Method and application of estimating fetal DNA concentration using paternal and maternal DNA

Abstract

The present invention discloses a method and application for evaluating fetal DNA concentration using the DNA of the biological father and the mother, belonging to the field of bioinformatics technology. The method comprises the following steps: S101: Sequencing the dimorphic sites of the biological father DNA sample and the pregnant woman's cell-free DNA sample respectively to obtain DNA data F and S; S102: Obtaining the site set X' in S that meets the predetermined requirements; S103: Calculating the probability P of a certain site on the site set X' according to formula I; S104: Using the maximum likelihood value method to obtain p when the cumulative probability h of the point set X' is the largest; S105: The concentration N of fetal DNA = 2p max ; S105: The concentration N of fetal DNA = 2p max . This method can evaluate the fetal DNA concentration. During paternity testing, it can be determined whether it is necessary to detect the white blood cells of the pregnant woman and whether it is necessary to resubmit the sample according to the evaluation value; thereby improving the accuracy of paternity testing.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioinformatics analysis, and particularly relates to a method and application of evaluating fetal DNA concentration using the DNA of the biological father and mother, which is used for auxiliary judgment of paternity testing. Background Art

[0002] Genes are functional fragments on DNA molecules that carry genetic information and are the material that transmits genetic information in organisms. DNA is increasingly used, such as in paternity testing. The quality of DNA samples will directly affect the success or failure of subsequent experiments.

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

[0004] When using the peripheral blood of pregnant women for paternity testing, if the fetal DNA concentration is low, accurate identification is usually not possible and needs to be re-tested; if the fetal DNA concentration is high, accurate identification is usually not possible and requires sequencing of the pregnant woman's white blood cells, which increases costs and workload. Therefore, it is necessary to evaluate the fetal DNA concentration before conducting a paternity test. Summary of the invention

[0005] In one aspect, an embodiment of the present invention provides a method for estimating fetal DNA concentration using paternal and maternal DNA, the method comprising the following steps:

[0006] S101: Sequencing the dimorphic sites of the biological father's DNA sample and the pregnant woman's free DNA sample to obtain DNA data F and S respectively, wherein the pregnant woman's free DNA sample is obtained by separating the pregnant woman's peripheral blood;

[0007] S102: Obtain a site set X' in S that meets the following requirements,

[0008] X'={x i |na i (S) / n i (S)≤0.2∩na i (F) / n i (F)≥0.9}∪{x i |nA i (S) / n i (S)≤0.2∩nA i (F) / n i (F)≥0.9},

[0009] Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, n = nA + na, k = na;

[0010] S103: Calculate the probability P of a certain site on the site set X' according to formula I,

[0011]

[0012] in, 0≤p≤0.5, Pm=0.4;

[0013] S104: Use the maximum likelihood method to obtain the point set X' when the cumulative probability h is the maximum. max ;

[0014] S105: Fetal DNA concentration N = 2p max .

[0015] Wherein, in step S101, the sequencing method includes:

[0016] S1011: construct probe;

[0017] S1012: extract DNA from samples;

[0018] S1013: component library;

[0019] S1014: using the probe in step S1011 to perform hybridization capture and sequencing on the target region of the library;

[0020] S1015: Sequencing data is split and quality value filtered to obtain sequencing data.

[0021] Wherein, the dimorphic site is selected from SNP site, INDEL site and / or STR site, and the population frequency of the dimorphic site is 0.05-0.95.

[0022] Specifically, in the SNP site, A represents the wild-type site and a represents the mutant site.

[0023] Preferably, the number of dimorphic sites is greater than 1000.

[0024] Wherein, in step S104: the cumulative probability h of all sites on the site set X' is calculated according to formula II, p is calculated at predetermined intervals, and the value of p when h takes the maximum value is p max ;

[0025]

[0026] Preferably, the predetermined interval is 0.0001.

[0027] On the other hand, an embodiment of the present invention also provides an application of the aforementioned method for evaluating the fetal DNA concentration using the DNA of the biological father and the mother. When N≥0.4, when performing paternity testing, it is necessary to sequence the white blood cells of the pregnant woman; when 0.004<N<0.4, paternity testing is performed according to the second-generation DNA paternity testing method; when N≤0.004, when performing paternity testing, if all sites match the father, the father is judged to be the biological father; if there is a site mismatch and the fetus is a male, it is judged based on the matching of the Y chromosome, and if there is a mismatch, it is judged to be not the biological father; if there is a site mismatch and the fetus is a female, the parent-child relationship cannot be determined, and the free DNA sample of the pregnant woman needs to be recollected.

