Method for paternity test of fetus through born child

Abstract

The invention discloses a method for paternity testing of a fetus through a born child, and belongs to the technical field of paternity testing. The method comprises the following steps: sequencing the dimorphic sites of the born child of a pregnant woman and the free DNA sample of the pregnant woman to obtain DNA data Z and S respectively; obtaining the typing site set X(S) containing fetal signals in the sample S, and obtaining the site set Y(Z) of the sample Z; counting the sites in the site set X(S)∩Y(Z) as valid sites, counting the mismatch sites of Z and S in the valid sites, and calculating the mismatch rate between the fetus and the born child according to the mismatch sites and the valid sites; judging whether the mismatch rate conforms to the mismatch rate distribution M1 under the half-sire and half-mother relationship obtained by simulation calculation or the mismatch rate distribution M2 under the same father and same mother relationship; if the mismatch rate conforms to M1, judging that the fetus and the biological father of the born child exclude the parent-child relationship; if the mismatch rate conforms to M2, judging that the fetus and the biological father of the born child are in the parent-child relationship.

Description

Technical Field

[0001] The present invention belongs to the technical field of paternity testing, and in particular relates to a method for performing paternity testing on a fetus through a born child. Background Art

[0002] DNA paternity testing uses the theories and techniques of forensic medicine, biology and genetics to analyze genetic characteristics based on the similarities in morphological structure or physiological function between offspring and parents, and to determine whether parents and children are biologically related. The most common paternity test is the paternity test of father-son relationship. The paternity test of father-son relationship usually requires a sample from the suspected father and a sample from the child or fetus, but due to working away from home or privacy issues, it is not convenient to directly obtain the sample from the suspected father. Summary of the invention

[0003] If a pregnant woman has a biological child who has been born and conceived naturally, by detecting enough SNP sites through high-throughput sequencing, the mismatch rate of these SNP sites can determine the same-sex relationship between the born child and the fetus, and then determine the parent-child relationship between the fetus and the father of the born child. Therefore, an embodiment of the present invention provides a method for performing paternity testing on a fetus through a born child, the method comprising the following steps:

[0004] S101: Sequencing the dimorphic sites of the pregnant woman's born child and the pregnant woman's free DNA sample to obtain DNA data Z and S respectively;

[0005] S102: Obtain the typing site set X(S) containing fetal signals in sample S, and obtain the site set Y(Z) of sample Z;

[0006] S103: Count the sites in the site set X(S)∩Y(Z) as valid sites, count the mismatch sites between Z and S in the valid sites, and calculate the mismatch rate between the fetus and the born child based on the mismatch sites and the valid sites;

[0007] S104: Determine whether the mismatch rate is consistent with the mismatch rate distribution M1 under the half-sibling relationship or the mismatch rate distribution M2 under the full-sibling relationship obtained by simulation calculation; if the mismatch rate is consistent with M1, it is determined that the fetus and the biological father of the born child do not have a parent-child relationship; if the mismatch rate is consistent with M2, it is determined that the fetus and the biological father of the born child are in a parent-child relationship.

[0008] Among them, M1 and M2 are obtained by the following method:

[0009] S201: Randomly generate paternal DNA sample F1, maternal DNA sample M and unrelated male DNA sample F2 through the frequency of the Chinese population; through Mendel's law of inheritance, F1 and M generate offspring Z1 and Z2, and F2 and M generate offspring Z2';

[0010] S202: Mix samples at intervals of 0.01 at fetal concentrations of 0-0.25, mix Z2 and M to obtain simulated maternal free DNA sample S2, mix Z2' and M to obtain simulated maternal free DNA sample S2', generate multiple samples for each fetal concentration, and simulate sequencing depth of 60X-150X; select autosomal SNP sites with dimorphic mutation frequencies between [0.05-0.95] on the sample genome as genetic markers; perform SNP typing according to the simulated sequencing depth;

[0011] S203: According to the method of steps S102 and S103, the mismatch rate of Z2' and Z1 is obtained from Z1 and S2', and the mismatch rate of Z2 and Z1 is obtained from Z1 and S2;

[0012] S204: The mismatching rates of Z2' and Z1 and the mismatching rates of Z2 and Z1 at each concentration are respectively obtained by using a statistical algorithm to obtain mismatching rate distributions M1 and M2.

