) for 66InDel loci duo ) At 0.999 739, triplet cumulative non-parent exclusion probability (CPE) trio ) 0.999 999 999 999 999 999 999 999,417. The results show that: the 66InDel gene loci have good genetic polymorphism in Chinese Han nationality people, and can be independently applied to forensic individual identification and paternity identification research.
The primer group provided by the invention has strong specificity and high sensitivity, and can realize amplification of the 66InDel loci.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a 66InDel locus allele frequency distribution.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides the use of a human chromosomal InDel locus in any of the following a) -b):
a) Individual identification;
b) Performing paternity test;
the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs 146109109109101, rs151142037, rs59258861, rs4053254, rs145041883, rs 12156094, rs 7430767, rs 36363676043, rs34812419, rs144650377, rs 338383838328, rs 914009127, rs 2737701, rs 444593, rs56110100, rs 6656375635799756974, rs 34785635563579975633794, rs 343556355633799756335634798, rs 335634563456345794, rs 3356345794, rs 335634563456345794, rs 33563456345633563356335633563356335633563356339753, rs 34563456345634563456345634975634975634975634975634975634569, rs 335634563457401, rs.
In some preferred embodiments, the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs4053254, rs145041883, rs56012194, rs3074767, rs 36043, rs 34241819, rs144650377, rs 92338328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs34564973, rs144581522, rs10656522, rs10623704, rs58303500, rs71102499, rs 230787807, rs 56053535353535353760, rs 955557, rs59306401, rs 530139962, rs10544053, rs35615387, rs71843136, rs34339565, rs 61315 and rs11471707.
In a second aspect, the invention provides a primer set for amplifying the InDel locus, wherein the sequence of the primer set is shown as SEQ ID No. 1-SEQ ID No. 144.
Primer pairs for amplification of the InDel locus described above are shown in the following table:
it should be noted that two primer pairs are designed for better amplification at the above-mentioned partial loci.
The inventor autonomously screens 66 autosomal InDel loci from a dbSNP database, and autonomously constructs a 66InDel multiplex amplification system based on a high-throughput sequencing system. Through DNA typing detection results of 251 Chinese Han nationality irrelevant individual samples, population genetic parameters of 66InDel in Chinese Han nationality population are analyzed, and basic data are provided for individual identification and paternity test.
In a third aspect, the present invention provides a reagent comprising the above primer set.
Because the reagent comprises the primer group, the kit can amplify the 66InDel loci.
In a fourth aspect, the present invention provides a kit comprising the primer set or the reagent.
Because the kit comprises the primer group, the kit can realize amplification of the 66InDel loci.
In some preferred embodiments, the kit further comprises a reaction buffer and a hot start Taq enzyme.
The reaction buffer and the hot start Taq enzyme are not particularly limited in the present invention, and reagents which are conventional in the art may be used.
In a fifth aspect, the present invention provides a primer set, reagent or kit for use in individual identification or paternity testing.
In a sixth aspect, the present invention provides a method for identifying individuals of a material to be tested, comprising the steps of:
a. extracting DNA of a material to be detected;
b. obtaining the typing result of the DNA InDel gene locus;
c. performing individual identification according to the genotype of the locus;
the InDel locus comprises: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs4053254, rs145041883, rs56012194, rs3074767, rs 36043, rs 34241819, rs144650377, rs 92338328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs34564973, rs144581522, rs10656522, rs10623704, rs58303500, rs71102499, rs 230787807, rs 56053535353535353760, rs 955557, rs59306401, rs 530139962, rs10544053, rs35615387, rs71843136, rs34339565, rs 61315 and rs11471707.
The method is simple, and can effectively realize the inference of unknown material sources at the gene level.
In some preferred embodiments, the b step further comprises the step of amplifying the InDel locus using a primer set.
