Method for paternity testing of a pregnant fetus without the father

Abstract

This invention discloses a method for paternity testing of a pregnant fetus without the need for a paternal parent, belonging to the field of paternity testing technology. It includes the following steps: S101: Sequencing the dimorphic sites of DNA samples from the alleged father's direct relatives and the pregnant woman's cell-free DNA sample to obtain DNA data Z and S respectively, wherein the direct relatives are related to the alleged father as either parent-child or full siblings; S102: Obtaining the set of genotyping sites X(S) containing fetal signals in sample S, and obtaining the set of sites Y(Z) in sample Z; S103: Statistically analyzing the sites in the set X(S)∩Y(Z) as valid sites, counting the mismatch sites between Z and S among the valid sites, and calculating the mismatch rate K between the alleged father's direct relatives and the fetus based on the mismatch sites and valid sites; S104: Determining whether the mismatch rate K conforms to the mismatch rate distribution calculated from a large number of simulated samples under the same kinship; if it does, then the alleged father and the fetus are related as parent-child.

Description

Technical Field

[0001] This invention belongs to the field of paternity testing technology, and specifically relates to a method for paternity testing of a pregnant fetus without the need for the father. Background Technology

[0002] DNA paternity testing utilizes theories and techniques from forensic medicine, biology, and genetics to analyze genetic characteristics based on morphological or physiological similarities between parents and offspring, determining whether a parent and child are biologically related. The most common type of paternity testing is determining parentage based on the father's relationship. This usually requires samples from both the alleged father and the child or fetus. However, due to factors such as working away from home or privacy concerns, directly obtaining samples from the alleged father is often inconvenient. Summary of the Invention

[0003] This method involves obtaining samples from the alleged father's biological parents or siblings to conduct paternity testing. The method includes the following steps:

[0004] S101: Sequencing the dimorphic sites of DNA samples from the suspected father's immediate family members and the pregnant woman's cell-free DNA sample yields DNA data Z and S, respectively. The immediate family members are either parent-child or full siblings of the suspected father.

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

[0006] S103: The sites in the statistical site set X(S)∩Y(Z) are taken as valid sites. The mismatch sites between Z and S in the valid sites are counted. The mismatch rate K between the suspected father's lineal relatives and the fetus is calculated based on the mismatch sites and valid sites.

[0007] S104: Determine whether the mismatch rate K conforms to the mismatch rate distribution calculated from a large number of simulated samples under the same kinship; if it does, then the suspected father and the fetus are related.

[0008] Specifically, if the direct relative and the suspected father are in a parent-child relationship and K conforms to the mismatch rate distribution KY calculated from a large number of simulated samples under the grandfather-grandson relationship, then the suspected father and the fetus are in a parent-child relationship; if the direct relative and the suspected father are full siblings and K conforms to the mismatch rate distribution KT calculated from a large number of simulated samples under the uncle-nephew relationship, then the suspected father and the fetus are in a parent-child relationship.

[0009] In this process, multiple cell-free DNA samples from pregnant women and DNA samples from the immediate family of the fathers were simulated at different concentrations. The fathers were only related to the fetuses corresponding to some of the cell-free DNA samples from pregnant women. The simulated mismatch rate between the immediate family of the fathers and the fetuses was calculated in steps S102 and S103. The simulated mismatch rate distribution was obtained by statistically analyzing the simulated mismatch rate when the fathers and fetuses were related.

[0010] KY and KT were obtained through the following method:

[0011] S201: Generate paternal DNA sample F1, parental DNA sample F2 of F1, full sibling DNA sample F3 of F1, maternal DNA sample M, and unrelated male DNA sample T by randomizing the frequency of the Chinese population; and generate offspring Z from F1 and M, and offspring Z' from T and M according to Mendel's laws of inheritance.

[0012] S202: Fetal concentrations of 0-25% were mixed in an isogradient manner. Z and M were mixed to obtain a simulated sample of cell-free DNA from the pregnant woman, S1; Z' and M were mixed to obtain a simulated sample of cell-free DNA from the pregnant woman, S2. Multiple simulated samples were generated for each simulated concentration. The simulated sequencing depth was 100X-200X. Autosomal SNP sites with a mutation frequency between [0.05-0.95] on the sample genome were selected as genetic markers. SNP genotyping was performed based on the simulated sequencing depth.

[0013] S203: According to the methods of steps S102 and S103, the simulated mismatch rate k1 of Z and F2 is obtained from S1 and F2, the simulated mismatch rate k2 of Z and F3 is obtained from S1 and F3, and the simulated mismatch rate k3 of Z' and F2 or F3 is obtained from S2 and F2 or F3.

