Method and application for evaluating fetal DNA concentration from maternal plasma-free and maternal DNA

Abstract

The present invention discloses a method and application for evaluating fetal DNA concentration using maternal cell-free DNA and maternal DNA, belonging to the field of bioinformatics technology. The method comprises the following steps: S101: sequencing the dimorphic sites of the maternal leukocyte DNA sample and the maternal cell-free DNA sample to obtain DNA data M and S respectively; S102: obtaining the site set X' that meets the predetermined requirements in S; 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; 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 maternal leukocytes 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 for evaluating fetal DNA concentration using free and maternal DNA of pregnant women, which is used for assisting in the judgment of paternity testing. Background Art

[0002] Genes are functional fragments on DNA molecules that carry genetic information and are the substances for organisms to transmit genetic information. The use of DNA is becoming increasingly widespread, 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 paternity testing. Compared with STR, SNPs are more widely distributed and more numerous in chromosomes, and the detection method is also more convenient and reliable.

[0004] When performing paternity testing using the peripheral blood of pregnant women, if the fetal DNA concentration is low, accurate identification usually cannot be achieved and it is necessary to resubmit the sample for testing; if the fetal DNA concentration is high, accurate identification usually cannot be achieved either, and it is necessary to sequence the white blood cells of the pregnant woman, which will increase the cost and workload. Therefore, it is necessary to evaluate the fetal DNA concentration before performing paternity testing. Summary of the Invention

[0005] On the one hand, an embodiment of the present invention provides a method for evaluating fetal DNA concentration using free and maternal DNA of pregnant women, the method comprising the following steps:

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

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

[0008] X' = {x i | 0 ≤ na i (M) / n i (M) < 0.1 ∪ 0 ≤ nA i (M) / n i (M) < 0.1},

[0009] X′ = {xi | 0 ≤ nai(M) / ni(M) < 0.1 ∪ 0 ≤ nAi(M) / ni(M) < 0.1}

[0010] wherein, nA and na respectively represent the observed values of dimorphic sites A and a, n = nA + na, and k = na;

[0011] S103: Calculate the probability P of a certain locus on the locus set X' according to formula I,

[0012]

[0013] wherein, 0 ≤ p ≤ 0.5, Pm = 0.4;

[0014] S104: Obtain p when the cumulative probability h of the point set X' is the largest by using the maximum likelihood value method max ;

[0015] S105: Fetal DNA concentration N = 2p max .

[0016] Wherein, in step S101, the sequencing method includes:

[0017] S1011: Construct a probe;

[0018] S1012: Extract DNA from the sample;

[0019] S1013: Construct a library;

[0020] S1014: Hybridize and capture and sequence the target region of the library using the probe in step S1011;

[0021] S1015: Split and perform quality value filtering on the sequencing data to obtain the sequencing data.

[0022] Wherein, the dimorphic locus is selected from SNP locus, INDEL locus and / or STR locus, and the population frequency of the dimorphic locus is 0.05 - 0.95.

[0023] Specifically, in the SNP locus, A represents the wild-type locus, and a represents the mutant locus.

[0024] Preferably, the number of the dimorphic loci is greater than 1000.

[0025] Wherein, in step S104: Calculate the cumulative probability h of all loci on the locus set X', p takes values at a predetermined interval to calculate the cumulative probability h, and the p value when h takes the maximum value is p max ;

[0026]

[0027] Preferably, the predetermined interval is 0.0001.

[0028] On the other hand, the embodiment of the present invention also provides an application of the foregoing method for evaluating fetal DNA concentration from free DNA and the DNA of the pregnant woman herself. When N ≥ 0.4, leukocytes of the pregnant woman need to be sequenced for paternity testing; when 0.004 < N < 0.4, paternity testing is performed according to the second-generation DNA paternity testing method; when N ≤ 0.004, for paternity testing, if all loci match the father, it is determined to be the biological father; if there is a locus mismatch and the fetus is male, it is determined according to the matching situation of the Y chromosome, and if there is one mismatch, it is determined not to be the biological father; if there is a locus mismatch and the fetus is female, the paternity relationship cannot be determined, and a free DNA sample of the pregnant woman needs to be collected again.

[0029] The evaluation method provided by the invention can evaluate the fetal DNA concentration. For paternity testing, it can be determined whether it is necessary to detect the leukocytes of the pregnant woman and whether it is necessary to resubmit the sample according to 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

[0030] Figure 1 is a flowchart of the method for evaluating fetal DNA concentration from free DNA and the DNA of the pregnant woman herself provided by the embodiment of the present invention;

[0031] Figure 2 is a flowchart of step S101;

[0032] Figure 3 is a curve graph of the cumulative probability h and the P value;

[0033] Figure 4 is a linear distribution graph of n-N in Example 3;

[0034] Figure 5 is a linear distribution graph of n-N in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] 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.