In some preferred embodiments, the sequences of the primer sets are shown in SEQ ID NO. 1-SEQ ID NO. 132.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
1 materials and methods
1.1InDel screening procedure
The research is based on a dbSNP database (https:// www.ncbi.nlm.nih.gov/snp /) data set with human GRCh37.Hg19 as a reference genome, and the InDel gene locus with higher identification efficiency in Chinese population is screened out by adopting VCFtools and plink software. Specific screening criteria are as follows: (1) A binary polymorphic insertion/deletion sequence located in an autosomal intron region with a Global Minor Allele Frequency (GMAF) between 0.4 and 0.5, yielding 17374 loci; (2) Satisfies Hardy-Weinberg equilibrium (HWE) law, P HWE >0.05, heterozygosity>0.4, resulting in 1263 loci; (3) Coincidence with chain equilibrium test (LE), r 2 <0.01, 987 loci were obtained; (4) Inter-population (east Asia, south Asia, europe, america, africa) population differentiation index (Fst)<0.06, 549 gene loci are obtained; (5) Segment length of InDel locus>1bp, obtaining 172 gene loci; (6) On the same chromosome, the physical distance between InDel loci is more than or equal to 10MB, and 93 loci are obtained. And (3) designing multiple primers for 93 loci obtained by the screening standard by adopting Primer Premier 5.0 and MFEprimer software, considering the Primer specificity, the Primer length, the annealing temperature, the Primer structure design and other factors to ensure that each pair of primers can be amplified uniformly, and finally determining 66InDel loci for composite amplification. 66. No literature reports relating to medically sensitive information are available for each InDel locus.
1.2 sample Collection and DNA extraction
According to the principle of informed consent, FTA blood cards prepared by collecting peripheral blood samples of 100 Chinese Han unrelated individuals and peripheral blood of 151 Chinese Han unrelated individuals comprise 68 Beijing Han nationality, 50 Henan Han nationality, 41 Shandong Han nationality, 21 inner Mongolian Han nationality, 18 Shanxi Han nationality, 16 Shenyang Han nationality, 16 Lanzhou Han nationality, 11 Jiangxi Han nationality and 10 Guangdong Han nationality. Peripheral blood samples were all obtained
DNA was extracted from the DNA Investigator Kit (Qiagen, germany). DNA quantification was performed using a Qubit 3.0 fluorescence quantifier (Thermo Fisher, USA). Storing at-20 deg.C for use. After the peripheral blood of 151 unrelated individuals is prepared into an FTA blood card, the FTA blood card is stored in a ventilated and dry place for later use.
1.3PCR amplification and library preparation
By using
Custom Panel (IGMU 258V 1) (Jiaxing Aijietakang Biotechnology Co., ltd.) (to be noted, the kit is designed for the 66InDel loci of the present invention, including the primer set provided by the present invention) to perform PCR multiplex amplificationThe total amplification system is 30 mu L, wherein Enhancer buffer NB (1N) is 3.5 mu L, enhance buffer M is 2.5 mu L, primer (including 144 primers provided by the invention, the sequences are shown in SEQ ID NO. 1-SEQ ID NO. 132) is 5 mu L, IGT-EM808 polymerase mixture is 10 mu L, template DNA is 5 mu L (1 ng/mu L) or FTA blood card sample DNA with the diameter of 1mm, and deionized water is added to 30 mu L. In the experiment process, 9947A and 9948 are used as positive controls, and deionized water is used as a negative control. Using ProFlex TM Amplification was performed by a PCR amplification apparatus (Applied Biosystems, USA), and the PCR amplification conditions were as follows: 95 ℃ 3min 30s; 2 s at 98 ℃, 4min at 60 ℃ and 18 cycles; finally, the mixture was stored at 72 ℃ for 10min and 4 ℃.
The PCR product was purified by AMPure XP magnetic beads (Beckman Coulter, USA), and 13.5. Mu.L of PCR product, 1. Mu.L of Index-i, 2.5. Mu.L of Enhance buffer M, and 2. Mu.L of deionized water were added. By ProFlex TM Amplification was performed by a PCR amplification apparatus (Applied Biosystems, USA), and the PCR amplification conditions were as follows: 95 ℃ 3min 30s; 20s at 98 ℃, 1min at 58 ℃, 30s at 72 ℃ and 9 cycles; finally, storing at 72 ℃ for 5min and 4 ℃.
