Method for noninvasive prenatal genetic relationship judgment by adopting two-state SNP (Single Nucleotide Polymorphism) sites

Abstract

The invention relates to the field of biological detection, in particular to the field of paternity test and detection, and particularly relates to a method for non-invasive prenatal genetic relationship judgment by adopting a two-state SNP (Single Nucleotide Polymorphism) site. According to the method, the minimum number of SNP sites required for judging the genetic relationship is calculated, the genetic relationship is judged by screening the sites containing fetal information in the free DNA sample of the pregnant woman and combining the indexes, the genotype of the pregnant woman sample does not need to be additionally detected, meanwhile, whether the parent-child relationship between a fetus and a suspected parent conforms to the parent-child relationship or not can be accurately judged, and the application range is wider.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, specifically to the field of paternity testing, and in particular to a method for non-invasive prenatal kinship determination using dimorphic SNP sites. Background Art

[0002] Parentage testing is the identification of controversial parent-child blood relationships through the detection of human genetic markers and analysis of genetic laws. The indicators or genetic markers used for paternity testing should be a simple genetic trait that is determined to be inherited according to Mendel's law, and has genetic polymorphisms as proven by population surveys, and has relatively high system efficiency.

[0003] In the field of kinship identification, there are a variety of genetic markers that are widely used. In the early days, DNA analysis used restriction fragment length polymorphism (RFLP) analysis technology to detect small satellite VNTR sites and construct DNA fingerprints. It is a first-generation genetic marker based on DNA molecular hybridization. Its disadvantage is that the analysis has relatively high requirements on the quality and quantity of DNA, and the entire processing process is relatively long, the steps are cumbersome, and there are certain technical difficulties. Therefore, it is limited in research that requires a large number of molecular markers. Later, PCR technology gradually replaced restriction fragment length polymorphism analysis and became the second-generation DNA typing technology. The corresponding genetic marker is STR (Short Tandem Repeats)-short tandem repeat sequences are also called microsatellites (microsatellite) or simple repeat sequences. The number of repetitions at a homologous STR site is different between different individuals. At present, STR is widely used in the field of forensic evidence, including individual identification, paternity testing, full sibling identification, non-skipped grandparent identification, etc.

[0004] The third generation genetic marker SNP refers to the DNA sequence polymorphism caused by the variation of a single base sequence in a specific part of the genome. It is the most abundant DNA sequence polymorphism in the human genome, with an average of one SNP per 1000bp. Due to the change in bases, it manifests as dimorphism, trimorphism or tetramorphism, among which dimorphic SNP is the most common. Compared with STR, although the amount of genetic information that a single SNP can provide is limited, because the number of its sites is several orders of magnitude more than that of the second generation genetic marker STR, on the whole, its polymorphism is higher. At the same time, the mutation rate of SNP genetic markers is lower than that of STR, and it has higher stability, which is more conducive to paternity testing.

[0005] At present, there are corresponding specifications for paternity testing or kinship testing of two individuals. There are no unified standards and corresponding regulations for paternity testing during pregnancy, that is, before birth. SNPs are often used as genetic markers in the field of prenatal paternity testing to identify the parent-child relationship between the fetus and the suspected father. In "A method for determining prenatal paternity using SNPs", Chen Hongliang et al. determined the parent-child relationship by selecting sites where both the pregnant woman and the father are all SNP homozygous. The utilization rate of SNPs is insufficient, and it can only be used to deny kinship, and it is impossible to accurately give a conclusion that conforms to the kinship. In "A non-invasive paternity test analysis method and device", Gao Shengjie et al. determined the parent-child relationship by calculating the cumulative paternity index CPI and / or cumulative exclusion probability CPE between the fetus and the suspected father. The premise is that the genotypes of multiple genetic marker sites of both the pregnant woman and the parents of the fetus need to be determined to infer the fetal genotype. The experiment is costly and difficult to analyze.