[0013] Specifically, the M1 is [48.49, 55.55], and the M2 is [21.71, 29.84].

[0014] Wherein, the dimorphic site is selected from a SNP site, and the population frequency of the dimorphic site is 0.05-0.95.

[0015] Preferably, the concentration of S is greater than 2%, and the number of effective sites is ≥180.

[0016] Wherein, in step S102, the typing site set X(S) containing fetal signals in sample S is obtained according to formula I,

[0017] X(S)={Xi|0 <m(a)i / (m+n)i<0.2∪Xi|0<n(A)i / (m+n)i<0.2}(I),

[0018] Where n(A) and m(a) represent the observed values ​​of dimorphic sites A and a, respectively.

[0019] The present invention provides a method for performing paternity testing on a fetus through a born child. No suspected father sample is required. The paternity testing between the fetus and the biological father of the born child can be achieved by providing that the pregnant woman has a biological child that has been born and conceived naturally. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a flow chart of a method for performing paternity testing on a fetus through a born child provided by an embodiment of the present invention;

[0021] Figure 2 It is a distribution diagram of the mismatch rate between Z2' and Z1 and the mismatch rate between Z2 and Z1 at various concentrations in Example 2. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Example 1

[0024] See also Figure 1 Embodiment 1 provides a method for performing paternity testing on a fetus through a born child, the method comprising the following steps:

[0025] S101: Sequencing the dimorphic sites of the born child of the pregnant woman and the free DNA sample of the pregnant woman to obtain DNA data Z and S respectively. The suspected father and the born child are biologically father and son, and the born child is the older brother or sister of the fetus.

[0026] S102: According to formula I, the typing site set X(S) containing fetal signals in sample S is obtained, and the site set Y(Z) of sample Z is obtained.

[0027] X(S)={Xi|0 <m(a)i / (m+n)i<0.2∪Xi|0<n(A)i / (m+n)i<0.2}(I),

[0028] Where n(A) and m(a) represent the observed values ​​of dimorphic sites A and a, respectively.

[0029] SNP typing method: After sequencing and analysis, each SNP site of each sample will have a total sequencing depth, as well as the depth of the "wild type" and "mutant" sites determined according to the human genome reference sequence. Ordinary samples: Taking a certain SNP site as an example, A represents the wild type site and a represents the mutant site. If the total depth of the site in the sequencing result is 100X, where A is 100X and a is 0X, then the site is a homozygous wild type site, recorded as AA; if A is 0X and a is 100X, then it is a homozygous mutant site, recorded as aa; if the sequencing depth of A and a is close to 1:1, then the site is heterozygous, recorded as Aa. Pregnant free DNA sample: If the sequencing depth ratio of A and a at this site is A / (A+a)<0.2, then the genotype of the pregnant woman herself is recorded as aa, and the fetus's genotype contains an A. The pregnant woman and the fetus are in a parent-child relationship, then the fetus is Aa at this site. Similarly, if the sequencing depth ratio of A and a at the site is a / (A+a)<0.2, then the fetus's genotype is detected to contain an a, and the fetus is Aa at the site.

[0030] S103: Count the sites in the site set X(S) ∩ Y(Z) as valid sites and mismatched sites. In this step, in the maternal plasma DNA sample S2, at each site, when 0 < m(a)i / (m + n)i < 0.2, it can be considered that the genotype of the mother at this site is AA, and the genotype of the fetus contains a. If the sample being compared can provide an a at this position, that is, the genotype is Aa or aa, then this site is recorded as a matching site, otherwise it is a mismatched site. When 0 < n(A)i / (m + n)i < 0.2, it can be considered that the genotype of the mother at this site is aa, and the genotype of the fetus contains A. If the sample being compared can provide an A at this position, that is, the genotype is Aa or AA, then this site is recorded as a matching site, otherwise it is a mismatched site. Count the mismatched sites between Z and S among the valid sites, and calculate the mismatch rate between the fetus and the born child according to the mismatched sites and the valid sites. The means of counting the number of mismatched sites are conventional techniques. The mismatch rate calculation formula is as follows:

