CN114574595A - 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 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 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 fields of individual identification and paternity testing due to its high sensitivity and strong identification ability. But STR mutation rate phaseFor higher, about 10-3There may be misjudgment in the paternity test; and the fragment is longer (100-400bp), and the detection success rate in the degraded test material is lower. Insertion/deletion polymorphisms (InDel) are a novel genetic marker, widely distributed in the human genome; the mutation rate is significantly lower than STR, about 10-8(ii) a Amplicons are shorter (<200bp) is more suitable for detecting highly degraded samples; the two-state structure is easier to type and detect.
At present, some researches design an InDel genetic marker multiplex amplification system aiming at different countries and different groups, and provide basic data for individual identification and paternity identification by evaluating group genetics related parameters of a gene locus. 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 30 InDel accumulated individual recognition rates (TDP) in the Beijing Han nationality population were 0.999999999985, the TDP in the Guizhou Gelao and Miao populations were 0.99999999934 and 0.99999999686, respectively, but the TDP in the Spain population was as high as 0.999999999999. The AGCU InDel 50kit is a commercialized typing kit developed in China, and comprises 47 autosomal InDel gene loci, 2Y chromosome InDel gene loci and 1 individual identification gene locus (Amelogenin). The cumulative individual recognition rate (TDP) and cumulative non-paternity exclusion probability (CPE) for 47 indels in the chinese population were 0.999999999999999 and 0.9997, respectively. Although 47 InDel identified more potent than TDP (0.99999999998) of conventional STR system, able to meet individual identification requirements, CPEtrio<0.9999, the paternity test was less effective and was in need of further improvement.
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 set 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 the 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, rs 146109109109101, rs151142037, rs59258861, rs4053254, rs145041883, rs 12156094, rs 7430767, rs 363636043, rs34812419, rs144650377, rs 33838383839, rs 9140027, rs 27701, rs 444593, rs56110100, rs 6656357997567997567841401, rs 34799756335633794, rs 3456355635563379975634794, rs 3456355635563356335633563356349794, rs 3456345634563456345634563456345794, rs 335634563456345633563356335633563356349794, rs 34563456345634563456345634563456345634563456345634563456345634563456345633563497569, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 33563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456.
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.
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 primer set or the 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 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, rs 146109109109101, rs151142037, rs59258861, rs4053254, rs145041883, rs 12156094, rs 7430767, rs 363636043, rs34812419, rs144650377, rs 33838383839, rs 9140027, rs 27701, rs 444593, rs56110100, rs 6656357997567997567841401, rs 34799756335633794, rs 3456355635563379975634794, rs 3456355635563356335633563356349794, rs 3456345634563456345634563456345794, rs 335634563456345633563356335633563356349794, rs 34563456345634563456345634563456345634563456345634563456345634563456345633563497569, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 33563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456.
As a further technical scheme, the step b further comprises the step of amplifying the InDel locus by adopting a primer group.
As a further technical scheme, the sequence of the primer group is shown as 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>0.000758), the loci are in linkage equilibrium. The average observed heterozygosity (Ho) of each locus was 0.482, the average expected heterozygosity (He) was 0.483, the average individual Discrimination (DP) was 0.612, the average Polymorphism Information Content (PIC) was 0.365, and the cumulative individual discrimination (TDP) was 0.99999999999999999999999999942818. Diad cumulative non-paternal exclusion probabilities (CPEs) for 66InDel lociduo) 0.999739, triplets accumulate non-parent exclusion probabilities (CPEs)trio) Is 0.999999999417. KnotThe 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.
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 those skilled in the art will understand 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 881, rs59027185, rs35908330, rs70954609, rs147779273, rs35829610, rs60570384, rs146109101, rs151142037, rs59258861, rs 405353535353535353254, rs 145883, rs 12156094, rs 7430767, rs 36101043, rs34812419, rs144650377, rs 3392838326, rs 9127, rs 152701, rs 444593, rs 1067967110100, rs 6656615661563277567781974, rs 7856341815635563256325632563256327753, rs 33778197563256325632563277401, rs 3377401, rs 335632563256325632563277401, rs 3377401, rs 33563256325632563256325632563277401, rs 3332563256325632563256325632563297401, rs 3332563256325632563256325632563256325632563277401, rs 3332563256325632563256325632563256325632848, rs 3332563256325632563256325632563256325632563256325632563256325632563256325632563284and 34975.