[0006] As described above, although various methods for prenatal kinship determination using SNPs have been disclosed in the prior art, there are generally problems such as difficulty in determining kinship or high identification costs. Summary of the invention

[0007] In order to address the deficiencies in the prior art, the present invention provides a method for non-invasive prenatal kinship identification based on dimorphic SNP sites, calculates the minimum number of SNP sites required for determining kinship, screens sites containing fetal information in free DNA samples of pregnant women, and determines kinship by combining various indicators without the need for additional genotype testing of pregnant women's samples. At the same time, it can accurately determine whether the fetus and the suspected father are in a parent-child relationship or not, and has a wider range of applications.

[0008] The present invention specifically discloses a method for determining kinship using dimorphic SNP sites, the specific steps of which include:

[0009] (1) Screening at least 292 autosomal dimorphic SNP sites with mutation frequencies between [0.4-0.6] in the sample genome as genetic markers, performing high-throughput sequencing, with a sequencing depth greater than 60X, and performing SNP typing based on the sequencing depth. In the present invention, 295 SNP sites that meet the requirements are designed as a paternity test panel;

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[0021] (2) The depth of sequencing at each site is used to obtain the informative site set of the suspected father sample and the fetal genotype;

[0022] (3) The mismatch rate of SNP sites in the genotypes of the suspected father and the fetus is calculated and used as one of the indicators for judging the relationship between the suspected father and the fetus.

[0023] Specifically, the result determination method is as follows:

[0024] 1. Determine the fetal concentration and parent-child relationship through concentration

[0025] The method and application of evaluating fetal DNA concentration using free DNA of pregnant women (CN113999900A) and the method and application of evaluating fetal DNA concentration using DNA of non-biological father or non-biological father and mother (CN113981062A, CN113889189A) are used to evaluate the fetal concentration and determine whether the parent-child or non-parent-child relationship is determined by whether the fetal concentrations calculated by the two methods are the same or twice as high.

[0026] 2. Determine parent-child relationship through matching rate and mismatch rate:

[0027] Using the above SNP locus panel, the loci that do not conform to Mendel's laws of inheritance are recorded as mismatched loci. When the fetus and the suspected father are in a parent-child relationship, all loci in the screened information locus set should conform to Mendel's laws of inheritance with the suspected father, the number of mismatched loci is 0, and the mismatch rate is 0. When the fetus and the suspected father are not in a parent-child relationship, the range of the mismatch rate in the patented method for paternity testing of pregnant fetuses without the need for a father (CN116240274A) is used to determine whether the fetus and the suspected father are not in a parent-child relationship.

[0028] 3. Determine the parent-child relationship through efficiency and inefficiency:

[0029] A method for paternity testing using homozygous loci (CN116580770A) is used to calculate the distribution of effective and inefficient results and determine the parent-child relationship.

[0030] By combining the above methods to determine the parent-child relationship between the suspected father sample and the fetus, the obtained results are accurate and low-cost, and are suitable for popularization and use.

[0031] More preferably, in order to obtain the minimum number of SNP sites required to exclude parent-child relationships, the present invention further discloses the following scheme:

[0032] The present invention only discusses dimorphic SNP sites. Taking a certain SNP site as an example, it is compared with the reference genome, and A represents the wild-type site, and the population frequency is recorded as p. a represents the mutant site, and the population frequency is recorded as q, and p+q=1.

[0033] (1) Calculate the optimal value of SNP population frequency

[0034] The peripheral blood of pregnant women contains fetal DNA. The sites at which the pregnant woman's genotype is AA and the fetus' genotype is Aa, or the pregnant woman's genotype is aa and the fetus' genotype is Aa, at a certain SNP site are recorded as information sites. The present invention uses information sites to determine kinship. The more information sites there are, the higher the system efficiency.

[0035] The probability that a SNP site in the peripheral blood sample of the gestational age is an information site is P:

[0036] P=P(AA(pregnant woman)+Aa(fetus) or aa(pregnant woman)+Aa(fetus))=p*p*q+q*q*p=p*q

[0037] When p is 0.5, the probability that a SNP site in the free peripheral blood sample of the pregnant woman is an information site is the highest, and the maximum value is 0.25, that is, theoretically, a maximum of 25% of the SNP sites measured in the free DNA sample of the pregnant woman are information sites.

[0038] That is, the optimal value of the population frequency of a dimorphic SNP site should be 0.5. When determining kinship, the population frequency of the selected SNP should be as close to 0.5 as possible.

