Method for parentage testing using homozygous sites

Abstract

This invention discloses a method for paternity testing using homozygous loci, belonging to the field of paternity testing technology. The method includes: S101: sequencing the pregnant woman's cell-free DNA sample S and the father's DNA sample F, and genotyping S and F based on sequencing depth; S102: calculating the inefficiency P of the two samples based on the genotyping results. 无 or inefficient P 无 With efficiency P 有 The ratio is used to perform paternity testing; the effectiveness P 有 The effective loci represent the proportion of the homozygous loci in the set X of opposite homozygous loci. Effective loci are those in S that conform to the set X of opposite homozygous loci and can detect fetal signals. Loci in S and F are selected according to Formula I to form the set X of opposite homozygous loci; inefficiency P 无 The percentage of invalid loci in the same homozygous locus set X' is defined as the proportion of invalid loci in S that conform to the same homozygous locus set X' and can detect fetal signals. Loci in S and F are selected according to Formula II to form the same homozygous locus set X'.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological information analysis, and particularly relates to a method for parentage identification by using homozygous sites. BACKGROUND

[0002] Genes are functional fragments carrying genetic information on DNA molecules, and are the material for biological transmission of genetic information. DNA is increasingly widely used, such as for parentage identification. The quality of the DNA sample will directly affect the success of subsequent experiments.

[0003] With the rapid development of high-throughput sequencing technology, single nucleotide polymorphism (SNP) as the third generation of genetic markers is increasingly becoming the latest detection means for individual and parentage identification. Compared with STR, SNP is more widely distributed in chromosomes, has a larger number, and is more convenient and reliable to detect.

[0004] In the prior art, the peripheral blood of a pregnant woman and a paternal sample can be used for parentage identification. A conventional method is to perform parentage identification according to mismatch rates and fetal concentration indicators. If the fetal DNA concentration is low, accurate identification is usually not achieved, and retesting is required. If the fetal DNA concentration is high, accurate identification is usually not achieved, and white blood cells of the pregnant woman need to be sequenced, which increases the cost and workload. In these scenarios, the mismatch rate and the fetal concentration indicator may not be standard, and other indicators need to be combined to assist in judgment. SUMMARY

[0005] The embodiments of the application provide a method for parentage identification by using homozygous sites, and provide another method for parentage identification by using the peripheral blood of a pregnant woman and a paternal sample, which is basically irrelevant to the fetal DNA concentration. The method can accurately obtain the result of parentage identification under various fetal concentrations. The method comprises the following steps:

[0006] S101: Sequencing the pregnant woman's free DNA sample S and the paternal sample F, and typing the sample S and the sample F according to the sequencing depth.

[0007] S102: Calculating the invalidity P of the two samples according to the typing result 无 or the ratio of the invalidity P 无 and the validity P 有 , and performing parentage identification according to the calculation result.

[0008] Wherein, the validity P 有 is the proportion of valid sites in the opposite homozygous site set X, and the valid site is a site in the sample S that meets the opposite homozygous site set X and can detect the fetal signal. The opposite homozygous site set X is selected according to formula I from the sites of the sample S and the sample F:

[0009] (I).

[0010] Inefficient P 无 The percentage of invalid loci in the same homozygous locus set X' is defined as the proportion of invalid loci in sample S that conform to the same homozygous locus set X' and can be detected by fetal signals. Loci from samples S and F are selected according to Formula II to form the same homozygous locus set X'.

[0011] (II)

[0012] Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, and n = nA + na.

[0013] In one embodiment of this patent, in step S102, the inefficiency P of sample S and sample F is calculated. 无 If there is no efficiency P 无 If the result is approximately 0, then it is determined that the fetus and sample F have a parent-child relationship.

[0014] In another embodiment of this patent, in step S102, the efficiency P of sample S and sample F is calculated. 有 and inefficiency P 无 According to P 无 / P 有 Conduct a paternity test.

[0015] Specifically, in step S102, if P 无 / P 有 If P ≈ 1, then it is determined that there is no parent-child relationship between the fetus and sample F; if P 无 / P 有 If the result is approximately 0, then it is determined that the fetus and sample F have a parent-child relationship.

[0016] Preferably, in step S102, if P 无 / P 有 =0.8-1.0, which indicates that there is no parent-child relationship between the fetus and sample F.

[0017] In step S102,

[0018] The total number of loci in the set of opposite homozygous loci X is denoted as T; the total number of valid loci is denoted as H; and the efficiency P is calculated according to Formula III. 有 :

[0019] (III).