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 the DNA typing detection results of 251 Chinese Han irrelevant individual samples, group genetic parameters of 66InDel in Chinese Han population are analyzed, and basic data are provided for individual identification and parental right identification.
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 above primer set or the above 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, rs 146109109109101, rs151142037, rs59258861, rs4053254, rs145041883, rs 12156094, rs 7430767, rs 363636043, rs34812419, rs144650377, rs 33838383839, rs 9140027, rs 27701, rs 444593, rs56110100, rs 6656357997567997567841401, rs 34799756335633794, rs 3456355635563379975634794, rs 3456355635563356335633563356349794, rs 3456345634563456345634563456345794, rs 335634563456345633563356335633563356349794, rs 34563456345634563456345634563456345634563456345634563456345634563456345633563497569, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 33563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456.
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 to SEQ ID NO. 132.
The present invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for illustrative purposes 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 study was based on the dbSNP database (https:// www.ncbi.nlm.nih.gov/snp /) dataset with human GRCh37.hg19 as the reference genome, and the InDel loci with higher identification potency among Chinese populations were screened using 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, PHWE>0.05, heterozygosity>0.4, resulting in 1263 loci; (3) coincidence with chain equilibrium test (LE), r2<0.01, yielding 987 loci; (4) inter-population (east Asia, south Asia, Europe, America, Africa) population differentiation index (Fst)<0.06, 549 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) carrying out multiplex Primer design on 93 loci obtained by the screening standard by adopting Primer Premier 5.0 and MFEprimer software, considering factors such as Primer specificity, Primer length, annealing temperature, Primer structure design and the like to ensure that each pair of primers can be amplified uniformly, and finally determining 66InDel loci for composite amplification. There are no reports in the literature relating to medically sensitive information for the 66InDel loci.
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 independent individuals and peripheral blood of 151 Chinese Han independent individuals comprise 68 cases of Beijing Han nationality, 50 cases of Henan Han nationality, 41 cases of Shandong Han nationality, 21 cases of inner Mongolian Han nationality, 18 cases of Shanxi Han nationality, 16 cases of Shenyang Han nationality, 16 cases of Lanzhou Han nationality, 11 cases of Jiangxi Han nationality and 10 cases of 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. 151 independent of each otherAnd (4) preparing the peripheral blood into an FTA blood card, and storing the FTA blood card in a ventilated and dry place for later use.
1.3PCR amplification and library preparation
By using
Custom Panel (IGMU258V1) (GJIETAIKANG Biotech Co., Ltd.) (it should be noted that the kit is designed for 66InDel loci of the present invention, and includes the Primer set provided by the present invention) to perform PCR multiplex amplification, the total amplification system is 30 μ L, wherein Enhancer buffer NB (1N)3.5 μ L, Enhance buffer M2.5 μ L, Primer (including 144 primers provided by the present invention, sequence shown in SEQ ID NO. 1-SEQ ID NO. 132) 5 μ L, IGT-EM808 polymerase mix 10 μ L, template DNA 5 μ L (1ng/μ L) or FTA blood card sample DNA with diameter of 1mm, and deionized water to 30 μ L. In the experiment process, 9947A and 9948 are used as positive controls, and deionized water is used as a negative control. Using ProFlexTMAmplification was performed using a PCR amplification apparatus (Applied Biosystems, USA) under the following conditions: 3min at 95 ℃ for 30 s; 20s at 98 ℃, 4min at 60 ℃ and 18 cycles; finally, the mixture is 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 the PCR product, Index-i 51. mu.L, Index-i 71. mu.L, Enhance buffer M2.5. mu.L, and deionized water 2. mu.L were added. By ProFlexTMAmplification was performed using a PCR amplification apparatus (Applied Biosystems, USA) under the following conditions: 3min at 95 ℃ for 30 s; 20s at 98 ℃, 1min at 58 ℃ and 30s at 72 ℃ for 9 cycles; finally, the mixture is stored 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 Inc., 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 Identify Known details from maps module. According to the standard GA/T1693-. 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 tests between loci were performed on each locus, and a Typical Paternity Index (TPI) was calculated. The identification power (DP), the cumulative identification power (TDP), and the Polymorphism Information Content (PIC) were calculated using the Modifie-Powerstates software package. Calculating cumulative diad non-parent exclusion probability (CPE) by using Cervus 3.0 software (http:// www.fieldgenetics.com/pages/download. jsp)duo) And cumulative triplet non-parent exclusion probability (CPE)trio)。
2 results
2.166-plex InDels gene locus genetic information and standard typing results
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 plus one 180bp, and the Agilent 2100 bioanalyzer has no non-specific amplification under the quality control. Specific InDel locus genetic information and positive standard (9947A, 9948) typing are shown in Table 1.