1.4 high throughput sequencing and InDel typing
The sample library was quality controlled and quantified using an Agilent 2100 bioanalyzer (Agilent, USA) and 7500 real-time fluorescent quantitative PCR (Thermo Fisher, USA), followed by high-throughput sequencing of the 10pM DNA mixed library on a MiSeq high-throughput sequencer (Illinia, USA) using the MiSeq v2 kit (2X 150 cycles) (Illinia, USA). Typing analysis was performed on 66InDel loci and Amel sex loci using CLC Genomics Workbench 20.0 (Qiagen, germany). Firstly, performing quality control on the fastq sequence through QC for sequencing Reads and Trim Reads functions, including quality evaluation and data trimming of original data (removing low-quality Reads and linker sequences with a quality score of less than 30 in original sequencing Reads). Importing a Structural Variant Call module according to the bed file configured in the amplification area; configuring a vcf file according to the InDel position and the variation information, and importing an identification Known details from maps module. According to the standard GA/T1693-2020 "second generation sequencing and Detection Specification for DNA in the court science", the "identification Known Mutations from maps" parameter Minimum coverage is set to be 100, and the Detection frequency is 20% of the software default. Typing results were obtained for each locus.
1.5 statistical analysis
Allele Frequencies (AF) of 66InDel loci, observed heterozygosity (Ho), expected heterozygosity (He) were calculated using GenAIEx 6.5 software, hardy-Weinberg equilibrium and linkage equilibrium test between loci were performed on each locus, and Typical Paternity Index (TPI) was calculated. The individual recognition power (DP), the cumulative individual recognition power (TDP), and the Polymorphism Information Content (PIC) were calculated using the Modifie-PowerState software package. Cumulative diad non-paternal exclusion probability (CPE) was calculated using Cervus 3.0 software (http:// www.fieldgenetics. Com/pages/download.jsp) duo ) And cumulative triplet non-parent exclusion probability (CPE) trio) 。
2 results
2.1 Genetic information of 66-plex InDels gene locus and standard typing result
The IGMU kit can effectively amplify 251 samples of Chinese Han nationality irrelevant individuals, the mass fraction Q30 of all the samples is more than 95 percent through the analysis of CLC Genomics Workbench 20.0 software, the average sequencing depth of an amplicon is 1550X, the main distribution range of the multiple PCR product fragments is 150-180bp, and the Agilent 2100 bioanalyzer is controlled by quality and has no non-specific amplification. Specific InDel locus genetic information and positive standard (9947A, 9948) typing are shown in Table 1.
TABLE 1 typing of genetic information and Positive Standard for 66InDel loci
Note: 1) rs # is the ID number of the InDel locus in the dbSNP database; 2) Physical location of the InDel locus on the human genome database (GRCh37. P19) chromosome 3) reference allel (Ref) was named 1in the subject line and alternative allel (Alt) was named 2 in the subject line.
2.2 Hardy-Weinberg equilibrium test and linkage equilibrium test in 66-plex InDels in Chinese Han population
Hardy-Weinberg balance test is carried out on 66 loci, and the genotype distribution of other 63 loci conforms to Hardy-Weinberg balance (P) except three loci of rs146109101, rs3061475 and rs2307807>0.05 Specific P values are shown in table 2. Corrected by the Bonferroni method, the P value was set to 0.000 758 (0.05/66), and the P values of 66InDel loci were all greater than 0.000 758, consistent with Hardy-Weinberg equilibrium. The 66 loci were combined pairwise to carry out linkage balance test, yielding 2145 (2 145= [ N × (N-1)]N is the number of loci studied) to obtain the linkage equilibrium P between loci. No pairwise linkage was found at 66 loci corrected by Bonferroni, P =0.000 023 3 (0.05/2145). Therefore, TDP and CPE can be performed duo 、CPE trio And (4) calculating.