[0020] In step S102,

[0021] Count the total number of homozygous loci in the set X', denoted as T'; count the total number of invalid loci, denoted as H', and calculate the inefficiency P_nil using formula IV:

[0022] (Ⅳ).

[0023] The sequencing depth of the pregnant woman's cell-free DNA sample S and the father's DNA sample F is 10x-100x, and the number of sequencing sites is greater than 1000.

[0024] Specifically, embodiments of the present invention provide a method for paternity testing using homozygous loci, the method comprising:

[0025] S101: Sequencing of cell-free DNA sample S from the pregnant woman and paternal parent F, and genotyping of sample S and sample F according to sequencing depth, wherein the sequencing depth of cell-free DNA sample S from the pregnant woman and paternal parent F is 10x-100x, and the number of sequencing sites is greater than 1000.

[0026] S102: Calculate the inefficiency P of the two samples based on the genotyping results. 无 If there is no efficiency P 无 If ≈0, then it is determined that the fetus and sample F have a parent-child relationship;

[0027] Alternatively, calculate the P-value of the two samples based on the typing results. 无 / P 有； If P 无 / P 有 =0.8-1.0, it can be determined that there is no parent-child relationship between the fetus and sample F; if P 无 / P 有 If the result is approximately 0, then it is determined that the fetus and sample F have a parent-child relationship.

[0028] This invention provides a method for paternity testing using homozygous loci, and another method for paternity testing using the pregnant woman's peripheral blood and the father's DNA, which is largely unrelated to fetal DNA concentration. Paternity test results can be accurately obtained under various fetal DNA concentrations, and the method is simple. The specific process is as follows: calculate the inefficiency P between the two samples based on the genotyping results. 无 If there is no efficiency P 无 If ≈0, then a parent-child relationship is determined between the fetus and sample F; or, the P-value between the two samples is calculated based on the typing results. 无 / P 有； If P 无 / P 有 =0.8-1.0, it can be determined that there is no parent-child relationship between the fetus and sample F; if P 无 / P 有 If the result is approximately 0, then it is determined that the fetus and sample F have a parent-child relationship. Attached Figure Description

[0029] Figure 1is a flow chart of the method for parentage identification by using homozygous sites provided by the embodiment of the present application;

[0030] Figure 2 is a distribution diagram of the efficiency under different concentrations and simulation conditions;

[0031] Figure 3 is a distribution diagram of the inefficiency under different concentrations and simulation conditions;

[0032] Figure 4 is a distribution diagram of the inefficiency / efficiency under different concentrations and simulation conditions;

[0033] Figure 5 is a distribution diagram of the inefficiency, efficiency and ratio of the two under different concentrations and simulation conditions;

[0034] Figure 6 is a distribution diagram of the inefficiency, efficiency and ratio of the two under different concentrations and simulation conditions. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings.

[0036] Referring to Figure 1 , the embodiment of the present application provides a method for parentage identification by using homozygous sites, which comprises the following steps:

[0037] S101: sequencing the pregnant woman's free DNA sample S and the paternal sample F, and typing the sample S and the sample F according to the sequencing depth.

[0038] The typing process is as follows: when the sequencing depth is 100x, the A 95x; a5x is detected on a certain SNP site of the sample S, then the fetal signal (which can be understood as a small signal, a5x) ratio = 5 / 100 = 0.05, so this site is recorded as the maternal AA homozygous genotype, and the fetal signal is present, and the fetal genotype is Aa. Similarly, if the A98x; a2x is detected in the F sample at a certain site, then the large signal (A98x) ratio = 98 / 100 = 0.98, so this site is recorded as the paternal AA homozygous genotype. The S and F are typed according to the above rules.

[0039] S102: calculating the inefficiency P 无 or the ratio of the inefficiency P 无 and the efficiency P 有 (which can be P 无 / P 有 or P 有 / P 无 , and the embodiment is P 无 / P 有 And perform paternity testing based on the calculation results.

[0040] Among them, the efficiency P 有 The effective loci represent the proportion of the homozygous loci in the set X of opposite homozygous loci. Effective loci are those in sample S that conform to the set X of opposite homozygous loci and can be detected by fetal signals. Loci from samples S and F are selected according to Formula I to form the set X of opposite homozygous loci:

[0041] (I).