[0028] The evaluation method provided by the invention can evaluate the fetal DNA concentration. During paternity testing, it can be determined whether it is necessary to test the pregnant woman's white blood cells and whether it is necessary to re-test based on the evaluation value. Within the empirical value range (0.004<N<0.4), the conventional second-generation DNA paternity testing method can be used for paternity testing, thereby improving the accuracy of paternity testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a flow chart of a method for evaluating fetal DNA concentration using paternal and maternal DNA provided by an embodiment of the present invention;

[0030] Figure 2 is a flowchart of step S101;

[0031] Figure 3 It is a graph of cumulative probability h and P value;

[0032] Figure 4 is a linear distribution diagram of nN in Example 3;

[0033] Figure 5 This is the linear distribution diagram of nN in Example 4. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1

[0036] Example 1 provides a method for estimating fetal DNA concentration using paternal and maternal DNA, the method comprising the following steps:

[0037] S101: Sequencing the dimorphic sites of the father's DNA sample and the pregnant woman's free DNA sample to obtain DNA data F and S respectively; wherein the pregnant woman's free DNA sample is obtained by separating the pregnant woman's peripheral blood (including fetal DNA). In this embodiment, the DNA data S and F are obtained by the second generation sequencing technology.

[0038] S102: Obtain a site set X' in S that meets the following requirements,

[0039] X'={x i |na i (S) / n i (S)≤0.2∩na i (F) / n i (F)≥0.9}∪{x i |nA i (S) / n i (S)≤0.2∩nA i (F) / n i (F)≥0.9},

[0040] Among them, nA and na represent the observed values ​​of dimorphic sites A and a respectively, n=nA+na, k=na; the value requirement of F means that for data S, the same point on F must meet the above value requirements.

[0041] S103: Calculate the probability P of a certain site on the site set X' according to formula I,

[0042]

[0043] in, 0≤p≤0.5, Pm=0.4; specifically, p takes discrete values ​​at predetermined intervals starting from 0.

[0044] S104: Use the maximum likelihood method to obtain the point set X' when the cumulative probability h is the maximum. max .

[0045] S105: Fetal DNA concentration N = 2p max .

[0046] Wherein, conventional second generation sequencing technology is used in step S101, and the sequencing method includes:

[0047] S1011: Construct a probe, wherein the probe is designed according to needs.

[0048] S1012: Extract DNA from the sample.

[0049] S1013: Component library.

[0050] S1014: Use the probe in step S1011 to perform hybridization capture and sequencing on the target region of the library.

[0051] S1015: Sequencing data is split and quality value filtered to obtain sequencing data.

[0052] The dimorphic loci are selected from SNP loci, INDEL loci and / or STR loci, etc., and the population frequency of the dimorphic loci is 0.05-0.95.

[0053] Among them, in the SNP site, A represents the wild-type site, and a represents the mutant site. Specifically, the site is compared with the human genome reference sequence, and the one that is consistent with the reference genome is called the wild type, recorded as A, and the opposite is the mutant, recorded as a.

[0054] Preferably, in order to ensure accuracy, the number of dimorphic sites is greater than 1000, such as 2693.

[0055] Among them, in step S104: the cumulative probability h of all sites on the site set X' is calculated according to formula II, p is calculated at predetermined intervals (from 0 to 0.5), and the p value when h takes the maximum value is p max ;

[0056]

[0057] Preferably, in order to ensure accuracy, the predetermined interval is 0.0001; of course, it may also be other values, such as 0.001, as required.

[0058] Example 2

[0059] The embodiment of the present invention also provides the application of the method disclosed in Example 1 for evaluating the concentration of fetal DNA with the DNA of the biological father and the mother. When N≥0.4, when performing paternity testing, it is necessary to sequence the white blood cells of the pregnant woman to obtain the SNP site genotype of the pregnant woman herself. When 0.004<N<0.4, the paternity test is performed according to the conventional second-generation DNA paternity testing method. When N≤0.004, when performing paternity testing, if all sites match the father, it is judged to be the biological father; if there is a site mismatch and the fetus is a male fetus, it is judged based on the matching of the Y chromosome, and if there is a mismatch, it is judged to be not the biological father; if there is a site mismatch and the fetus is a female fetus, the parent-child relationship cannot be determined, and the sample needs to be re-submitted. When applying, you don’t need to worry about whether the father is the biological father or not.

[0060] In addition, using this method, the biological father and the non-biological father are calculated using the same algorithm (steps S101-S104 are the same, and step S105 uses N=2p max ) is different from the fetal DNA concentration obtained by combining it with other algorithms to obtain the fetal DNA concentration, which can determine whether the father is the biological father.

