Method for identifying the parentage between an assisted reproduction embryo and a sperm or egg donor

Abstract

This invention discloses a method for identifying the parentage between assisted reproductive embryos and sperm or egg donors, belonging to the field of paternity testing technology. The method includes the following steps: S101: Obtaining a single-cell amplification product sample S and sperm / egg donor samples F / M; S102: Sequencing the dimorphic loci of the two samples to obtain DNA data N(S) and N(F / M); S103: Using the loci in the statistical locus set N(S)∩N(F / M) as valid loci; S104: Calculating the mismatch rate and correlation between the two samples based on the valid loci; S105: Determining the parentage between the two samples based on the mismatch rate and correlation; if the correlation ∈ [0.45, 0.60] and the mismatch rate ∈ [0, 3.125%], then the two samples are determined to be parent-child. This patent can accurately determine the parentage between the embryo and the sperm / egg donor even when the genome may be lost.

Description

Technical Field

[0001] This invention belongs to the field of paternity testing technology, and specifically relates to a method for identifying the parentage between an assisted reproductive embryo and a sperm or egg donor. Background Technology

[0002] In assisted reproductive technology (ART), embryologists retrieve 1-2 blastomeres from the embryo on day 3 of in-vitro fertilization (IVF) or some trophoblast cells on day 5 for preimplantation genetic testing (PGT). This assesses for abnormalities and selects healthy embryos for transfer, reducing the risk of passing on genetic diseases to offspring. However, this only determines embryo health and does not establish paternity between the embryo and the sperm or egg donor. Errors can occur during artificial insemination, leading to incorrect sperm selection. Furthermore, during single-cell amplification, some heterozygotes may randomly lose alleles, resulting in misclassified homozygotes in the final sequencing results, complicating paternity testing between the embryo and the sperm / egg donor. Summary of the Invention

[0003] This patent is based on NGS sequencing of single-cell amplification results from early-stage single embryonic cells. Even with potential genome loss, it can still accurately determine the parentage between the embryo and the sperm / egg donor. The method is as follows:

[0004] This invention provides a method for identifying the parentage between an assisted reproductive embryo and a sperm or egg donor, the method comprising the following steps:

[0005] S101: Obtain single-cell amplification product sample S and sperm donor / egg donor sample F / M;

[0006] S102: Sequencing of the dimorphic sites in the two samples yielded DNA data N(S) and N(F / M);

[0007] S103: Sites in the statistical site set N(S)∩N(F / M) are considered as valid sites;

[0008] S104: Calculate the mismatch rate and correlation between two samples based on valid sites;

[0009] S105: Determine the parent-child relationship between two samples based on the mismatch rate and correlation; if the correlation ∈ [0.45, 0.60] and the mismatch rate ∈ [0, 3.125%], then determine that the two samples are parent-child.

[0010] Specifically, the correlation γ is calculated according to Formula I:

[0011] (I);

[0012] Where x is the ratio of the depth of S at the SNP site A to the sum of the depths of A and a, denoted as: x （S） =A （S） / (A+a) （S） ; y is the ratio of the depth of F / M at the SNP site A to the sum of the depths of A and a, denoted as: y （F / M） =A （F / M） / (A+a) （F / M） A represents the wild-type site, and a represents the mutant site.

[0013] Specifically, the mismatch rate is calculated according to Formula II:

[0014] (II)

[0015] Among them, autosomal SNP loci with a mutation frequency between [0.05-0.95] on the sample genome were selected as genetic markers, and the depth of each effective locus was not less than 20X. Attached Figure Description

[0016] Figure 1 This is a flowchart of a method for identifying the parentage between assisted reproductive embryos and sperm or egg donors, provided in an embodiment of the present invention. Detailed Implementation

[0017] 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.

[0018] Example 1

[0019] See Figure 1 This invention provides a method for identifying the parentage between an assisted reproductive embryo and a sperm or egg donor, the method comprising the following steps:

[0020] S101: Obtain single-cell amplification product sample S and sperm donor / egg donor sample F / M; specifically, sperm donor sample is F, and egg donor sample is M.

[0021] S102: Sequencing of dimorphic sites in two samples yielded DNA data N(S) and N(F / M). The sequencing process involved using next-generation sequencing (NGS) technology to obtain sequencing data from both samples. Thousands of highly conserved dimorphic SNP sites on the human genome were selected as genetic markers for paternity testing. After obtaining the samples, nucleic acid extraction was performed on the target samples, followed by whole-genome library construction. During library construction, barcode sequences representing numbers, sequencing adapters for high-throughput sequencing, and other necessary sequences were added to the F / M and S DNA sequences, and whole-genome amplification was performed. After library construction, a set of probe sequences was used to capture thousands of SNP sites using liquid-phase hybridization, followed by high-throughput sequencing and bioinformatics analysis.

[0022] S103: Sites in the statistical site set N(S)∩N(F / M) are considered as valid sites, and the depth of each valid site is not less than 20X.

[0023] S104: Calculate the mismatch rate and correlation between two samples based on valid sites.

[0024] Specifically, each valid site in each sample will have a total sequencing depth, as well as the sequencing depths of "wild-type" and "mutant" sites determined based on the human genome reference sequence. Taking a specific SNP site as an example, let A represent the wild-type site and a represent the mutant site. The sequenced data is aligned to the human reference genome, and the sequencing depths of A and a for each sample at each SNP site are calculated. The correlation γ is calculated according to Formula I:

[0025] (I).

[0026] Where x is the ratio of the depth of S at the SNP site A to the sum of the depths of A and a, denoted as: x （S） =A （S） / (A+a) （S） ; y is the ratio of the depth of F / M at the SNP site A to the sum of the depths of A and a, denoted as: y （F / M） =A （F / M） / (A+a) （F / M） .