[0042] Inefficient P 无 The percentage of invalid loci in the same homozygous locus set X' is defined as the proportion of invalid loci in sample S that conform to the same homozygous locus set X' and can be detected by fetal signals. Loci from samples S and F are selected according to Formula II to form the same homozygous locus set X'.

[0043] (II)

[0044] Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, and n = nA + na.

[0045] In this patent, only the inefficiency P can be calculated. 无 If we can obtain the inefficiency P 无 If ≈0, then a parent-child relationship is determined between the fetus and sample F, and P does not need to be calculated. 无 / P 有 If there is no efficiency P 无 If it is not approximately equal to 0, then calculate P again. 无 / P 有 This can effectively reduce the amount of computation.

[0046] Of course, P can also be calculated directly. 无 / P 有 Directly based on P 无 / P 有 Make a judgment.

[0047] In one embodiment of this patent, in step S102, the inefficiency P of sample S and sample F is calculated. 无 If there is no efficiency P 无 If the result is approximately 0, then it is determined that the fetus and sample F have a parent-child relationship.

[0048] In another embodiment of this patent, in step S102, the efficiency P of sample S and sample F is calculated. 有 and inefficiency P 无 According to P 无 / P 有 Conduct a paternity test.

[0049] Specifically, in step S102, if P 无 / P 有 ≈1, it is determined that the fetus and the sample F have no parentage; if P 无 / P 有 ≈0, it is determined that the fetus and the sample F have parentage.

[0050] Preferably, in step S102, if P 无 / P 有 = 0.8-1.0, it is determined that the fetus and the sample F have no parentage.

[0051] In step S102,

[0052] The total number of sites of the homozygous site set X is counted and recorded as T; the total number of effective sites is counted and recorded as H, and the efficiency P is calculated according to formula III 有 :

[0053] (III).

[0054] The total number of sites of the homozygous site set X' is counted and recorded as T'; the total number of ineffective sites is counted and recorded as H', and the inefficiency P is calculated according to formula IV

[0055] (IV).

[0056] The sequencing depth of the pregnant woman's free DNA sample S and the father F is 10x-100x, and the number of sequencing sites is greater than 1000.

[0057] Specifically, the embodiment of the present application provides a method for parentage identification by using homozygous sites, which comprises:

[0058] S101: sequencing the pregnant woman's free DNA sample S and the father F, and typing the sample S and the sample F according to the sequencing depth. The sequencing depth of the pregnant woman's free DNA sample S and the father F is 10x-100x, and the number of sequencing sites is greater than 1000.

[0059] S102: calculating the inefficiency P 无 of the two samples according to the typing result; if the inefficiency P 无 ≈0, it is determined that the fetus and the sample F have parentage.

[0060] Alternatively, the P 无 / P 有； of the two samples is calculated according to the typing result; if P 无 / P 有 = 0.8-1.0, it is determined that the fetus and the sample F have no parentage; if P 无 / P 有≈0, it is determined that the fetus and the sample F have a parent-child relationship.

[0061] Whether to calculate P 无 / P 有 According to the result, if the result of parentage identification has been obtained, only P 无 ; if P 无 The identification result cannot be obtained, P 无 / P 有 .

[0062] In this patent, the effective rate P 有 It can be understood as follows: selecting a site where both parents are homozygous (opposite genotypes). Assuming that there are 500 such sites, theoretically, the pregnant woman's free DNA sample will appear fetal signals at these 500 sites. The effective rate is 1. However, due to low sequencing depth or low concentration, some signal sites cannot be measured, resulting in only 400 signal sites, and the effective rate is 0.8.

[0063] The invalid rate P 无 It can be understood as follows: selecting a site where both parents are homozygous (same genotype). Theoretically, the pregnant woman's free DNA sample should not appear fetal signals at this site, and the invalid rate is 0. In fact, due to sequencing errors, some sites will appear fetal signals, resulting in an actual invalid rate greater than 0 but close to 0.

[0064] Example 2: Data simulation

[0065] The process is as follows:

[0066] 1. Randomly generate true parent DNA sample F, maternal DNA sample M and random parent DNA sample F' by Chinese population frequency. Simulated sequencing depth: 50x-100x. Generate offspring Z by Mendelian inheritance law, from 0-0.4 every interval 0.01, mix the sample of offspring Z and maternal M, and the simulated pregnant woman's free DNA sample S can be obtained. Each proportion mixture generates 10 samples, and the sample number of S sample set is 400. Each sample in S sample set contains more than 1000 sites, and contains biallelic types including SNP and INDEL.