[0014] S204: Statistically calculate k1 at different simulated concentrations and use a statistical algorithm to obtain the mismatch rate distribution KY; statistically calculate k2 at different concentrations and use a statistical algorithm to obtain the mismatch rate distribution KT; statistically calculate k3 at different concentrations and use a statistical algorithm to obtain the mismatch rate distribution KW.

[0015] Specifically, KY is [11.34%, 16.53%], KT is [11.41%, 16.46%], and KW is [24.29%, 32.08%].

[0016] Specifically, the gradient difference for equal gradients is 0.5% or 1%, 50 samples are generated for each simulated concentration, and more than 2,000 SNP sites are selected from the sample genome.

[0017] The dimorphic loci are selected from SNP loci, and the population frequency of the dimorphic loci is 0.05-0.95.

[0018] In step S102, the set of genotyping sites containing fetal signals is obtained according to formula I.

[0019] X(S)={X i |0 <na i (S) / n i (S)<0.2∪0 <nA i (S) / n i(S)<0.2}(I),

[0020] Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, and n = nA + na.

[0021] This invention provides a method for paternity testing of a pregnant fetus without the need for a paternal sample. It indirectly determines the paternity relationship between the fetus and the suspected father by accurately determining the kinship between the fetus and the suspected father's direct relatives. Attached Figure Description

[0022] Figure 1 This is a flowchart of a method for performing paternity testing on a pregnant fetus without the father, provided in an embodiment of the present invention;

[0023] Figure 2 This is a graph showing the relationship between fetal concentration and mismatch rate across different kinship groups;

[0024] Figure 3 This is a graph showing the relationship between fetal concentration and mismatch rate in a grandfather-granddaughter relationship.

[0025] in, Figure 2 In the coordinate system, the lower part represents the mismatch rate under grandfather-grandson and uncle-nephew relationships, with the mismatch rates of the two relationships partially overlapping; the upper part represents the mismatch rate under no-kinship relationships. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] See Figure 1 Example 1 provides a method for paternity testing of a pregnant fetus without the father's biological parentage. The method includes the following steps:

[0029] S101: DNA data Z and S were obtained by sequencing the dimorphic sites in DNA samples from the suspected father's immediate family members and the pregnant woman's cell-free DNA sample. The immediate family members are either parent-child or full siblings of the suspected father; more specifically, the immediate family member is the suspected father's father or mother; or the suspected father's older brother, older sister, younger sister, or younger brother. The dimorphic sites were selected from SNP sites, with a population frequency of 0.05-0.95.

[0030] S102: According to Formula I, obtain the set of genotyping sites X(S) containing fetal signals in sample S, and obtain the set of sites Y(Z) in sample Z;

[0031] X(S)={X i |0 <na i(S) / n i (S)<0.2∪0 <nA i (S) / n i (S)<0.2}(I).

[0032] SNP genotyping method: After sequencing and analysis, each SNP locus in each sample will have a total sequencing depth, as well as the depth of "wild-type" and "mutant" loci determined based on the human genome reference sequence. For ordinary samples: Taking a specific SNP locus as an example, let A represent the wild-type locus and a represent the mutant locus. If the total depth of this locus in the sequencing results is 100X, where A is 100X and a is 0X, then this locus is a homozygous wild-type locus, denoted as AA; if A is 0X and a is 100X, then it is a homozygous mutant locus, denoted as aa; if the sequencing depth of A and a is close to 1:1, then this locus is heterozygous, denoted as Aa. For cell-free DNA samples from pregnant women: If the sequencing depth ratio of A and a at this locus is A / (A+a) < 0.2, then the pregnant woman's genotype is denoted as aa. If the fetus's genotype contains one A, and the pregnant woman and fetus are related, then the fetus at this locus is denoted as Aa. Similarly, if the sequencing depth ratio of A and a at this locus is a / (A+a)<0.2, then the fetal genotype contains one a, and the fetus at this locus is Aa.

