Screening method and application of human low-recombination-rate region micro-haplotype genetic marker

Abstract

The invention provides a screening method and application of a human low-recombination-rate region micro-haplotype genetic marker. The screening method comprises the following steps: dividing a human genome into a low-recombination-rate region and a high-recombination-rate region; identifying a haplotype which is less than 200bp in length and contains 2-5 SNPs from a low recombination rate region, and taking the haplotype as a micro haplotype; calculating forensic medicine parameters of each micro haplotype; the forensic parameters comprise individual identification capability, non-father exclusion rate and effective allele number; screening out micro haplotypes with forensic parameters within a preset range; and evaluating the paternity identification efficiency of the screened micro-haplotypes by using the contribution degree of the product, and sequencing. According to the invention, the genetic marker is screened from a low recombination region, so that the interference of exchange recombination between homologous chromosomes in a cell meiosis (passage) process on a paternity identification result can be effectively avoided; meanwhile, the paternity identification efficiency of the genetic marker is evaluated through the product contribution degree, the individual identification ability and the paternity exclusion rate can be comprehensively considered, and a favorable basis is provided for selection of the genetic marker.

Description

Technical Field

[0001] The present invention relates to the field of forensic medicine technology, and in particular to a method for screening micro-haplotype genetic markers in a low recombination rate region of humans and its application. Background Art

[0002] At present, the most widely used genetic marker in the field of forensic evidence is short tandem repeat (STR), but it has disadvantages such as high mutation rate and low detection accuracy. Genetic markers composed of single nucleotide polymorphisms (SNPs) have advantages such as low mutation rate and carrying ancestral genetic information, but the identification efficiency of a single SNP is insufficient. Microhaplotype, as a new type of genetic marker, has the advantages of low mutation rate, high polymorphism and short amplified fragment, and has shown unique advantages in the field of forensic medicine. However, in the screening process of microhaplotypes, the influence of recombination rate is not sufficiently considered. Microhaplotypes in high recombination rate areas are susceptible to meiotic recombination interference due to linkage disequilibrium, resulting in deviations in paternity test results. Therefore, developing a method for screening microhaplotype genetic markers from low recombination rate areas is of great significance for improving the accuracy and reliability of forensic identification. Summary of the Invention

[0003] To solve the above problems, the object of the present invention is to provide a method for screening micro-haplotype genetic markers in low recombination rate regions of humans and its application.

[0004] Screening methods for microhaplotype genetic markers in low recombination rate regions of humans, including:

[0005] Step 1: Divide the human genome into low recombination rate regions and high recombination rate regions;

[0006] Step 2: Identify haplotypes less than 200 bp in length and containing 2-5 SNPs from the low recombination rate region as microhaplotypes;

[0007] Step 3: Calculate the forensic parameters of each microhaplotype; the forensic parameters include: individual identification ability, non-paternal exclusion rate, and effective allele number;

[0008] Step 4: Screen out microhaplotypes whose forensic parameters are within the preset range;

[0009] Step 5: Use product contribution to evaluate the paternity identification performance of the selected micro-haplotypes and rank them.

[0010] Preferably, in step 2, the method further comprises filtering haplotypes with an allele frequency AF < 0.1 and haplotypes in repetitive sequence regions.

[0011] Preferably, in step 3, the calculation formula of individual recognition ability is:

[0012]

[0013] Among them, PD is the individual recognition ability, n is the number of alleles, p i is the frequency of the ith allele at the locus.

[0014] Preferably, in step 3, the calculation formula of the non-parent exclusion rate is:

[0015]

[0016] Where PE is the non-paternal exclusion rate, n is the number of alleles, and p i and p j represent the frequencies of the i-th and j-th alleles, respectively.

[0017] Preferably, in step 3, the calculation formula for the effective number of alleles is:

[0018]

[0019] Where Ae is the effective number of alleles, n is the number of alleles, and p i is the frequency of the ith allele.

[0020] Preferably, in step 4, the screening thresholds are set as: PD>0.75, PE>0.62, Ae>3.5.

[0021] Preferably, in step 5, the product contribution calculation formula is adopted:

[0022] CI=PD×PE

[0023] Calculate the paternity testing efficiency of the micro-haplotype after screening; where CI represents the contribution of the micro-haplotype after screening to the paternity testing efficiency, PD is the individual recognition ability, and PE is the non-paternity exclusion rate.

[0024] The present invention also provides a human low recombination rate region micro-haplotype genetic marker combination, including 30 micro-haplotypes located on autosomes, the SNP site composition of the 30 micro-haplotypes and the chromosome numbers on which they are located are:

[0025]

[0026]

[0027] The present invention also protects the use of a combination of human low recombination rate region micro-haplotype genetic markers in forensic identification.