[0067] 2. SNP typing of S, F and F' according to sequencing depth.

[0068] 3. Selecting S and F' as opposite homozygous sites, forming site set X, and counting the total number of sites, denoted as T.

[0069] 4. Selecting S and F' as same homozygous sites, forming site set X', and counting the total number of sites, denoted as T'.

[0070] The selection methods of X and X' are as follows:

[0071] (I) ;

[0072] (II).

[0073] 5. Calculate the effective rate P 有 : If S has fetal signal in the site of the site set X, it is recorded as an effective site, and the total number of effective sites is counted, denoted as H. The effective rate P 有 The calculation method is as follows:

[0074] .

[0075] 6. Calculate the ineffective rate P 无 : If S has fetal signal in the site of the site set X', it is recorded as an ineffective site, and the total number of ineffective sites is counted, denoted as H'. The ineffective rate P 无 The calculation method is as follows:

[0076] .

[0077] With the concentration as the horizontal axis, P 有 as the Y axis, draw the distribution diagram of the effective rate P 有 under the mismatch condition, as shown in Figure 2 ; With the concentration as the horizontal axis, P 无 as the Y axis, draw the distribution diagram of the ineffective rate P 无 under the mismatch condition, as shown in Figure 3 ; With the concentration as the horizontal axis, P 无 / P 有 as the Y axis, draw the distribution diagram of P 无 / P 有 under the mismatch condition, as shown in Figure 4 .

[0078] 7. Similarly, as above, taking S and F as the analysis object, calculate the effective rate P 有 and the ineffective rate P 无 under the matching condition, and analyze the site set as follows:

[0079] (III) ;

[0080] (IV).

[0081] According to the calculation method of steps 5 and 6, calculate the effective rate P 有 and the ineffective rate P 无 under the matching condition. With the concentration as the horizontal axis, P 有 as the Y axis, draw the distribution diagram of the effective rate P 有 under the matching condition, as shown in Figure 2Figure 3 shows the distribution of P 无 efficiency with concentration for matched samples, where P 无 efficiency is plotted on the y-axis and concentration is plotted on the x-axis. Figure 3 Figure 4 shows the distribution of P 无 / P 有 efficiency with concentration for matched samples, where P 无 / P 有 efficiency is plotted on the y-axis and concentration is plotted on the x-axis. Figure 4

[0082] Example 3: Theoretical calculations

[0083] The process is as follows:

[0084] 1. Randomly generate a true father DNA sample F and a mother DNA sample M by Chinese population frequency, with a simulated sequencing depth of 50x-100x. Generate offspring Z by Mendelian inheritance law, with a mixture of 0-0.4 every 0.01 interval. Mix the samples of offspring Z and mother M to obtain simulated pregnant woman free DNA samples S, with 10 samples generated for each proportion. The sample set S contains 400 samples, each containing more than 1000 sites and containing biallelic types including SNPs and INDELs.

[0085] 2. Generate a random father DNA sample F’ by Chinese population frequency.

[0086] 3. Select S and F’ as opposite homozygous sites, and count the total number of sites, denoted as T, using the following selection method:

[0087] (Ⅴ).

[0088] 4. Calculate the efficiency P 有 : Mark the sites in S that appear fetal signals as 1. Mark the sites that do not appear as 0. If the genotype of S at a certain site is AA, and the genotype of F’ at the corresponding site is aa, and the site appears fetal signals, then the efficiency of a corresponding site is calculated as follows:

[0089] , where p(aa) represents the frequency of aa genotype of F’ at the site.

[0090] If the genotype of S at a certain site is AA, and the genotype of F’ at the corresponding site is aa, and the site does not appear fetal signals, then the efficiency of a corresponding site is calculated as follows:

[0091] .

[0092] Add the efficiency of each site to calculate the total efficiency, using the following method: ​

[0093] .

[0094] where i is the locus corresponding to the locus set X', pi is the frequency of the genotype corresponding to the locus in F', and ai is the fetal signal at the corresponding locus, 1 if present, otherwise 0.

[0095] 5. Calculate the inefficiency P 无 : The locus in S where the fetal signal is present is recorded as 1. The locus where the fetal signal is not present is recorded as 0. If the genotype of S at a locus is AA, the genotype of F' at the corresponding locus is AA, and the fetal signal is present at the locus, the calculation method of the inefficiency corresponding to a locus is as follows:

[0096] .