[0033] S103: Sites in the statistical site set X(S)∩Y(Z) are considered as valid sites and mismatch sites. In this step, in the pregnant woman's cell-free DNA sample S2, at each site, when 0 <na i (S) / n i When (S) < 0.2, the maternal genotype at this locus can be considered to be AA, and the fetal genotype contains 'a'. If the sample being compared can provide 'a' at this location, i.e., the genotype is Aa or aa, then this locus is recorded as a matching locus; otherwise, it is a mismatch locus. When 0 <nA i (S) / n i When (S) < 0.2, the maternal genotype at this locus is considered to be aa, and the fetal genotype contains A. If the compared sample provides an A at this locus, i.e., the genotype is Aa or AA, then this locus is recorded as a matched locus; otherwise, it is a mismatch locus. The number of mismatched loci (Z and S) in the valid loci is counted, and the mismatch rate between the fetus and the born child is calculated based on the mismatched loci and the valid loci. The method for counting mismatched loci is a standard technique. The formula for calculating the mismatch rate is as follows:

[0034]

[0035] S104: Determine whether the mismatch rate K conforms to the mismatch rate distribution calculated from a large number of simulated samples under the same kinship relationship; if it does, then the suspected father and the fetus are related as parents. Specifically, if the direct relative and the suspected father are related as parents and K conforms to the mismatch rate distribution KY calculated from a large number of simulated samples under the grandfather-grandson relationship, then the suspected father and the fetus are related as parents; if the direct relative and the suspected father are full siblings and K conforms to the mismatch rate distribution KT calculated from a large number of simulated samples under the uncle-nephew relationship (of course, it can also be calculated using a large number of actual samples, but obtaining the samples is more difficult), then the suspected father and the fetus are related as parents.

[0036] In this study, multiple cell-free DNA samples from pregnant women and DNA samples from the immediate family of the fathers were simulated at different concentrations. The fathers were only related to the fetuses corresponding to some of the pregnant women's cell-free DNA samples. The simulated mismatch rate between the fathers' immediate family members and the fetuses was calculated in steps S102 and S103, and the mismatch rate distribution was obtained by statistically analyzing the simulated mismatch rate under the condition that the father and fetus were related. Specifically, KY was [11.34%, 16.53%], KT was [11.41%, 16.46%], and KW was [24.29%, 32.08%]. KY, KT, and KW can be used as constants and directly used in each judgment. Specifically, if the direct relative and the suspected father are related as parents and K matches the mismatch rate distribution calculated from a large number of simulated samples under the grandfather-grandson relationship [11.34%, 16.53%], then the suspected father and the fetus are related as parents; if the direct relative and the suspected father are full siblings and K matches the mismatch rate distribution calculated from a large number of simulated samples under the uncle-nephew relationship [11.41%, 16.46%], then the suspected father and the fetus are related as parents; if K matches [24.29%, 32.08%], then the fetus and the suspected father's direct relative are not related, and thus it is determined that the suspected father and the fetus are not related as parents.

[0037] Example 2

[0038] Example 2 provides that KY and KT are obtained through the following method:

[0039] S201: Generate paternal DNA sample F1, parental DNA sample F2 of F1, full sibling DNA sample F3 of F1, maternal DNA sample M, and unrelated male DNA sample T by randomizing the frequency of the Chinese population; and generate offspring Z from F1 and M, and offspring Z' from T and M, according to Mendel's laws of inheritance.

[0040] S202: Fetal concentrations of 0-25% were mixed using an isogradient method, with each gradient being 1%; specifically 0%, 1%, 2%...25%. Cell-free DNA simulation samples S1 were obtained by mixing Z and M, and S2 were obtained by mixing Z' and M. 50 simulation samples were generated for each concentration, resulting in 1250 samples for S1 and S2, and 50 samples for F2 and F3. The simulated sequencing depth was 100X-200X. 2500 autosomal SNP loci with bimorphic mutation frequencies between [0.05-0.95] were selected as genetic markers. SNP data were obtained from: ftp: / / ftp.ncbi.nlm.nih.gov / snp / .redesign / .archive / b155 / VCF / GCF_000001405.39.gz. SNP genotyping was performed based on the simulated sequencing depth.

[0041] S203: According to the methods in steps S102 and S103, the simulated mismatch rate k1 of Z and F2 is obtained from S1 and F2, the simulated mismatch rate k2 of Z and F3 is obtained from S1 and F3, and the simulated mismatch rate k3 of Z' and F2 or F3 is obtained from S2 and F2 or F3.

[0042] S204: Calculate the mismatch rate distribution KY for different simulated concentrations of k1 using a statistical algorithm; calculate the mismatch rate distribution KT for different concentrations of k2 using a statistical algorithm; and calculate the mismatch rate distribution KW for different concentrations of k3 using a statistical algorithm. Specifically, the statistical algorithm can be calculated using 95% confidence intervals: [mean - 1.96*sd, mean + 1.96*sd]. The calculated values ​​are: KY = [11.34%, 16.53%], KT = [11.41%, 16.46%], and KW = [24.29%, 32.08%].