[0028] The present invention also protects the use of a combination of micro-haplotype genetic markers in a low recombination rate region of humans in identification of kinship.

[0029] The present invention also protects the beneficial effect of the method for screening micro-haplotype genetic markers in low recombination rate regions of humans: compared with the existing technology, the present invention evaluates the paternity identification efficiency of genetic markers by multiplying the contribution degree, can comprehensively consider individual recognition ability and non-father exclusion rate, avoid the selection difficulties based on experience judgment in traditional methods, and make the evaluation of genetic markers more comprehensive.

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 A flow chart of the method for screening microhaplotype genetic markers in low recombination rate regions of humans provided by the present invention;

[0033] Figure 2 A schematic diagram showing the comparison of forensic parameters of microhaplotypes in low and high recombination regions provided by the present invention;

[0034] Figure 3 A schematic diagram comparing the mutation rates of all minor haplotypes in the low and high recombination regions provided by the present invention;

[0035] Figure 4 A comparison chart of the CPI calculation results of 30 microhaplotype genetic markers screened from low and high recombination regions in the triplet family provided by the present invention;

[0036] Figure 5 This is a graph showing the results of the simulation data validation provided by the present invention. A represents the log10(CPI) value of the microhaplotype in the low-recombination region calculated in the positive and negative data sets; B represents the log10(CPI) value of the microhaplotype selected from the low- and high-recombination regions calculated in the positive data set. DETAILED DESCRIPTION

[0037] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0038] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature identified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0039] In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "connect," "fixed," etc. should be understood broadly. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediary; or internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0040] See also Figure 1 , screening methods for micro-haplotype genetic markers in low recombination rate regions of humans, including:

[0041] Step 1: Divide the human genome into low recombination rate regions and high recombination rate regions;

[0042] In this embodiment of the present invention, based on the SNP data of the GRCh38 version of the 1000 Genomes Project and combined with the high-resolution recombination rate map (Halldorsson et al., 2019), the genome is divided into low recombination rate regions (RecRate < 0.1 cM / Mb) and high recombination rate regions (RecRate > 5 cM / Mb).

[0043] Step 2: Identify haplotypes less than 200 bp in length and containing 2-5 SNPs from the low recombination rate region as microhaplotypes;

[0044] In step 2, the Plink tool was used to identify haplotypes less than 200 bp in length and containing 2-5 SNPs from regions with low recombination rates. If a low-frequency haplotype allele appears at a haplotype locus, its heterozygosity is low and it is not suitable for use as a genetic marker for paternity testing. Furthermore, repetitive sequence regions, especially simple repeats, have a high mutation rate, which can affect paternity testing results. Therefore, low-frequency haplotypes (AF < 0.1) and haplotypes in repetitive sequence regions were filtered out. As a control, microhaplotypes were identified from regions with high recombination rates using the same method.

[0045] Step 3: Calculate the forensic parameters of each microhaplotype; the forensic parameters include: individual identification ability, non-paternal exclusion rate, and effective allele number;

[0046] Step 4: Screen out microhaplotypes whose forensic parameters are within the preset range;

[0047] Parameters such as individual discrimination ability (PD), non-paternal exclusion rate (PE), and effective allele number (Ae) were calculated for each microhaplotype, and the screening thresholds were set as PD>0.75, PE>0.62, and Ae>3.5.

[0048] PD is the probability that two individuals will have different genotypes when randomly selected from a survey population. It is an indicator for evaluating the effectiveness of a genetic marker system in identifying different individuals. The formula for calculating the individual identification ability of a haplotype is as follows:

[0049]

[0050] Where n is the number of alleles, p i is the frequency of the ith allele at the locus.

[0051] PE is used to assess the probability that a man who is not the biological father of a child can be excluded by a certain genetic marker system. The calculation method for the non-father exclusion probability of autosomal SNP haplotypes used in triplet paternity testing is as follows:

[0052]

[0053] Among them, p i and p j represent the frequencies of the i-th and j-th alleles, respectively.

[0054] The PI is the likelihood ratio of the two conditional probabilities required to determine paternity: the ratio of the probability (X) that the alleged father (also called the disputed father) is the biological father of the child to the probability (Y) that a random man is the biological father of the child (PI = X / Y). The formula for calculating the PI for a haplotype that does not undergo mutation is as follows:

[0055]

[0056] Among them, d i and d j Respectively represent the number of times i and j appear in the two alleles of the suspected father, d' i and d' j Respectively represent the number of times genes i and j appear in the two alleles of the mother, p i and p j represents the frequency of i and j in the population.