2.3 Population genetic parameter investigation of 66-plex InDels in Chinese Han population
According to statistic analysis of GenAIEx 6.5 software, except that the Minor Allele Frequency (MAF) of rs79444593 locus is lower than 0.1, the minor allele frequencies of the other 65 InDel loci are distributed between 0.28 and 0.5, the average value is 0.4366, the allele frequency distribution of the InDel loci is relatively balanced in Han people in China, and the allele frequency distribution of 66InDel loci is shown in figure 1. Statistics show that the minimum values of Ho, he, DP, PIC, PE and TPI occur at the locus rs 79444593. Except rs79444593, the observed heterozygosity (Ho) of other 65 loci is 0.390-0.562, the expected heterozygosity (He) is 0.409-0.500, and the difference between the observed heterozygosity (Ho) and the expected heterozygosity (He) is smaller; the higher the individual recognition rate (DP) is between 0.562 and 0.653, the higher the DP is, the more effective the genetic marker is in recognizing different individuals; the Polymorphism Information Content (PIC) is 0.325-0.375, and the higher the PIC is, the more the information content of the genetic marker is; the cumulative paternity index (TPI) is 0.820-1.141. The cumulative individual identification probability (TDP) was 0.999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 428. Specific locus parameters are shown in table 2. Except the rs79444593 locus, other loci have higher polymorphism in Chinese Han population, and 66-plex InDels is suitable for individual identification research of Chinese Han population.
TABLE 2 66InDel loci forensic genetics parameters (n = 251)
2.4 Application of 66-plex InDels in individual identification research
2.5 Application of 66-plex InDels in paternity test case
Sample information: FTA blood cards prepared by collecting peripheral blood samples of 100 Chinese Han independent individuals and peripheral blood of 151 Chinese Han independent individuals comprise 68 Beijing Han nationality, 50 Henan Han nationality, 41 Shandong Han nationality, 21 inner Mongolian Han nationality, 18 Shanxi Han nationality, 16 Shenyang Han nationality, 16 Lanzhou Han nationality, 11 Jiangxi Han nationality and 10 Guangdong Han nationality.
The experimental steps are as follows: refer to steps 1.2-1.5.
For case analysis of paternity test, use is made of
DIPplex system, dual non-father exclusion probability (CPE) of cantonese han nationality of china duo ) At 0.942 342, triple non-parent exclusion probability (CPE) trio ) Is 0.994 748.AGCU InDel 50kit triplet non-parent exclusion probability (CPE) in Guangzhou Han nationality trio ) And was 0.999. Triple non-paternal exclusion probability (CPE) for two commercial kits trio ) All are less than 0.999, the efficiency for identifying the diad case is lower, and the method cannot be independently applied to paternity identification cases. As shown in Table 3, 66-plex InDels bigeminal non-parent exclusion Probability (PE) duo ) Is composed of0.0155-0.125, triple non-father exclusion Probability (PE) trio ) 0.1415-0.2812, the higher the probability of excluding non-paternal loci, the higher the efficiency of excluding non-paternal parents when performing paternity test analysis. CPE in 66-plex InDels for 251 unrelated individual samples analyzed by Cervus software duo At 0.999 739,CPE trio 0.999 999 999 999 999 999 999 999,417. CPE for 66-plex InDels trio More than 20 STR loci (CPE) trio =0.999 999 996 (see: yang Xiu, mu Rui, cai Lu Wei, N.E.E, li Chun Xiang, xia Chen, liu Hua Jie, ma Wen Hua, xinjiang Stone river area Kazak nationality, hui nationality, han nationality people 20 STR locus genetic polymorphism research [ J]Laser biology newspaper, 2021,30 (04): 339-347.)) has stronger identification efficiency, which indicates that 66-plex InDels can be independently applied to the analysis of triplet paternity test cases, but the system efficiency of the dyad case does not meet the requirement of forensic paternity test, and other commercial kits are needed for combined use. The four systems are compared in the identifying efficacy of Chinese Han population, as shown in Table 4.
TABLE 3 Duplex non-paternal exclusion probabilities and triplet non-paternal exclusion probabilities for the 66InDel loci (n = 251)
TABLE 4 comparison of the identifying potencies of different systems
Discussion of the related Art
STR is the most common polymorphic genetic marker in the field of forensic physical evidence identity identification at present, and the detection length of a multiplex amplification system is generally distributed between 100 and 500bp, so that the STR is not suitable for detecting highly degraded and low-copy samples. Indels are used as the genetic markers of the two alleles, and the amplified segments of the genetic markers are all smaller than 200bp, so that the method is suitable for the typing of degradation test materials. The study autonomously constructed an Illumina sequencing platform-based multiplex amplification system (66-plex InDels) comprising 66 autosomal Indel loci and one Amel sex locus.