[0031]

[0032] S104: Determine whether the mismatch rate conforms to the mismatch rate distribution M1 under the relationship of half - siblings or the mismatch rate distribution M2 under the relationship of full - siblings obtained by simulation calculation; if the mismatch rate conforms to M1, then determine that the biological father of the fetus and the born child excludes the paternity relationship; if the mismatch rate conforms to M2, then determine that the biological father of the fetus and the born child is in a paternity relationship. Among them, M1 and M2 can be obtained by the simulation calculation in Example 2; specifically, M1 is [48.49, 55.55], and M2 is [21.71, 29.84].

[0033] Among them, the dimorphic sites are selected from SNP sites, and the population frequency of the dimorphic sites is 0.05 - 0.95.

[0034] Preferably, the concentration of S is greater than 2%, and the number of valid sites ≥ 180 to ensure the accuracy rate.

[0035] Example 2

[0036] Example 2 provides the calculation process for simulating the mismatch rate density distribution ranges M1 and M2, and the process is as follows:

[0037] S201: Randomly generate a paternal DNA sample F1, a maternal DNA sample M, and an unrelated male DNA sample F2 according to the Chinese population frequency; through Mendel's genetic law, generate offspring Z1 and Z2 from F1 and M, then Z1 and Z2 are full - siblings; generate offspring Z2' from F2 and M, then Z1 and Z2' are half - siblings.

[0038] S202: Mix samples at intervals of 0.01 at fetal concentrations of 0-0.25, mix Z2 and M to obtain simulated maternal free DNA sample S2, mix Z2' and M to obtain simulated maternal free DNA sample S2', generate multiple samples for each fetal concentration (specifically, 50 samples, then the number of samples S2' and S2 are both 1250), and the simulated sequencing depth is 60X-150X; select 2500 (SNP data from: ftp: / / ftp.ncbi.nlm.nih.gov / snp / .redesign / .archive / b155 / VCF / GCF_000001405.39.gz) autosomal SNP sites with a mutation frequency between [0.05-0.95] on the sample genome as genetic markers; perform SNP typing according to the simulated sequencing depth.

[0039] S203: According to the method of steps S102 and S103, the typing site set X(S2) containing fetal signals in sample S2 and the typing site set X(S2') containing fetal signals in sample S2' are obtained according to formula I, and the detection site set Y(Z1) of sample Z1 is obtained. The number of sites in the site set X(S2)∩Y(Z1) and X(S2')∩Y(Z1) is counted as effective sites, and the mismatch rate between the fetus and the born child is calculated based on the mismatch sites and the corresponding effective sites. That is, the mismatch rate of Z2' and Z1 is obtained from Z1 and S2', and the mismatch rate of Z2 and Z1 is obtained from Z1 and S2.

[0040] S204: The mismatch rate between Z2' and Z1 and the mismatch rate between Z2 and Z1 at each concentration are calculated using a statistical algorithm (such as [mean-1.96*sd, mean+1.96*sd] according to the 95% confidence interval) to obtain the mismatch rate distributions M1 and M2 respectively. Figure 2 As shown, with proportional concentration as the horizontal axis and mismatch rate as the vertical axis, the mismatch rate of Z2' and Z1 and the mismatch rate of Z2 and Z1 at each concentration are plotted in the same coordinate system. The lower part of the coordinate system is the same father and the same mother, and the upper part of the coordinate system is the same mother and the different fathers. It can be seen from the figure that the mismatch rate of Z2' and Z1 is mainly concentrated in the range of [48.49,55.55], and the mismatch rate of Z2 and Z1 is mainly concentrated in the range of [21.71,29.84].

[0041] Example 3

[0042] Example 3 discloses a theoretical calculation method for the mismatch rate between a fetus and a born child:

[0043] High-throughput sequencing is used to detect the dimorphic sites of the DNA samples of the born children, which are denoted as S1; the dimorphic site fetal DNA data of the free DNA samples of the pregnant women is S2, and the free DNA samples of the pregnant women are separated by the peripheral blood of the pregnant women. After sequencing and analysis, each SNP site of each sample will have a total sequencing depth, as well as the depth of the "wild type" and "mutant" sites determined according to the human genome reference sequence. The present invention only discusses dimorphic sites, and the dimorphic sites are represented by A and a respectively. Taking a certain SNP site as an example, A represents the wild-type site, and the population frequency is denoted as p, a represents the mutant site, and the population frequency is denoted as q, and p+q=1 is satisfied. Then the AA genotype frequency is p 2 ; aa genotype frequency is q 2 ; The frequency of Aa genotype is 2pq.