[0039] (2) Calculate the probability Px that a single SNP site is an informative site and a random paternal match

[0040] The random paternal matching probability refers to the probability that the fetus' genotype and the random paternal genotype conform to Mendel's laws of inheritance at a certain information site. The genotype distribution of a certain SNP site that conforms to Mendel's laws of inheritance is shown in the table below.

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[0042] That is, when the maternal genotype is AA, the fetal genotype is Aa, and the paternal genotype is Aa or aa, the site conforms to Mendel's law of inheritance; similarly, when the maternal genotype is aa, the fetal genotype is Aa, and the paternal genotype is AA or Aa, the site also conforms to Mendel's law of inheritance.

[0043] The probability of a single SNP information site randomly matching is recorded as Pi, and Pi is calculated as follows:

[0044] Pi=P(AA(pregnant woman), Aa(fetus), father=Aa, aa)+P(aa(pregnant woman), Aa(fetus), father=AA, Aa)

[0045] Pi={ppq*(2pq+q^2)+qqp*(2pq+p^2)} / (ppq+qqp)=pq*(p*(2pq+qq)+q*(2pq+pp)) / pq

[0046] =2ppq+pqq+2pqq+ppq=pq(2p+q+2q+p)=3pq

[0047] Under the premise of p+q=1, Pi=3pq<=3*1 / 4<=3 / 4<=0.75

[0048] When p is the optimal value of 0.5, the theoretical probability that a single SNP information site randomly conforms to Mendelian inheritance is 75%, and the theoretical probability that a single SNP information site does not conform to Mendelian inheritance is 25%.

[0049] The probability that a single SNP locus is an informative locus and the random paternal match is recorded as Px, and Px is calculated as follows: Px = P(AA(pregnant woman), Aa(fetus), paternal = Aa, aa) + P(aa(pregnant woman), Aa(fetus), paternal = AA, Aa)

[0050] Px={ppq*(2pq+q^2)+qqp*(2pq+p^2)}

[0051] =pq*(p*(2pq+qq)+q*(2pq+pp))=p^2q^2(2p+q+2q+p)=p^2q^2(2p+q+2q+p)=3p^2q^2

[0052] Under the premise of p+q=1, Px=3p^2q^2<=3*(1 / 4) 2 <=3 / 16<=0.1875

[0053] When p is the optimal value of 0.5, the theoretical probability that a single SNP is an informative site and matches a random father is 18.75%

[0054] (3) Calculate the minimum number of SNPs required for kinship identification

[0055] Based on China's population of 1.4 billion, the probability that a random father and fetus are in a parent-child relationship is less than 1 / 1.4 billion.

[0056] The number of SNP information sites required for the probability of a random father and a fetus being in a parent-child relationship less than 1 / 1.4 billion is recorded as m, and the total number of corresponding SNP sites is recorded as n. m and n are calculated according to the following formula:

[0057] 0.75^m<=1 / 1400 million=1 / 14*10^-8

[0058] m*ln 0.75<=ln(1 / 14)-8*ln 10

[0059] m> = (ln (1 / 14) - 8 * ln 10) / ln 0.75 = (-2.64 - 8 * 2.3) / -0.288 = 73 sites

[0060] n=m / pq>=73 / pq>=73 / 0.25=292

[0061] Conclusion: At least 292 SNP sites need to be detected in the free peripheral blood samples of pregnant women to make the probability of the random father and fetus being in a parent-child relationship less than 1 / 1.4 billion, that is, to exclude the parent-child relationship between the two.

[0062] Preferably, in step (1), a trimorphic SNP site, a tetramorphic SNP site, a short tandem repeat sequence, or a methylation site can also be selected as a genetic marker. The polymorphic sites can be divided into two groups so that the difference in the population frequency and the population frequency of the two groups is minimized. One group is used as a wild-type allele A, and the other group is used as another mutant allele a. According to the above principle, the minimum number of sites required to exclude parent-child relationships is calculated by population frequency.