TABLE 166 InDel locus genetic information and positive standard typing
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.
2.266-plex InDels Hardy-Weinberg equilibrium test and linkage equilibrium test in Chinese Han population
Hardy-Weinberg equilibrium test is carried out on 66 loci, and the genotype distribution of other 63 loci conforms to Hardy-Weinberg equilibrium (P) except three loci of rs146109101, rs3061475 and rs2307807>0.05), the specific P values are shown in Table 2. Corrected by the Bonferroni method, the P value is 0.000758 (0.05/66), and the P values of 66InDel loci are all more than 0.000758, which accords with Hardy-Weinberg balance. Combining 66 loci in pairs for linkage equilibrium test to yield 2145 (2145 [. sub.N × (N-1))]N is the number of loci studied) to obtain the linkage equilibrium P between loci. No pairwise linkage was found at any of the 66 loci, corrected by Bonferroni, at 0.0000233 (0.05/2145). Therefore, TDP and CPE can be performedduo、CPEtrioThe calculation of (2).
2.366-Plex InDels population genetics parameter investigation in Chinese Han population
According to the statistical analysis of GenAIEx 6.5 software, except that the Minor Allele Frequency (MAF) of the rs79444593 locus is lower than 0.1, the minor allele frequency distribution of the rest 65 InDel loci is between 0.28 and 0.5, the average value is 0.4366, the allele frequency distribution 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 heterozygosity (Ho) of the other 65 loci is 0.390-0.562, the expected heterozygosity (He) is 0.409-0.500, and the difference between the heterozygosity (Ho) and the expected heterozygosity (He) is small; the individual recognition rate (DP) is 0.562-0.653, and the higher the DP is, the stronger the efficacy of the genetic marker in recognizing different individuals is; 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) is 0.99999999999999999999999999942818. Specific locus parameters are shown in table 2. Except the rs79444593 locus, the polymorphism of other loci in Chinese Han population is high, and 66-plex InDels is suitable for the individual identification research of Chinese Han population.