The genetic parameters of 66-plex InDels were evaluated by genetic polymorphism investigation of 251 unrelated individuals in Han nationality of China. The result shows that 66InDel loci are distributed on 18 chromosomes, and the average allele frequency of the InDel loci in Chinese Han population is 0.5242; the average observed heterozygosity is 0.482; the average expected heterozygosity is 0.483; the average match probability is 0.388; the average individual recognition rate is 0.612; the average polymorphism information content is 0.365; the average cumulative paternity index was 0.974. All loci, after Bonferroni correction, obey the Hardy-Weinberg equilibrium law (P >0.000 758) and there is no linkage disequilibrium between two loci (P >0.000 023 3). The gene locus rs79444593 has a MAF of 0.4385 in the east Asia population of the International thousand-people genome project, but has a MAF of 0.09761 in the Chinese Han population of the research, which indicates that the locus has low genetic polymorphism and poor identification efficiency in the Chinese Han population. In subsequent experiments, random sampling errors may be eliminated as much as possible by increasing the sample size, considering that random sampling may also cause errors.
Previous studies have shown that the individual identification efficacy of about 60 InDel loci can be equivalent to the commercial STR kit commonly used in current forensic identification (e.g., the Sinofiler kit contains 15 STR loci). DP is used as an important index for forensic individual identification, DP is the probability that two random individual phenotypes are different, and the larger the DP is, the stronger the individual identification efficiency is. The cumulative individual identification probability (TDP) for 66-plex InDels was calculated to be 0.999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 428, and the cumulative non-parent exclusion probability (CPE) was 0.999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 999 417.TDP higher than DNA type 21 TM The identification efficiency of 20 autosomal STRs in the kit is high, and the CPE is high>0.999 Therefore, 66-plex InDels can be independently applied to individual identification and paternity identification, and can also be used as a supplementary gene locus of an existing commercial kit to carry out complex genetic relationship identification.
The research has the defects that the genetic polymorphism of an individual gene locus (rs 79444593) in Chinese Han population is poor, the site can be replaced by optimizing a multiple multiplex amplification system, and more InDel gene loci can be added to improve the system efficiency of 66-plex InDels. Meanwhile, the subsequent research can analyze the genetic polymorphism of other ethnic groups of Chinese population, evaluate the forensic application value of 66-plex InDels in different groups in China, carry out the balance research of genetic parameters among groups and explore the universality of 66-plex InDels in Chinese population.
The research obtains 66 autosomal InDel gene locus multiplex amplification systems, and by the forensic population genetic analysis of 66-plex InDels in Chinese Han population, provides genetic background data for individual identification and paternity test research of the InDel locus in Chinese population.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> military medical research institute of military science institute of people's liberation force of China
Application of <120> human chromosome InDel gene locus, primer group and product thereof, and individual identification method of test material
<160> 144
<170> PatentIn version 3.5
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acaagagtag gataggtgcc acttt 25
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ttgcttgtgg tatccatggt gac 23
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ggccccattt gttgcttatt acc 23
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tggagaaatt ctcagcacac ctac 24
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tggaagcggg gaaaacaaga agt 23
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gagcaacttg gtgcttcttg ga 22
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aaccttcaca acaaccctgt gaa 23
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actccagagc ctacatgttt aacc 24
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gtgagacagg atgaacagtc ttcccc 26
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acacgtgtct ggagagtcta gagaag 26
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ttctaaccat tgccctggtg ag 22
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ttccatgcag gtcacacaag atc 23
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ttacccagtc ttgcccttca tg 22
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atctctaaga tgaactccat ttggctt 27
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cagccatctt tatcaacttc ccatg 25
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tagatattgt tgctcttaca agaggcaa 28