[0063] Preferably, in step (1), the specific steps of high-throughput sequencing are as follows: first, DNA is extracted from the free DNA sample of the peripheral blood of the pregnant woman and the sample of the suspected father to be tested, and a whole genome library is constructed, followed by whole genome amplification. After the library construction is completed, a probe SNP-panel for capturing the SNP site sequence is designed, the SNP site is captured by liquid phase hybridization, and high-throughput sequencing is performed to obtain sequencing data of the sample to be tested.

[0064] More preferably, in high-throughput sequencing, a barcode sequence representing the number, a sequencing adapter that can be used for high-throughput sequencing, and other necessary sequences are added to the DNA sequence of each sample during the library construction process, and then whole genome amplification is performed.

[0065] Wherein, the gestational age of the pregnant woman is required to be greater than 5 weeks.

[0066] Preferably, in step (2), the specific steps of obtaining the SNP typing information of the suspected father sample are as follows: A represents the wild-type site and a represents the mutant site; the sequencing data is subjected to bioinformatics analysis, requiring that the total depth DP of each SNP site is greater than 30X, if the sequencing depth ratio of the site A and a is DP(A) / DP(A+a)>0.9, then the genotype of the suspected father sample is recorded as AA; if the sequencing depth ratio of the site A and a is DP(a) / DP(A+a)>0.9, then the genotype of the suspected father sample is recorded as aa. If the sequencing depth of A and a is close to 1:1, then the genotype of the suspected father sample is recorded as a heterozygous, recorded as Aa.

[0067] More preferably, the above-mentioned bioinformatics analysis includes raw data quality control, reads alignment, reads screening, site annotation, mathematical statistics, etc.

[0068] Preferably, in step (2), the specific steps for obtaining the SNP typing information of the pregnant woman's genotype and the fetal genotype are as follows: for the pregnant woman's free DNA sample, if the sequencing depth ratio of the SNP sites A and a is 0<DP(A) / DP(A+a)<0.2, then the pregnant woman's genotype is recorded as aa, and the fetus's genotype contains A and its genotype is Aa; if the sequencing depth ratio of the sites A and a is 0<DP(a) / DP(A+a)<0.2, then the pregnant woman's genotype is recorded as AA, and the fetus's genotype contains a and its genotype is Aa.

[0069] Compared with the prior art, the present invention has the following significant effects:

[0070] (1) The technical solution of the present invention can accurately determine whether the fetus and the suspected father are in a parent-child relationship or not, and has a wide range of applications;

[0071] (2) The technical solution of the present invention requires only the detection of 292 dimorphic SNP sites with a population frequency of about 0.5 to determine the parent-child relationship between the fetus and the suspected father, and the identification cost is low. DETAILED DESCRIPTION

[0072] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0073] Embodiment 1:

[0074] A family numbered QZ54182 sent the maternal peripheral blood sample and the suspected father's blood sample for identification, which were numbered QZ54182S and QZ54182F respectively. The panel containing 295 SNPs designed by this patent was used for hybridization capture, and the SNP typing results of QZ54182S and QZ54182F were obtained after sequencing. The SNP mismatch rate between the fetus and the suspected father was calculated to be 0%, and the suspected father was judged to be the true father, and the suspected father and the fetus were in a parent-child relationship. The fetal concentrations were calculated to be 14.78% and 15.22% respectively by the method of evaluating the fetal DNA concentration by the free DNA of the pregnant woman using the existing fetal concentration index and the method of evaluating the fetal DNA concentration by the suspected father's DNA and the mother's DNA, and the suspected father and the fetus were judged to be in a parent-child relationship; the effective rate was calculated to be 0.978 by the method of using the existing indicators to perform paternity testing using homozygous sites; the invalid rate was 0, which was consistent with the distribution of the effective and inefficient (matching) when the parent-child relationship was in place, and the suspected father and the fetus were judged to be in a parent-child relationship. In summary, based on the dimorphic SNP sites, the suspected father and the fetus of this family can be accurately identified as the parent-child relationship through various indicators.