[0043] Example 3

[0044] Example 3 discloses a theoretical calculation method for the mismatch rate between a pregnant fetus and its grandparents:

[0045] (1) If fetal signal 'a' appears in the cell-free DNA sample of the pregnant woman, compared with the true father's parent (hereinafter referred to as grandfather): the fetus has genotype 'a' and is related to the mother. According to Mendel's laws of inheritance, the fetus's 'a' must be provided by the true father. Therefore, the true father's genotype is Aa or aa, with a probability of p(aa) = q; p(Aa) = p. Let the total number of fetal signal loci be n, of which the number of fetal signal loci with the fetal signal A is m; and the number of fetal signal loci with the fetal signal a is k. Based on this, if the true father's genotype is aa, he can definitely provide a 'a'. Under this condition, the grandfather's locus matches the fetus's. If the true father's genotype is Aa, and the grandfather's genotype is one of AA, aa, or Aa, only when the grandfather is AA will the fetus's locus be mismatched with the grandfather's, with a mismatch rate of p*p. 2 =p 3 .

[0046] (2) Similarly, if fetal signal A appears in the pregnant woman's cell-free DNA sample, the mismatch rate is q. 3 .

[0047] (3) The total mismatch rate is:

[0048]

[0049] When q = p = 0.5, P is 12.5%, which is within the simulation range [11.34%, 16.53%].

[0050] Example 4

[0051] Example 4 discloses a theoretical calculation method for the mismatch rate between a pregnant fetus and its paternal uncle or aunt:

[0052] (1) If fetal signal 'a' appears in the cell-free DNA sample of the pregnant woman, compared with the full siblings of the true father: the fetus has genotype 'a' and is related to the mother, according to Mendel's laws of inheritance, the fetus's 'a' must be provided by the true father, so the true father's genotype is Aa or aa, with probabilities p(aa) = q; p(Aa) = p; based on this, if the true father's genotype is aa, then the grandfather must provide 'a', under this condition, the probability that the grandfather is Aa is p; the probability that the grandfather is aa is q. When the grandfather is Aa, the probability that the grandfather's offspring (the true father's full siblings) are AA is q*(1 / 2P*1 / 2P) = 1 / 4q*p 2 When the grandfather's genotype is aa, the offspring will definitely carry 'a', and this is a match. If the true father's genotype is Aa, and the grandfather's genotype is one of AA, aa, or Aa, with probabilities p, p, and p respectively. 2 q 2 2pq; When the grandfather is AA, the probability that the grandfather's offspring (all siblings of the true father) are AA is: P*P 2 *1 / 2p=1 / 2P4 When the grandfather is Aa, the probability that his offspring (all siblings of the true father) are AA is: p*1 / 2*2pq*P=qp 3 ,

[0053] (2) If fetal signal A is present in the pregnant woman's cell-free DNA sample,

[0054] (3) The total mismatch rate is:

[0055]

[0056] When q = p = 0.5, P is 12.5%, which is within the simulation range [11.41%, 16.46%].

[0057] Example 5:

[0058] Example 5 discloses a specific instance of paternity testing using the method of the present invention, as follows: A family numbered RT5081 submitted three nail samples, numbered RTZ5081S1, RTZ5081S3, and RTZ5081M. Sequencing analysis revealed a parent-child relationship between samples RTZ5081S1 and RTZ5081S3; a parent-child relationship between samples RTZ5081M and RTZ5081S3; and a grandparent-grandchild relationship between samples RTZ5081M and RTZ5081S3. Consultation confirmed that RTZ5081S3 is the mother of RTZ5081S1, and RTZ5081M is the grandmother of RTZ5081S1. Based on this, samples were mixed at 0.01 increments from 0 to 0.25, with each concentration mixed 50 times. By mixing samples RTZ5081S3 and RTZ5081S1, a simulated cell-free DNA sample S from a pregnant woman could be obtained. The analysis site set was obtained according to the method in the simulation, and the mismatch rate of sample S and RTZ5081M was calculated. The results are shown in Table 3, which are consistent with [11.34%, 16.53%].

[0059] Example 6:

[0060] Example 6 discloses a specific instance of paternity testing using the method of the present invention, as follows: A family numbered QZ21457 submitted a peripheral blood sample from a pregnant woman, numbered QZ21457S1, and a toothbrush sample from the fetus's true father's brother, numbered QZY21457F, for identification. SNP genotyping results for QZ21457S1 and QZY21457F were obtained through sequencing analysis. The analysis locus sets for the two samples were obtained according to the method steps in the data simulation, and the mismatch rate was calculated. The detection conclusion: the mismatch rate between the two samples was 12.24% (consistent with [11.41%, 16.46%]), which is within the mismatch rate density distribution range for full siblings of the fetus and true father according to the present invention, supporting the relationship between QZ21457S1 and QZY21457F as uncle and nephew.