Table 266 InDel loci forensic genetics parameters (n 251)
Application of 2.466-plex InDels in individual recognition research
Application of 2.566-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 chinaduo) 0.942342, triple non-parent exclusion probability (CPE)trio) Is 0.994748. AGCU InDel 50kit triplet non-parent exclusion probability (CPE) in Guangzhou Han nationalitytrio) Is 0.999732. Triple non-paternal exclusion probability (CPE) for two commercial kitstrio) All are less than 0.9999, the efficiency for identifying the diad case is lower, and the method cannot be independently applied to paternity test cases. As shown in Table 3, 66-plex InDels bigeminal non-parent exclusion Probability (PE)duo) 0.0155-0.125, triple non-father exclusion Probability (PE)trio) 0.1415-0.2812, the higher the probability of excluding non-parents of a locus, the higher the efficacy of excluding non-parents when performing paternity testing analysis. CPE in 66-plex InDels for 251 unrelated individual samples analyzed by Cervus softwareduoIs 0.999739, CPEtrioIs 0.999999999417. CPE for 66-plex InDelstrioMore than 20 STR loci (CPE)trio0.999999996 (see: Yang Xiu, mu Rui, Cao Rad Wei, Wang Wu, Li Chun Xiang, Cai Jun Cheng, Liu Hua Jie, Mawenhua, Xinjiang Stone river region Kazak nationality, Hui nationality, Han 20 STR loci genetic polymorphism research [ J]Laser biology newspaper, 2021,30(04):339-347.)) has stronger identification efficacy, which indicates that 66-plex InDels can be independently applied to the analysis of triplet paternity test cases, but other commercialized kits are needed for the combined use when the system efficacy of the dyad case does not meet the requirement of forensic paternity test. The four systems are compared in the identifying efficacy of Chinese Han population, as shown in Table 4.
Table 366 InDel loci bivalent non-paternal exclusion probabilities and tripartite non-paternal exclusion probabilities (n 251)
TABLE 4 comparison of the identifying Performance of different systems
Discussion of the preferred embodiments
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 composite amplification system is generally distributed between 100-500bp, 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 results show 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 was 0.612; the average polymorphism information content is 0.365; the average cumulative paternity index was 0.974. All loci, after Bonferroni correction, obey Hardy-Weinberg equilibrium law (P > 0.000758) and no linkage disequilibrium (P > 0.0000233) between each two loci. The rs79444593 locus has an MAF of 0.4385 in the east Asian population of the International thousand-people genome project, but the MAF of the locus is 0.09761 in the Chinese Han population in the research, which shows 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 currently used in 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. A calculation was made for 66-plex InDels with a cumulative individual identification probability (TDP) of 0.99999999999999999999999999942818 and a cumulative non-parent exclusion probability (CPE) of 0.999999999417. TDP higher than DNA type 21TMThe identification efficiency of 20 autosomal STRs in the kit is high, and the CPE is high>0.9999, 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 (rs79444593) 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 populations in China, carry out the balance research of genetic parameters among the populations and explore the universality of 66-plex InDels in Chinese population.
The research obtains 66 autosomal InDel gene locus multiplex amplification systems, and provides genetic background data for individual identification and paternity identification research of the InDel gene locus in Chinese population through forensic population genetic analysis of 66-plex InDels 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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<213> Artificial sequence
<400> 110
atgtcccctg gcttatgtcc ta 22
<210> 111
<211> 25
<212> DNA
<213> Artificial sequence
<400> 111
ccccccattt cttagtgtag atggc 25
<210> 112
<211> 24
<212> DNA
<213> Artificial sequence
<400> 112
gacagtcaca gagcatagag cact 24
<210> 113
<211> 22
<212> DNA
<213> Artificial sequence
<400> 113
accttgccct gtctatgtct tc 22
<210> 114
<211> 21
<212> DNA
<213> Artificial sequence
<400> 114
gcagagtcta agcatgccct t 21
<210> 115
<211> 26