[0044] The selection condition of S1 is based on the genotype of each site in the leukocyte DNA (denoted as M) of the pregnant woman to be tested and the depth distribution of each site in the peripheral blood free DNA. When the sequencing depth is higher than the threshold (>60X), there are at least two polymorphic sites at the site, and the corresponding leukocyte DNA is homozygous (M genotype is AA / aa), that is, the S2 genotype is Aa. Among them, the dimorphic site is selected from SNP sites, INDEL sites and / or STR sites, and the population frequency of the dimorphic site is 0.05-0.95.

[0045] If S1 and S2 are related by the same father and mother, assuming that M's genotype is AA and S2's genotype is Aa. F is a normal individual, and the frequency of the dimorphism distribution at a certain SNP site is: P (A) = p, P (a) =q. Since S2, F and M all have a parent-child relationship at this SNP site, the genotype of F must carry a. Therefore, the distribution probability of the F genotype at this SNP site is: P (Aa) =p and P (aa) =q. From this, we can infer that since S1 and F, M (AA) meet the parent-child relationship at this SNP site, the probability of S1 genotype distribution at this site is: P (AA) =p 2 , P (Aa) =(q 2 +2pq). When the genotype of S1 is Aa, S2(Aa) can provide an a allele, that is, S1(Aa) and S2(Aa) are judged as a match at this site; while S2(AA) cannot provide an a allele at this site, it is judged as a mismatch. Therefore, at this SNP site, if S1 and S2 are of the same parent, the mismatch rate is p 2 .

[0046] If S1 and S2 are half-siblings, assuming the mother's genotype is AA, S2's genotype is Aa, and F is a normal individual, the frequency of the dimorphism distribution at a certain SNP site is: P (A) =p,P (a) =q. Since F and S2 do not conform to the parent-offspring relationship, the distribution frequency of the F genotype at this site is P (AA) =p 2 , P (aa) =q 2 , P (Aa) =2pq, and the genotype of M is AA, then the genotype of S1 at this SNP site is P (AA) =p and P (Aa) =q. When the genotype of S1 is Aa, S2(Aa) can provide an a allele, that is, S1(Aa) and S2(Aa) are judged as a match at this site; while S2(AA) cannot provide an a allele at this site, it is judged as a mismatch. Therefore, at this SNP site, if S1 and S2 are in a half-sister relationship, the mismatch rate is p.

[0047] In the normal population, p is approximately equal to q, so the mismatch rate p 2 and p are 25% and 50% respectively. However, in actual cases, a set of valid sites is selected. The values ​​of p in these sets may vary greatly, but they conform to the normal distribution, so the actual mismatch rate is different from 25% and 50%.

[0048] If S1 and S2 are of the same father and mother, the theoretical mismatch rate is about 0.25, corresponding to the simulated mismatch rate density distribution range of [21.71, 29.84]; if S1 and S2 are of the same mother and half father, the theoretical mismatch rate is about 0.5, corresponding to the simulated mismatch rate density distribution range of [48.49, 55.55].

[0049] Embodiment 4:

[0050] Example 4 discloses a method for paternity testing of a fetus through a born child, including: it is known that A is a born child of pregnant woman M1, B is a fetus of pregnant woman M1 during pregnancy, and A and B are of the same father and mother. Blood from A and M is drawn and marked as samples CS294 and CS295, respectively, for full sibling (same father and same mother) relationship identification. The sequencing depth of CS295 is 74.5X, and the concentration of B in free DNA is 14.874%, which meets the requirements. The SNP typing results of CS294 and CS295 are obtained by sequencing analysis. According to the rules of Example 1, a total of 561 valid sites for the two samples are obtained, and the mismatch rate is calculated.