[0061] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for paternity testing of a pregnant fetus without requiring the father's biological father, characterized in that... The method includes the following steps: S101: Sequencing the dimorphic sites of DNA samples from the suspected father's immediate family members and the pregnant woman's cell-free DNA sample yields DNA data Z and S, respectively. The immediate family members are either parent-child or full siblings of the suspected father. S102: Obtain the set of genotyping sites X(S) containing fetal signals in sample S, and obtain the set of sites Y(Z) in sample Z; S103: The sites in the statistical site set X(S)∩Y(Z) are taken as valid sites. The mismatch sites between Z and S in the valid sites are counted. The mismatch rate K between the suspected father's lineal relatives and the fetus is calculated based on the mismatch sites and valid sites. S104: Determine whether the mismatch rate K conforms to the mismatch rate distribution calculated from a large number of simulated samples under the same kinship relationship; if the direct relative and the suspected father are in a parent-child relationship and K conforms to the mismatch rate distribution KY calculated from a large number of simulated samples under the grandfather-grandson relationship, then the suspected father and the fetus are in a parent-child relationship; if the direct relative and the suspected father are full siblings and K conforms to the mismatch rate distribution KT calculated from a large number of simulated samples under the uncle-nephew relationship, then the suspected father and the fetus are in a parent-child relationship. The KY percentage is [11.34%, 16.53%], and the KT percentage is [11.41%, 16.46%]; The KY and KT are obtained through the following method: S201: Generate paternal DNA sample F1, parental DNA sample F2 of F1, full sibling DNA sample F3 of F1, maternal DNA sample M, and unrelated male DNA sample T by randomizing the frequency of the Chinese population; and generate offspring Z from F1 and M, and offspring Z' from T and M according to Mendel's laws of inheritance. S202: Fetal concentrations of 0-25% were mixed in an isogradient manner. Z and M were mixed to obtain a simulated sample of cell-free DNA from the pregnant woman, S1. Z' and M were mixed to obtain a simulated sample of cell-free DNA from the pregnant woman, S2. Multiple simulated samples were generated for each simulated concentration. The simulated sequencing depth was 100X-200X. Autosomal SNP sites with a mutation frequency between [0.05-0.95] on the sample genome were selected as genetic markers. SNP genotyping was performed based on the simulated sequencing depth. S203: According to the methods of steps S102 and S103, the simulated mismatch rate k1 of Z and F2 is obtained from S1 and F2, the simulated mismatch rate k2 of Z and F3 is obtained from S1 and F3, and the simulated mismatch rate k3 of Z' and F2 or F3 is obtained from S2 and F2 or F3. S204: Statistically calculate k1 at different simulated concentrations and use a statistical algorithm to obtain the mismatch rate distribution KY; statistically calculate k2 at different concentrations and use a statistical algorithm to obtain the mismatch rate distribution KT; statistically calculate k3 at different concentrations and use a statistical algorithm to obtain the mismatch rate distribution KW.

2. The method for paternity testing of a pregnant fetus without the need for a father, as described in claim 1, is characterized in that... Multiple cell-free DNA samples from pregnant women and DNA samples from the first-degree relatives of the fathers were simulated at different concentrations. The fathers were only related to the fetuses corresponding to some of the cell-free DNA samples from pregnant women. The simulated mismatch rate between the first-degree relatives of the fathers and the fetuses was calculated in steps S102 and S103. The simulated mismatch rate distribution was obtained by statistically analyzing the simulated mismatch rate when the fathers and fetuses were related.

3. The method for paternity testing of a pregnant fetus without the need for paternal parentage, as described in claim 1, is characterized in that... The KW is [24.29%, 32.08%].

4. The method for paternity testing of a pregnant fetus without the need for a paternal parent, as described in claim 1, is characterized in that... The gradient difference for equal gradients is 0.5% or 1%, and 50 samples are generated for each simulated concentration, selecting more than 2000 SNP sites on the sample genome.

5. The method for paternity testing of a pregnant fetus without the need for a father, as described in claim 1, is characterized in that... The dimorphic loci are selected from SNP loci, and the population frequency of the dimorphic loci is 0.05-0.

95.

6. The method for paternity testing of a pregnant fetus without the need for paternal parentage, as described in claim 1, is characterized in that... In step S102, the set of genotyping sites containing fetal signals is obtained according to Formula I. X(S)={X i |0<na i (S) / n i (S)<0.2∪0<nA i (S) / n i (S)<0.2}(I), Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, and n = nA + na.

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