<212> DNA
<213> Artificial sequence
<400> 115
gtcctgaatg gtattgccta ggtttt 26
<210> 116
<211> 25
<212> DNA
<213> Artificial sequence
<400> 116
ccgcttttca agagtgatgt ggtac 25
<210> 117
<211> 24
<212> DNA
<213> Artificial sequence
<400> 117
tggcttaaga gaaagcacac cttt 24
<210> 118
<211> 24
<212> DNA
<213> Artificial sequence
<400> 118
atctgttcct cattctgcac ctca 24
<210> 119
<211> 27
<212> DNA
<213> Artificial sequence
<400> 119
gtcctttgcc atttttctat tgacatg 27
<210> 120
<211> 30
<212> DNA
<213> Artificial sequence
<400> 120
tggcaataac gtaaaaatgt ttggtactag 30
<210> 121
<211> 25
<212> DNA
<213> Artificial sequence
<400> 121
cctcttcctc ataaatctgg ctgtc 25
<210> 122
<211> 25
<212> DNA
<213> Artificial sequence
<400> 122
aggtagcctc tttcttcagg aatcg 25
<210> 123
<211> 23
<212> DNA
<213> Artificial sequence
<400> 123
ttactgagga tggaggcagg acg 23
<210> 124
<211> 21
<212> DNA
<213> Artificial sequence
<400> 124
ctaccagtgt gccatgtcag t 21
<210> 125
<211> 21
<212> DNA
<213> Artificial sequence
<400> 125
agcggcagaa aatgagtgac a 21
<210> 126
<211> 22
<212> DNA
<213> Artificial sequence
<400> 126
tggagaattc ccagcatgga ac 22
<210> 127
<211> 21
<212> DNA
<213> Artificial sequence
<400> 127
aaaagtgcct gctgtgggaa t 21
<210> 128
<211> 22
<212> DNA
<213> Artificial sequence
<400> 128
tcatgctgat agcccatcac ac 22
<210> 129
<211> 23
<212> DNA
<213> Artificial sequence
<400> 129
gaaatgttga cgctagggac tga 23
<210> 130
<211> 24
<212> DNA
<213> Artificial sequence
<400> 130
gagatccaaa agacagaggc atgg 24
<210> 131
<211> 26
<212> DNA
<213> Artificial sequence
<400> 131
ccaacggtgt gtaagtattc ctcttt 26
<210> 132
<211> 25
<212> DNA
<213> Artificial sequence
<400> 132
gacctgacca aactctatga actga 25
<210> 133
<211> 26
<212> DNA
<213> Artificial sequence
<400> 133
gtaataggag cagcagtacg gatttt 26
<210> 134
<211> 21
<212> DNA
<213> Artificial sequence
<400> 134
gggattacag gtgctgccac a 21
<210> 135
<211> 23
<212> DNA
<213> Artificial sequence
<400> 135
caatgcctgt cctctctgca tct 23
<210> 136
<211> 22
<212> DNA
<213> Artificial sequence
<400> 136
tcaacgacgg gaaatacgtc ct 22
<210> 137
<211> 23
<212> DNA
<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 (10)
1. Use of a human chromosomal InDel locus in any one 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.
2. The use of claim 1, wherein 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 363636043, rs34812419, rs144650377, rs 33838383839, rs 9140027, rs 27701, rs 444593, rs56110100, rs 6656357997567997567841401, rs 34799756335633794, rs 3456355635563379975634794, rs 3456355635563356335633563356349794, rs 3456345634563456345634563456345794, rs 335634563456345633563356335633563356349794, rs 34563456345634563456345634563456345634563456345634563456345634563456345633563497569, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 33563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456.
3. 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 to SEQ ID No. 144.
4. A reagent comprising the primer set according to claim 3.
5. A kit comprising the primer set according to claim 3 or the reagent according to claim 4.
6. The kit of claim 5, further comprising a reaction buffer and a hot start Taq enzyme.
7. Use of the primer set of claim 3, the reagent of claim 4, or the kit of claim 5 or 6 for individual identification or paternity testing.
8. 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 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 363636043, rs34812419, rs144650377, rs 33838383839, rs 9140027, rs 27701, rs 444593, rs56110100, rs 6656357997567997567841401, rs 34799756335633794, rs 3456355635563379975634794, rs 3456355635563356335633563356349794, rs 3456345634563456345634563456345794, rs 335634563456345633563356335633563356349794, rs 34563456345634563456345634563456345634563456345634563456345634563456345633563497569, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 33563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 3356345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456300, rs 335634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456345634563456.
9. The individual identification method according to claim 8, wherein the b-step further comprises the step of amplifying the InDel locus using a primer set.
10. The individual identification method according to claim 9, wherein the sequences of the primer sets are shown in SEQ ID No.1 to SEQ ID No. 144.
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