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tttatgctct gtcccctcgt gaa 23
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ttgcttcagt tctcagcaag ttcc 24
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tgaggtactg tgtcctaaac cctat 25
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ttcagaagca agagtctacc aacc 24
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ccgtggtgcc atattttgag gta 23
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tcagaagcaa gagtctacca acc 23
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tcaactgcat cacagcttac ctg 23
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aggtcttcct aggatagata gcttg 25
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cttcatttca actgcatcac agctt 25
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aaaggtcttc ctaggataga tagcttg 27
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<400> 27
ctgcctctct ttcccctaat tgac 24
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aacctccact ttcctaccag aagttc 26
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ttttccaaca aatgatgctg ggaaa 25
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actggattta accatggttt cttgg 25
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ttcatacccc agtgtacctg gaat 24
<210> 32
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aattctcata ccagcacagc agtc 24
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agtagatcta cagtttgatc cagcaatc 28
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gaaacaacct aagtgcctat tgacca 26
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aacatcacgt tgtatacttt aaatatacac aa 32
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gccattactt cctcattctt tgcttcc 27
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gcaagcaagt cctgaactgg tata 24
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ggccctttag gccactttaa gacct 25
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cacagcaagc aagtcctgaa c 21
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ggccctttag gccactttaa gac 23
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gcgagactcc atctcaaaaa cac 23
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agtagaaaga gcatgaaacc tccatg 26
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aacacaggct tgaggccaac c 21
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gggactgcta atgcaaacag tg 22
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aggtggcttc tacatggtgt tgg 23
<210> 46
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ctctgttaga gcagtgcaga agg 23
<210> 47
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attggcacat gggtggtatc tgaa 24
<210> 48
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<400> 48
cacaggaatg gctaacagca gt 22
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<400> 49
gagaaaaggc tgacttcccc tt 22
<210> 50
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<400> 50
ctgcctgtta cacatgtgct agga 24
<210> 51
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<400> 51
accatgccta gcctcagcat a 21
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<400> 52
taacattggc catatgggtc tctg 24
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<400> 53
tttctctctc aactggggct tga 23
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<400> 54
tcagctgttc tccgtctact ctga 24
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<400> 55
tattccaagc tgtggtggct acag 24
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<400> 56
gttccaagcc ttggctattg gatg 24
<210> 57
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<400> 57
tgagggaagg tatctgaagg tcaga 25
<210> 58
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<400> 58
tacacatata tacagcatat atacacacat aaactg 36
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<400> 59
catcccattt ctcaggtccc aaat 24
<210> 60
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<400> 60
tcactgttct gccctctgta ttctg 25
<210> 61
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<400> 61
gtttatttaa tccctggctc ccatgc 26
<210> 62
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<400> 62
tctcactcca tctaaacaac gaaacc 26
<210> 63
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<400> 63
ttgtgaggtc agtcctgagg aa 22
<210> 64
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<400> 64
aatagggaac acctcctggg tt 22
<210> 65
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<400> 65
aggatggaga gtctgtgctg tg 22
<210> 66
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<400> 66
accctttcta ggtgtacagt tctgtg 26
<210> 67
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<400> 67
ctcacaagcc ctttttatgg tgaca 25
<210> 68
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<400> 68
cagtgttcag ggaacaggat atgc 24
<210> 69
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<400> 69
gggagaaatt caagcccaat cca 23
<210> 70
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<400> 70
ggcctagaag gaaaaatgat tttgttg 27
<210> 71
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<400> 71
actcctctct catgcacagt ca 22
<210> 72
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<400> 72