[0036] Example 1

[0037] Example 1 provides a method for evaluating fetal DNA concentration from free DNA and the DNA of the pregnant woman herself, and the method includes the following steps:

[0038] S101: Sequence the dimorphic sites of the pregnant woman's leukocyte DNA sample and the pregnant woman's cell-free DNA sample (a mixed signal of the pregnant woman's own DNA and the fetus's DNA) to obtain DNA data M (the pregnant woman's own DNA data) and S respectively. Among them, the pregnant woman's cell-free DNA sample is obtained by separating the pregnant woman's peripheral blood (including the fetus's DNA). Among them, the DNA data M and S in this embodiment are obtained by next-generation sequencing technology.

[0039] S102: Obtain the site set X' in S that meets the following requirements.

[0040] X' = {x i | 0 ≤ na i (M) / n i (M) < 0.1 ∪ 0 ≤ nA i (M) / n i (M) < 0.1},

[0041] where nA and na respectively represent the observed values of the dimorphic sites A and a, n = nA + na, and k = na. The value requirement of M means that for the data S, the same positions on M should meet the above value requirements.

[0042] S103: Calculate the probability P of a certain site on the site set X' according to formula I.

[0043]

[0044] where 0 ≤ p ≤ 0.5, Pm = 0.4; specifically, p takes discrete values at a predetermined interval starting from 0.

[0045] S104: Use the maximum likelihood value method to obtain the p when the cumulative probability h of the point set X' is the largest. max .

[0046] S105: The fetal DNA concentration N = 2p max .

[0047] Among them, in step S101, a conventional next-generation sequencing technology is adopted, and this sequencing method includes:

[0048] S1011: Construct a probe, where the probe is designed as needed.

[0049] S1012: Extract the DNA in the sample.

[0050] S1013: Construct a library.

[0051] S1014: Use the probe in step S1011 to hybridize and capture and sequence the target region of the library.

[0052] S1015: Split and filter the quality values of the sequencing data to obtain the sequencing data.

[0053] Among them, the dimorphic sites are selected from SNP sites, INDEL sites, and / or STR sites, etc., and the population frequency of the dimorphic sites is 0.05 - 0.95.

[0054] Among them, in the SNP site, A represents the wild-type site, and a represents the mutant site. Specifically, this site is aligned with the human genome reference sequence. The one that is aligned with the reference genome is called the wild-type and denoted as A, and the opposite is the mutant type, denoted as a.

[0055] Preferably, to ensure accuracy, the number of dimorphic sites is greater than 1000, such as taking 2693.

[0056] Among them, in step S104: Calculate the cumulative probability h of all sites on the locus set X' according to formula II. p takes values at a predetermined interval (ranging from 0 - 0.5) to calculate the cumulative probability h. The p value when h takes the maximum value is p max ;

[0057]

[0058] Preferably, to ensure precision, the predetermined interval is 0.0001; of course, it can also be other values according to needs, such as 0.001.

[0059] Example 2

[0060] The embodiment of the present invention also provides an application of the method for evaluating the fetal DNA concentration from the free and own DNA of the pregnant woman disclosed in Example 1. When N ≥ 0.4 and paternity testing is carried out, it is necessary to sequence the white blood cells of the pregnant woman to obtain the SNP site genotypes of the pregnant woman herself. When 0.004 < N < 0.4, paternity testing is carried out according to the conventional second-generation DNA paternity testing method. When N ≤ 0.004 and paternity testing is carried out, if all sites match the father, it is judged as the biological father; if there are sites that do not match and the fetus is male, then it is judged according to the matching situation of the Y chromosome. If there is one mismatch, it is judged as non-biological father; if there are sites that do not match and the fetus is female, then the paternity relationship cannot be judged, and it is necessary to resample.

[0061] Example 3

[0062] Randomly generate the biological father DNA sample F and the biological mother DNA sample M according to the Chinese population frequency. Generate the offspring Z according to Mendel's genetic law. Mix the samples at intervals of 0.01 from 0 to 0.4. Mixing the samples of the offspring Z and the biological mother M can obtain the simulated cell-free DNA sample S of the pregnant woman. For each mixing ratio, 10 samples are generated, so the number of samples in the S sample set is 400. Among them, the dimorphic types contained in the M and S sample sets include SNPs and INDELs.

[0063] Obtain a partial subset of the polymorphic sites in the Chinese population as the detection site set X. In this embodiment, the number of dimorphic SNP sites with a population frequency belonging to 0.05 - 0.95 is more than 1000. Obtain the polymorphic distributions of each site xi in the detection site set X of the samples M and S.