[0075] Embodiment 2:

[0076] A family coded as QZ54406 sent the maternal peripheral blood sample and the suspected father's blood sample for identification, which were coded as QZ54406S and QZ54406F respectively. The SNP typing results of QZ54406S and QZ54406F were obtained by sequencing analysis. The SNP mismatch rate between the fetus and the suspected father was calculated to be 25.42%, which was in line with the error rate of the fetus and the random paternal population [24.52%, 32.69%], and it was judged that the suspected father and the fetus were not in a parent-child relationship. The fetal concentrations were calculated to be 15.08% and 7.41% respectively by using the existing fetal concentration index to evaluate the fetal DNA concentration using the free DNA of the pregnant woman and the fetal DNA using the DNA of the suspected father and the mother. The suspected father was judged to be a non-biological father, and the suspected father and the fetus were not in a parent-child relationship. The effective rate was calculated to be 0.472 by using the existing index to perform paternity testing using homozygous loci. The invalid rate was 0.469, and the ineffectiveness / effectiveness = 0.99≈1, which was consistent with the distribution of ineffectiveness / effectiveness when the non-parent-child relationship (mismatch) was met, and the suspected father and the fetus were judged to be not in a parent-child relationship. In summary, based on the dimorphic SNP loci, the suspected father and the fetus of this family can be accurately identified as non-parent-child relationship through various indicators.

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for determining kinship using dimorphic SNP sites, characterized in that: include: (1) Screening the sample genome for at least 292 autosomal dimorphic SNP loci with a mutation frequency between [0.4-0.6] as genetic markers, performing high-throughput sequencing, with a sample sequencing depth greater than 60X, and performing SNP typing based on the sequencing depth; specifically designing 295 SNP loci that meet the conditions as a panel for paternity testing; (2) The depth of sequencing at each locus was used to obtain the informative loci set containing the suspected father's sample and the fetal genotype; (3) Calculate the mismatch rate of SNP sites in the genotypes of the suspected father and the fetus, and use the mismatch rate as one of the indicators to judge the relationship between the suspected father and the fetus.

2. The method according to claim 1, characterized in that The dimorphic SNP loci set used in step (1) is selected from a panel of 295 SNP loci.

3. The method according to claim 1, characterized in that In step (1), a trimorphic SNP site, a tetramorphic SNP site, a short tandem repeat sequence, or a methylation site may also be selected as a genetic marker.

4. The method according to claim 1, wherein In step (1), the specific steps of high-throughput sequencing are as follows: first, DNA is extracted from the free DNA sample of the peripheral blood of the pregnant woman and the sample of the suspected father to be tested, and a whole-genome library is constructed, followed by whole-genome amplification. After the library construction is completed, a probe SNP-panel is designed to capture the SNP site sequence, and the SNP site is captured by liquid phase hybridization and high-throughput sequencing is performed. The sequencing depth DP is greater than 60X to obtain sequencing data of the sample to be tested.

5. The method according to claim 4, characterized in that The gestational age of the pregnant woman is required to be greater than 5 weeks.

6. The method according to claim 1, wherein In step (2), the specific steps for obtaining the SNP typing information of the suspected father's sample are as follows: A represents the wild-type site and a represents the mutant site; the sequencing data is subjected to bioinformatics analysis, requiring that the total depth DP of each SNP site is greater than 30X. If the sequencing depth ratio of sites A and a is DP(A) / DP(A+a)>0.9, the genotype of the suspected father's sample is recorded as AA; if the sequencing depth ratio of sites A and a is DP(a) / DP(A+a)>0.9, the genotype of the suspected father's sample is recorded as aa. If the sequencing depth of A and a is close to 1:1, the genotype of the suspected father's sample is recorded as heterozygous, recorded as Aa.

7. The method according to claim 6, characterized in that In step (2), the specific steps for obtaining the SNP typing information of the pregnant woman's genotype and the fetus's genotype are as follows: for the pregnant woman's free DNA sample, if the sequencing depth ratio of the SNP sites A and a is 0<DP(A) / DP(A+a)<0.2, it is recorded as the pregnant woman's own genotype is aa, and the fetus's genotype contains A and its genotype is Aa; if the sequencing depth ratio of the sites A and a is 0<DP(a) / DP(A+a)<0.2, it is recorded as the pregnant woman's own genotype is AA, and the fetus's genotype contains a and its genotype is Aa.

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