[0061] Example 3

[0062] The father's DNA sample F and the mother's DNA sample M were randomly generated by the Chinese population frequency, and the offspring Z was generated by Mendel's law of inheritance. The samples were mixed from 0-0.4 at intervals of 0.01. The samples of the offspring Z and the mother M were mixed to obtain the simulated pregnant woman's free DNA sample S. Ten samples were generated for each ratio, and the number of samples in the S sample set was 400. Among them, the F and S sample sets contain dimorphic types including SNP and INDEL.

[0063] A partial subset of polymorphic sites of the Chinese population is obtained as the detection site set X. This embodiment uses 2692 dimorphic SNP sites with a population frequency of 0.05-0.95 and 1 INDEL, totaling 2693. The polymorphic distribution of each site xi of the detection site set X of samples F and S is obtained.

[0064] The concentration of the fetus in the S sample is calculated from the detection site set of samples F and S according to the method of Example 1. Taking the simulated concentration of 0.2 as an example, the relationship between the cumulative probability h and the p value is as follows: Figure 3 As shown in the figure, it can be seen that when p is at a certain value (0.1), h can reach a maximum value. At the same time, it has been verified that the p value at this time is just 1 / 2 of the simulated concentration. With the simulated concentration n as the X-axis and the calculated N(p2) as the Y-axis, we can obtain a linear distribution diagram of nN, N = 1.0099126*n, r 2 =0.9997, the distribution diagram is as attached Figure 4 As shown. The lower oblique line is the actual simulated concentration, and the upper oblique line is the value calculated using the method of this patent. It can be seen from the figure that the concentration calculated using the method of this patent is very different from the simulated concentration, that is, the evaluation method of this patent has a very high accuracy.

[0065] Example 4

[0066] The polymorphic sites of the paternal DNA sample F, the maternal DNA sample M, and the offspring DNA sample Z were obtained by experimental sequencing analysis. The simulated free DNA sample S of the pregnant woman can be obtained by mixing the samples Z and M according to the known ratio p in Example 3. The concentration of the fetus in the S sample is calculated according to the method of Example 1 from the detection site concentration of samples F and S. With the simulated concentration n as the X-axis and the calculated N(p2) as the Y-axis, a linear distribution diagram of nN can be obtained, where N=n+0.0044, r 2 =0.9939483, the distribution diagram is as attached Figure 5 As shown. The lower oblique line is the actual simulated concentration, and the upper oblique line is the value calculated using the method of this patent. It can be seen from the figure that the concentration calculated using the method of this patent is very different from the simulated concentration, that is, the evaluation method of this patent has a very high accuracy.

[0067] 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 estimating fetal DNA concentration using paternal and maternal DNA, characterized in that: The method comprises the following steps: S101: Sequencing the dimorphic sites of the biological father's DNA sample and the pregnant woman's free DNA sample to obtain DNA data F and S respectively, wherein the pregnant woman's free DNA sample is obtained by separating the pregnant woman's peripheral blood; S102: Obtain a site set X' in S that meets the following requirements, ， Where nA and na represent the observed values ​​of dimorphic sites A and a, respectively, n=nA+na, k=na; S103: Calculate the probability P of a certain site on the site set X' according to formula I, (I) in, , 0≤p≤0.5, Pm=0.4; S104: Use the maximum likelihood method to obtain the point set X' when the cumulative probability h is the maximum. max ; S105: Fetal DNA concentration N=2p max ; The dimorphic site is selected from a SNP site and / or an INDEL site, and the population frequency of the dimorphic site is 0.05-0.95; A represents the wild-type site, and a represents the mutant site; The number of dimorphic sites is greater than 1000; In step S104: the cumulative probability h of all sites on the site set X' is calculated according to formula II, and the cumulative probability h is calculated at predetermined intervals. The value of p when h takes the maximum value is p max ; (II); The predetermined interval is 0.0001.

2. The method for estimating fetal DNA concentration using paternal and maternal DNA according to claim 1, characterized in that: In step S101, the sequencing method includes: S1011: construct probe; S1012: extract DNA from samples; S1013: component library; S1014: using the probe in step S1011 to perform hybridization capture and sequencing on the target region of the library; S1015: Splitting and quality value filtering the sequencing data to obtain sequencing data.

3. Use of the method for evaluating fetal DNA concentration using paternal and maternal DNA as described in any one of claims 1 to 2, characterized in that: When N≥0.4, the paternity test requires sequencing of the pregnant woman's white blood cells; when 0.004<N<0.4, the paternity test is performed according to the second-generation DNA paternity test method; when N≤0.004, when the paternity test is performed, if all sites match the father's, the father is determined to be the biological father; if there is a mismatch in the site and the fetus is a boy, the judgment is made based on the matching of the Y chromosome, and if there is a mismatch, the father is determined to be not the biological father; if there is a mismatch in the site and the fetus is a girl, the parent-child relationship cannot be determined, and a new free DNA sample from the pregnant woman needs to be collected.

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