[0097] where p(AA) represents the frequency of AA genotype of F' at the locus.

[0098] If the genotype of S at a locus is AA, the genotype of F' at the corresponding locus is AA, and the fetal signal is not present at the locus, the calculation method of the inefficiency corresponding to a locus is as follows:

[0099] .

[0100] Add the inefficiencies of each locus to calculate the total inefficiency, as follows:

[0101] .

[0102] where i is the locus corresponding to the locus set X', pi is the frequency of the genotype corresponding to the locus in F', and ai is the fetal signal at the corresponding locus, 1 if present, otherwise 0.

[0103] The concentration is taken as the horizontal axis, and the index value (P 有 , P 无 , or P 无 / P 有 ) is taken as the Y axis. The distribution graphs of the existence of parent-child relationship and non-parent-child relationship are drawn, respectively, as shown in Figure 5 and Figure 6 .

[0104] In Figure 2 , the theoretical random and mismatched curves overlap a lot (the two curves below the distribution graph, and before 0.1 and after 0.3 of the concentration, will change dramatically with the change of the concentration); and the matched curve (the curve above the distribution graph) is very different from the theoretical random and mismatched curves and before 0.1 of the concentration, will change dramatically with the change of the concentration. Therefore, it is impossible to determine the relationship of the two samples according to P 有 .

[0105] In Figure 3 , the theoretical random and mismatched curves largely coincide (the two curves above the distribution graph, and before the concentration 0.1 and after 0.25, will change dramatically with the concentration); while the matched curve (the curve below the distribution graph) is very different from the theoretical random and mismatched curves and does not change with the concentration, P 无 may be used to determine that the two samples are in a matched relationship (the straight line at the bottom of the distribution graph).

[0106] In Figure 4 , the theoretical random and mismatched curves largely coincide (the two curves above the distribution graph) and do not change with the concentration; while the matched curve (the straight line below the distribution graph) is very different from the theoretical random and mismatched curves and does not change with the concentration, P 无 / P 有 may be used to determine that the two samples are in a matched (the straight line at the bottom of the distribution graph) and non-matched relationship (in the interval of 0.8-1.0).

[0107] In Figure 5 , under the parent-child relationship, P 无 / P 有 and P 无 (the two straight lines below the distribution graph) and do not change with the concentration. That is, P 无 / P 有 =0 or P 无 =0, it can be determined that there is a parent-child relationship.

[0108] In Figure 6 , under the non-parent-child relationship, P 无 / P 有 (the curve above the distribution graph) and does not change with the concentration. That is, P 无 / P 有 ≈1, it can be determined that there is a non-parent-child relationship

[0109] Example 4: Actual sequencing data

[0110] The process is as follows:

[0111] 1. Obtain the polymorphic sites of the maternal DNA sample M, the offspring DNA sample Z, the true father DNA sample F, and other random father DNA samples F' by high-throughput sequencing experiments.

[0112] 2. Mix Z and M samples every 0.01 from 0-0.4 to obtain the simulated pregnant woman free DNA sample S.

[0113] 3. SNP typing of S, F and F' according to the sequencing depth.

[0114] 4. According to steps 3-7 in Example 1. The distribution of efficiency and inefficiency of S and F in the presence of parentage and S and F' in the absence of parentage was obtained. The results are shown in Figures Figure 4 and Figure 5

[0115] The results show that when the actual sample has parentage, the inefficiency is almost 0, and the ratio of inefficiency / efficiency is also close to 0 (excluding the case where the fetal concentration is particularly low), which meets the judgment index. When the samples are not in parentage, the efficiency and inefficiency values are similar, close to 1, which also meets the judgment index.

[0116] In summary, the efficiency, inefficiency and inefficiency / efficiency distribution of Example 2-3 and Example 4 are almost the same. When the fetus and the father have parentage (match): the inefficiency is close to 0; the ratio of inefficiency / efficiency is close to 0.

[0117] When the fetus and the father do not have parentage: inefficiency / efficiency ≈ 1.

[0118] In summary, the following judgment index can be obtained:

[0119] When the ratio of inefficiency / efficiency between two identification samples is approximately 0 or the inefficiency is close to 0, it can be determined that the two have parentage.

[0120] When the ratio of inefficiency / efficiency between two identification samples is approximately 1 (the ratio is around 0.8-1.0), it can be determined that the two do not have parentage.