[0057] Ae is the reciprocal of the expected value of homozygosity, that is This reflects the interaction between alleles and is another indicator of gene heterozygosity. The greater the number of effective alleles, the higher the heterozygosity and the greater the degree of genetic variation.

[0058] Step 5: Use product contribution to evaluate the paternity identification performance of the selected micro-haplotypes and rank them.

[0059] The present invention defines a new indicator (i.e., product contribution CI = PD × PE) to evaluate the paternity testing efficacy of genetic markers. This indicator provides a basis for screening SNP markers for paternity testing. It can comprehensively consider individual recognition ability and non-paternal exclusion rate, thereby avoiding the selection difficulties based on empirical judgment in traditional methods, while ensuring the comprehensive efficacy of the screened SNPs.

[0060] The 30 genetic markers with the highest contribution screened out by the present invention are shown in Table 1.

[0061] Table 1 Forensic parameters of 30 microhaplotype genetic markers in low recombination rate regions

[0062]

[0063]

[0064] (1) Figure 2 As shown in the results, the paternity identification efficiency of the microhaplotypes in the low recombination rate region screened by the present invention is significantly better than that of the microhaplotypes in the high recombination rate region.

[0065] (2) High stability: The microhaplotypes screened in low recombination rate regions are less affected by meiotic recombination and have a lower mutation rate than those in high recombination rate regions (see Figure 3 ).

[0066] (3) High sensitivity: In 580 pairs of true triplet families, the cumulative paternity index (CPI) was significantly higher than that of high recombination region markers (see Figure 5 In the simulation data verification, the cumulative paternity index (CPI) of the positive set (i.e., the simulated real paternity data) is significantly higher than that of the negative set (i.e., the simulated fake paternity data) (see Figure 5A), and the accuracy of genetic markers in low recombination regions for positive sets is higher than that of markers in high recombination regions (see Figure 5 B).

[0067] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technical solution that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for screening microhaplotype genetic markers in a low recombination rate region of humans, characterized in that: include: Step 1: Divide the human genome into low recombination rate regions and high recombination rate regions; Step 2: Identify haplotypes less than 200 bp in length and containing 2-5 SNPs from the low recombination rate region as microhaplotypes; Step 3: Calculate the forensic parameters of each microhaplotype; the forensic parameters include: individual identification ability, non-paternal exclusion rate, and effective allele number; Step 4: Screen out microhaplotypes whose forensic parameters are within the preset range; Step 5: Use product contribution to evaluate the paternity identification performance of the selected micro-haplotypes and rank them.

2. The method for screening human low recombination rate region microhaplotype genetic markers according to claim 1, characterized in that: In the step 2, it also includes: filtering haplotypes with an allele frequency AF<0.1 and haplotypes in the repetitive sequence region.

3. The method for screening human low recombination rate region microhaplotype genetic markers according to claim 1, characterized in that: In step 3, the calculation formula of individual recognition ability is: Among them, PD is the individual recognition ability, n is the number of alleles, p i is the frequency of the ith allele at the locus.

4. The method for screening human low recombination rate region microhaplotype genetic markers according to claim 1, characterized in that: In step 3, the non-father exclusion rate is calculated as: Where PE is the non-paternal exclusion rate, n is the number of alleles, and p i and p j represent the frequencies of the i-th and j-th alleles, respectively.

5. The method for screening human low recombination rate region microhaplotype genetic markers according to claim 1, characterized in that: In step 3, the effective number of alleles is calculated as: Where Ae is the effective number of alleles, n is the number of alleles, and p i is the frequency of the ith allele.

6. The method for screening human low recombination rate region microhaplotype genetic markers according to any one of claims 3 to 5, characterized in that: In step 4, the screening thresholds were set as: PD>0.75, PE>0.62, and Ae>3.

5.

7. The method for screening human low recombination rate region microhaplotype genetic markers according to claims 3-5, characterized in that: In step 5, the product contribution calculation formula is used: CI=PD×PE Calculate the paternity testing efficiency of the micro-haplotype after screening; where CI represents the contribution of the micro-haplotype after screening to the paternity testing efficiency, PD is the individual recognition ability, and PE is the non-paternity exclusion rate.

8. A human low recombination rate region microhaplotype genetic marker combination, characterized in that: Including 30 micro-haplotypes located on the autosomes, the SNP site composition of the 30 micro-haplotypes and the chromosome numbers on which they are located are:

9. Use of the human low recombination rate region microhaplotype genetic marker combination according to claim 8 in forensic identification.

10. Use of the human low recombination rate region microhaplotype genetic marker combination according to claim 8 in kinship identification.

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