[0064] Calculate the concentration of the fetus in the S sample from the detection site sets of the samples M and S according to the method of Example 1. Taking the simulated concentration of 0.2 as an example, the relationship diagram between the cumulative probability h and the p-value obtained is as Figure 3 shown. It can be seen from the figure that when p is at a certain value (0.1), h can take the maximum value. At the same time, it is verified that the p-value at this time is exactly 1 / 2 of the simulated concentration. Taking the simulated concentration n as the X-axis and the calculated N(p1') as the Y-axis to plot a graph, a linear distribution graph of n-N can be obtained, p1' = 1.0098308 * n, r 2 = 0.9997, and the distribution graph is as shown in the appendix Figure 4 shown. Among them, the lower diagonal line is the actual simulated concentration, and the upper diagonal line is the value calculated by using the method of this patent. It can be seen from the figure that the difference between the concentration calculated by using the method of this patent and the simulated concentration is very small, that is, the evaluation method of this patent has very high accuracy.

[0065] Example 4

[0066] Obtain the polymorphic sites of the biological mother DNA sample M and the offspring DNA sample Z through experimental sequencing analysis. Mix the samples of Z and M according to the known ratio p in Example 3 to obtain the simulated cell-free DNA sample S of the pregnant woman. Calculate the concentration of the fetus in the S sample from the detection site sets of the samples M and S according to the method of Example 1. Taking the simulated concentration n as the X-axis and the calculated N(p1') as the Y-axis to plot a graph, a linear distribution graph of n-N can be obtained, p1' = n - 0.0077, and the distribution graph is as shown in the appendix Figure 5 shown. Among them, the upper diagonal line is the actual simulated concentration, and the lower diagonal line is the value calculated by using the method of this patent. It can be seen from the figure that the difference between the concentration calculated by using the method of this patent and the simulated concentration is very small, that is, the evaluation method of this patent has very high accuracy.

[0067] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Method for evaluating fetal DNA concentration using pregnant women's free and own DNA, Characterized in that, The method comprises the following steps: S101: Sequencing the biallelic loci of the pregnant woman's leukocyte DNA sample and the pregnant woman's free DNA sample respectively to obtain DNA data M and S, wherein the pregnant woman's free DNA sample is obtained by separating the pregnant woman's peripheral blood; S102: Obtaining the locus set X' in S that meets the following requirements, X' = {x i | 0 ≤ na i (M) / n i (M) < 0.1 ∪ 0 ≤ nA i (M) / n i (M) < 0.1}, Wherein, nA and na respectively represent the observed values of biallelic locus A and a, n = nA + na, k = na; S103: Calculating the probability P of a certain locus on the locus set X' according to formula I, Among them, 0 ≤ p ≤ 0.5, Pm = 0.4; S104: Obtain p when the cumulative probability h of the point set X' is maximized by using the maximum likelihood value method max ; S105: Fetal DNA concentration N = 2p max ; The biallelic loci are selected from SNP loci and / or INDEL loci, and the population frequency of the biallelic loci is 0.05 - 0.95; A represents the wild-type locus, and a represents the mutant locus; The number of the biallelic loci is greater than 1000; In step S104: Calculate the cumulative probability h of all sites on the site set X' according to formula II. p takes values at a predetermined interval to calculate the cumulative probability h, and the p value when h reaches the maximum value is p max ; The predetermined interval is 0.0001.

2. The method for evaluating fetal DNA concentration using pregnant women's free and own DNA according to claim 1, Characterized in that, In step S101, the sequencing method comprises: S1011: Constructing a probe; S1012: Extracting DNA from the sample; S1013: Constructing a library; S1014: Using the probe of step S1011 to hybridize and capture and sequence the target region of the library; S1015: Splitting and quality value filtering the sequencing data to obtain the sequencing data.

3. Application of the method for evaluating fetal DNA concentration using pregnant women's free and own DNA according to any one of claims 1 - 2, Characterized in that, When N ≥ 0.4, for paternity testing, the leukocytes of the pregnant woman need to be sequenced; when 0.004 < N < 0.4, paternity testing is carried out according to the second-generation DNA paternity testing method; when N ≤ 0.004, for paternity testing, if all loci match the father, it is judged as the biological father; if there is a locus mismatch and the fetus is male, then it is judged according to the matching situation of the Y chromosome, and if there is one mismatch, it is judged as non-biological father; if there is a locus mismatch and the fetus is female, then the paternity relationship cannot be judged and a new pregnant woman's free DNA sample needs to be collected.

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