ttcctgtctt acttttccct ccacac 26
<210> 73
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<400> 73
ccacacctgg ctaatttttc tattagtaga 30
<210> 74
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<400> 74
ggcccatgag agcctttaat aatgg 25
<210> 75
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<400> 75
accctcagct gcattctagc tac 23
<210> 76
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<400> 76
actgggcccg tattactact ca 22
<210> 77
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ctctgccttt tcaagaacaa ttctgt 26
<210> 78
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<400> 78
actgtgattt atcaagttaa catcactct 29
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<400> 79
tagcctggtc tggaaatgtg gtt 23
<210> 80
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<400> 80
catgtgccat tacctgtgtg gt 22
<210> 81
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<400> 81
ccttaggtag atgtatgtat gtactgagtg cc 32
<210> 82
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<400> 82
cagtcattta catcaatgtg tggattaaat cc 32
<210> 83
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<400> 83
actgtggtta tgttagagga atgcatg 27
<210> 84
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<400> 84
aacaatcgtt gaagataggt gatgga 26
<210> 85
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<400> 85
ggctttgatt ctttctacag cgattt 26
<210> 86
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<400> 86
tgaaaaattc tctccactgg gagga 25
<210> 87
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<400> 87
cagtgatgtg aatagtttca atgggtt 27
<210> 88
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<400> 88
gtatgtgtga gcataaatta gtgtcctgt 29
<210> 89
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<400> 89
ctaattttgt atttttagta gagatggggt ctca 34
<210> 90
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<400> 90
acgttcttaa aacaacaata ttggtttata ggagt 35
<210> 91
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<400> 91
ggggtgttat gtgtggtgtg tg 22
<210> 92
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<400> 92
tgtgtggggg tatggatgtg gta 23
<210> 93
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<400> 93
gaaaagcttg ggtggagtct cat 23
<210> 94
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<400> 94
tggttgagca ttacaagcag tgtac 25
<210> 95
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<400> 95
aaatctcatc cccctgctgt ac 22
<210> 96
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<400> 96
tttccctgct atatccccag ca 22
<210> 97
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<400> 97
aaatacagca gccaggggag at 22
<210> 98
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<400> 98
aaggtcactg gaactcacac ttc 23
<210> 99
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<400> 99
ggaccacagg tgtgcactgc 20
<210> 100
<211> 21
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<400> 100
gagccattgt gtctggcctc a 21
<210> 101
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<400> 101
ggctgctatg ctgagtagat gtag 24
<210> 102
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<400> 102
ttgggaggaa gaactaagga cctag 25
<210> 103
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<400> 103
ctggttaaga tgctaagacc cagtct 26
<210> 104
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<400> 104
tctgatgatg caataaagtg ttattttcac a 31
<210> 105
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<400> 105
taccatgcat caagtggcct tct 23
<210> 106
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<400> 106
cagggccatg cagtaagatt tcc 23
<210> 107
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<400> 107
caaagtaacg ctgggaccac t 21
<210> 108
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<400> 108
caatcctcgc tctacaaccc cat 23
<210> 109
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ctggtgaatt tccgtccaga caa 23
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<400> 110
atgtcccctg gcttatgtcc ta 22
<210> 111
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<400> 111
ccccccattt cttagtgtag atggc 25
<210> 112
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<400> 112
gacagtcaca gagcatagag cact 24
<210> 113
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accttgccct gtctatgtct tc 22
<210> 114
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<400> 114
gcagagtcta agcatgccct t 21
<210> 115
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<400> 115
gtcctgaatg gtattgccta ggtttt 26
<210> 116
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<400> 116
ccgcttttca agagtgatgt ggtac 25
<210> 117
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<400> 117
tggcttaaga gaaagcacac cttt 24
<210> 118
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<400> 118
atctgttcct cattctgcac ctca 24
<210> 119
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<400> 119
gtcctttgcc atttttctat tgacatg 27
<210> 120
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<400> 120
tggcaataac gtaaaaatgt ttggtactag 30
<210> 121
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<400> 121
cctcttcctc ataaatctgg ctgtc 25