[0121] Example 5

[0122] Example 5 discloses a specific example of parentage identification using the method of the present application, as follows: a family numbered QZ40969, a pregnant woman's peripheral blood sample numbered QZ40969S1 and a blood sample of a suspected father numbered QZX40969F were sent for identification. The SNP typing results of QZ40969S1 and QZX40969F were obtained by sequencing analysis, and according to the mismatch rate and fetal concentration index (using the prior art), it can be determined that the fetus of the family and the suspected father are in parentage. According to the steps of the present patent, the efficiency and inefficiency were calculated. The efficiency is 0.950; the inefficiency is 0.018, and the inefficiency is 0.018≈0. It meets the distribution of efficiency and inefficiency when the parentage is matched.

[0123] Example 6

[0124] ​Example 6 discloses a specific example of paternity identification using the method of the present application as follows: a family numbered QZ40972, a pregnant woman's peripheral blood sample numbered QZ40972S1 and a hair sample of the suspected father numbered QZM40972F were sent for identification. The SNP typing results of QZ40972S1 and QZM40972F were obtained by sequencing analysis, and according to the mismatch rate and fetal concentration index (using the prior art), it can be determined that the fetus of the family and the suspected father are not in a parent-child relationship. According to the steps of the present patent, the efficiency and inefficiency are calculated. The efficiency is 0.440; the inefficiency is 0.405; the inefficiency / efficiency=0.92≈1. There is a distribution of inefficiency / efficiency when it is not a parent-child relationship (mismatch).

[0125] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for parentage testing using homozygous loci, characterized in that, The method comprises: S101: sequencing the pregnant woman's free DNA sample S and the paternal F, and typing the sample S and the sample F according to the sequencing depth; S102: Calculate the effective rate P of two samples according to the typing results 有 and the ineffective rate P 无 , according to P 无 / P 有 to carry out parentage identification; wherein the efficiency P 有 is the proportion of effective sites in the opposite homozygote site set X, the effective site is a site in the sample S that meets the opposite homozygote site set X and can detect the fetal signal, and the site set X of the sample S and the sample F is selected according to formula I: (Ⅰ)； Efficiency P 无 The invalidity rate P is the ratio of invalidity sites in the same homozygous site set X' in the sample S. The invalidity sites are sites in the sample S that meet the same homozygous site set X' and can detect the fetal signal. The same homozygous site set X' is selected according to formula II by selecting sites of the sample S and the sample F. (Ⅱ)； Wherein, nA and na respectively represent the observed values of the two-state sites A and a, n = nA + na; In step S102, if P 无 / P 有 = 0.8-1.0, it is determined that the fetus and the sample F do not have the parent-child relationship; if P 无 / P 有 is greater than 0 and close to 0, it is determined that the fetus and the sample F have the parent-child relationship.

2. The method for parentage testing using a homozygous site according to claim 1, characterized in that , in step S102, The total number of sites of the opposite homozygous site set X is counted and recorded as T; the total number of effective sites is counted and recorded as H, and the efficiency P is calculated according to formula III 有 : (Ⅲ)。 3. The method for parentage testing using a homozygous site according to claim 1, characterized in that , in step S102, The total site number of the same homozygote site set X' is counted and recorded as T'; the total number of invalid sites is counted and recorded as H', and the invalidity P is calculated according to formula IV 无 : (Ⅳ)。 4. The method for parentage testing using a homozygous site according to claim 1, characterized in that The sequencing depth of the pregnant woman's free DNA sample S and the paternal F is 10x-100x, and the number of sequenced sites is greater than 1000.

5. The method for parentage testing using a homozygous site according to claim 1, characterized in that The method comprises: S101: sequencing the pregnant woman's free DNA sample S and the paternal F, and typing the sample S and the sample F according to the sequencing depth, the sequencing depth of the pregnant woman's free DNA sample S and the paternal F is 10x-100x, and the number of sequenced sites is greater than 1000; S102: Calculate the inefficiency P of the two samples according to the typing results 无 ; if the inefficiency P 无 is greater than 0 and close to 0, it is judged that the fetus and the sample F have a parent-child relationship; Or, according to the typing result, calculate P of two samples 无 / P 有； If P 无 / P 有 =0.8-1.0, it can be judged that the fetus and sample F do not exist parent-child relationship; if P 无 / P 有 is greater than 0 and close to 0, it is judged that the fetus and sample F exist parent-child relationship.

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