[0051] Detection conclusion: There are 135 mismatch sites, and the calculated mismatch rate is 24.1%, which is consistent with the mismatch rate density distribution range of this patent [21.71, 29.84]. This supports that CS294 and CS295 are of the same father and mother, that is, the biological father of CS294 and CS295 is in a parent-child relationship.

[0052] Embodiment 5:

[0053] Example 5 discloses a method for paternity testing of a fetus through a born child, including: it is known that C is a born child of pregnant woman M2, D is a fetus of pregnant woman M2 during pregnancy, and C and D are in a half-sibling relationship. Blood from C and M is drawn and marked as samples CS296 and CS297, respectively, for full sibling (same father and same mother) relationship identification. The sequencing depth of CS296 is 108X, and the concentration of D in free DNA is 12.101%, which meets the requirements. The SNP typing results of CS296 and CS297 are obtained by sequencing analysis. According to the rules of Example 1, a total of 605 valid sites for the two samples are obtained, and the mismatch rate is calculated.

[0054] Detection conclusion: There are 304 mismatch sites, and the mismatch rate is calculated to be 50.2%, which is consistent with the mismatch rate density distribution range of this patent [48.49, 55.55]. This supports that CS296 and CS297 are half-siblings, that is, the biological father of CS296 and CS297 excludes the parent-child relationship.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for performing paternity testing on a fetus through a born child, characterized in that: The method comprises the following steps: S101: Sequencing the dimorphic sites of the pregnant woman's born child and the pregnant woman's free DNA sample to obtain DNA data Z and S respectively; S102: Obtain the typing site set X(S) containing fetal signals in sample S, and obtain the site set Y(Z) of sample Z; S103: Count the sites in the site set X(S)∩Y(Z) as valid sites, count the mismatch sites between Z and S in the valid sites, and calculate the mismatch rate between the fetus and the born child based on the mismatch sites and the valid sites; S104: Determine whether the mismatch rate conforms to the mismatch rate distribution M1 under the half-sibling relationship or the mismatch rate distribution M2 under the same-sibling relationship obtained by simulation calculation; if the mismatch rate conforms to M1, it is determined that the fetus and the biological father of the born child are excluded from the parent-child relationship; if the mismatch rate conforms to M2, it is determined that the fetus and the biological father of the born child are in the parent-child relationship; The dimorphic site is selected from a SNP site, and the population frequency of the dimorphic site is 0.05-0.95; The concentration of S is greater than 2%, and the number of effective sites is ≥180; In step S102, the typing site set X(S) containing fetal signals in sample S is obtained according to formula I, X(S)={Xi|0 <m(a)i / (m+n)i<0.2∪Xi|0<n(A)i / (m+n)i<0.2}(I), Where n(A) and m(a) represent the observed values ​​of dimorphic sites A and a, respectively; The M1 and M2 are obtained by the following method: S201: Randomly generate paternal DNA sample F1, maternal DNA sample M and unrelated male DNA sample F2 through the frequency of the Chinese population; through Mendel's law of inheritance, F1 and M generate offspring Z1 and Z2, and F2 and M generate offspring Z2'; S202: Mix samples at intervals of 0.01 at fetal concentrations of 0-0.25, mix Z2 and M to obtain simulated maternal free DNA sample S2, mix Z2' and M to obtain simulated maternal free DNA sample S2', generate multiple samples for each fetal concentration, and simulate sequencing depth of 60X-150X; select autosomal SNP sites with dimorphic mutation frequencies between [0.05-0.95] on the sample genome as genetic markers; perform SNP typing according to the simulated sequencing depth; S203: According to the method of steps S102 and S103, the mismatch rate of Z2' and Z1 is obtained from Z1 and S2', and the mismatch rate of Z2 and Z1 is obtained from Z1 and S2; S204: The mismatching rates of Z2' and Z1 and the mismatching rates of Z2 and Z1 at each concentration are respectively obtained by using a statistical algorithm to obtain mismatching rate distributions M1 and M2.

2. The method for paternity testing of a fetus through a born child according to claim 1, characterized in that: The M1 is [48.49, 55.55], and the M2 is [21.71, 29.84].

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