<210> 122
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<400> 122
aggtagcctc tttcttcagg aatcg 25
<210> 123
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<400> 123
ttactgagga tggaggcagg acg 23
<210> 124
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<400> 124
ctaccagtgt gccatgtcag t 21
<210> 125
<211> 21
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<400> 125
agcggcagaa aatgagtgac a 21
<210> 126
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<400> 126
tggagaattc ccagcatgga ac 22
<210> 127
<211> 21
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<400> 127
aaaagtgcct gctgtgggaa t 21
<210> 128
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<213> Artificial sequence
<400> 128
tcatgctgat agcccatcac ac 22
<210> 129
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<400> 129
gaaatgttga cgctagggac tga 23
<210> 130
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<400> 130
gagatccaaa agacagaggc atgg 24
<210> 131
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<400> 131
ccaacggtgt gtaagtattc ctcttt 26
<210> 132
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<400> 132
gacctgacca aactctatga actga 25
<210> 133
<211> 26
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<400> 133
gtaataggag cagcagtacg gatttt 26
<210> 134
<211> 21
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<213> Artificial sequence
<400> 134
gggattacag gtgctgccac a 21
<210> 135
<211> 23
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<213> Artificial sequence
<400> 135
caatgcctgt cctctctgca tct 23
<210> 136
<211> 22
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<213> Artificial sequence
<400> 136
tcaacgacgg gaaatacgtc ct 22
<210> 137
<211> 23
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<213> Artificial sequence
<400> 137
tgtccaatgc ctgtcctctc tgc 23
<210> 138
<211> 23
<212> DNA
<213> Artificial sequence
<400> 138
tcactcaacg acgggaaata cgt 23
<210> 139
<211> 24
<212> DNA
<213> Artificial sequence
<400> 139
ggagggaggg atagcattag gaaa 24
<210> 140
<211> 34
<212> DNA
<213> Artificial sequence
<400> 140
aaaacatctg ataattgaat taaaagatga gcaa 34
<210> 141
<211> 23
<212> DNA
<213> Artificial sequence
<400> 141
tcatatggtg cagctgctat gga 23
<210> 142
<211> 23
<212> DNA
<213> Artificial sequence
<400> 142
ccaaatggtg aaaccccgtc tct 23
<210> 143
<211> 23
<212> DNA
<213> Artificial sequence
<400> 143
ctgcctagca aaaggtcaat gct 23
<210> 144
<211> 24
<212> DNA
<213> Artificial sequence
<400> 144
gtgggtttga cagttggatg cttg 24
Claims (9)
1. Use of a reagent for detecting the human chromosomal InDel locus in any one of the following a) -b):
a) Individual identification;
b) Performing paternity test;
the InDel locus is: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs 405353254, rs 145883, rs 12156094, rs 30767, rs36119043, rs34812419, rs144650377, rs33928328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs34564973, rs144581522, rs10656522, rs10623704, rs58303500, rs71102499, rs 230787807, rs 56053535353535353760, rs 955557, rs59306401, rs 530139962, rs10544053, rs35615387, rs71843136, rs34339565, rs 61315 and rs11471707.
2. A primer set for amplifying the InDel locus according to claim 1, wherein the sequence of the primer set is shown as SEQ ID No. 1-144.
3. A reagent comprising the primer set according to claim 2.
4. A kit comprising the primer set according to claim 2 or the reagent according to claim 3.
5. The kit of claim 4, further comprising a reaction buffer and a hot start Taq enzyme.
6. Use of the primer set of claim 2, the reagent of claim 3, or the kit of claim 4 or 5 for individual identification or paternity testing.
7. An individual identification method for a material to be tested is characterized by comprising the following steps:
a. extracting DNA of a material to be detected;
b. obtaining the typing result of the DNA InDel gene locus;
c. performing individual identification according to the genotype of the locus;
the InDel locus is: rs56113354, rs66493016, rs34947838, rs10562732, rs10555133, rs3028599, rs34105738, rs10541877, rs3030108, rs10535391, rs142380122, rs5849187, rs59715790, rs139940865, rs35052276, rs10562881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs 405353254, rs 145883, rs 12156094, rs 30767, rs36119043, rs34812419, rs144650377, rs33928328 rs4009127, rs372779701, rs79444593, rs56110100, rs142281120, rs34822234, rs367897009, rs145635184, rs6143946, rs76989317, rs34825333, rs34399561, rs10667259, rs139101426, rs3061475, rs200055056, rs 344973, rs144581522, rs10656522, rs 1062323704, rs58303500, rs71102499, rs 230787807, rs 560535353535353, rs 955557, rs 13959401, rs 530530962, rs10544053, rs35615387, rs71843136, rs34339565, rs 6161315 and rs11471707.
8. The method of claim 7, wherein step b further comprises the step of amplifying the InDel locus using a primer set.
9. The individual identification method according to claim 8, wherein the sequences of the primer set are shown in SEQ ID No.1 to SEQ ID No. 144.
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