[0027] The mismatch sites between S and F / M are counted among the valid SNP sites. At each SNP site, if two samples share at least one A / a, they are considered a match; otherwise, they are considered a mismatch. The mismatch rate is calculated using Formula II:

[0028] (II)

[0029] S105: The parent-child relationship between two samples is determined based on the mismatch rate and correlation. If the correlation ∈ [0.45, 0.60] and the mismatch rate ∈ [0, 3.125%], then the two samples are determined to be parent-child. Conversely, if the correlation ∉ [0.45, 0.60] or the mismatch rate ∉ [0, 3.125%], then the parent-child relationship between the two samples cannot be determined. Specifically, correlation is the primary criterion, and its judgment results are accurate and stable. However, within the range of [0.45, 0.60], both parent-child and full sibling relationships are satisfied. Therefore, this patent introduces another criterion: the mismatch rate. When the correlation ∈ [0.45, 0.60] and the mismatch rate ∈ [0, 3.125%], the parent-child relationship between the two samples can be determined. Verification by other methods shows that the results of this method are correct.

[0030] Example 2

[0031] Example 2 illustrates the process of obtaining the correlation and mismatch rate thresholds:

[0032] The process of obtaining the correlation threshold is as follows: Through extensive sample testing, it was found that in conventional paternity testing, if two individuals are related, their correlation should be within the range of [0.45, 0.60]. Since the embryo is also related to the sperm and egg donors, the correlation between the two samples should also be within the range of [0.45, 0.60]. A correlation within the range of [0.45, 0.60] can be used as one of the criteria for determining whether a single-cell amplification product supports a paternity relationship with the sperm / egg donor.

[0033] The process for obtaining the mismatch rate threshold is as follows:

[0034] In conventional paternity testing, since offspring inherit half their genetic material from the mother and half from the father, all valid loci detected in two matching samples are correct, resulting in a mismatch rate of 0%. However, during single-cell amplification, some heterozygotes are lost, causing them to appear as homozygotes in the sequencing results. This leads to a small number of mismatches between the embryonic single-cell amplification product matching the paternity and the valid loci set of the sperm / egg donor. For example, if the embryonic cell has the genotype Aa at a certain SNP locus, and the sperm / egg donor has the genotype aa, due to the loss of heterozygosity during single-cell amplification, the sequencing results will show that the embryonic single-cell amplification product has the genotype AA at that SNP locus, which is determined to be a mismatch after comparison with the sperm / egg donor. The following is a theoretical calculation of the mismatch rate:

[0035] The selected SNPs are all dimorphic (i.e., only A and a are possible), and the gene frequencies of A and a are p and q, respectively. Therefore, the mother's (M) genotype is (AA(p...). 2), Aa(2pq), aa(q) 2 Similarly, the father's (F) genotype is (AA(p)). 2 ), Aa(2pq), aa(q) 2 Assuming the loss of heterozygosity rate of single-cell amplification products is m; that is, the percentage of lost heterozygotes out of the total heterozygotes is m; then the F, M, embryo (S) genotypes and the results of loss of heterozygosity are shown in Tables 1-3:

[0036] Table 1

[0037]

[0038] Table 2

[0039]

[0040] Table 3

[0041]

[0042] As shown in Table 1-3, after heterozygous deletion of single-cell amplification products, the probability of mismatch between embryo (S) and F is: P(F and S mismatch) = 1 / 2∙m∙p 3 q+1 / 2∙m∙p 2 q 2 +1 / 2∙m∙p 2 q 2 +1 / 2∙m∙pq 3 =1 / 2∙m∙pq∙(p+q) 2 Since p+q=1 in the population, that is, P(F mismatch with S) = 1 / 2∙m∙pq. Similarly, the probability of embryo (S) mismatching with M is: P(M mismatch with S) = 1 / 2∙m∙p. 2 q 2 +1 / 2∙m∙p 3 q+1 / 2∙m∙pq 3 +1 / 2∙m∙p 2 q 2 =1 / 2∙m∙pq. Therefore, the mismatch rate between sperm donor / egg donor and embryo is 1 / 2∙m∙pq. Among them, the probability of heterozygote loss during single-cell amplification and sequencing is 0%-25%, and in the population, p is approximately equal to q. Therefore, if the sperm donor / egg donor and embryo are parent-child, the mismatch rate threshold range is [0, 3.125%].

[0043] Example 4:

[0044] A is known to be a single-cell amplification product of an embryo, and B is the egg donor. DNA was extracted from A and B for paternity testing. The sequencing depth of A was 77X, and that of B was 31X. Sequencing analysis yielded SNP genotyping results for A and B, identifying 1488 valid SNP loci and 21 mismatch loci. Correlation and mismatch rate were calculated. The results showed a correlation of 0.534, consistent with the correlation distribution range of this patent [0.45, 0.60]; and a mismatch rate of 1.411%, consistent with the mismatch rate density distribution range of this patent [0, 3.125%]. Therefore, the results support the paternity relationship between A and B.

[0045] Example 5:

[0046] C is known to be a single-cell amplification product of an embryo, and D is the sperm donor. DNA was extracted from C and D for paternity testing. The sequencing depth of C was 83X, and that of D was 151X. Sequencing analysis yielded SNP genotyping results for C and D, identifying 1738 valid SNP sites and 5 mismatch sites. Correlation and mismatch rate were calculated. The results showed a correlation of 0.48, consistent with the correlation distribution range of this patent [0.45, 0.60]; and a mismatch rate of 0.288%, consistent with the mismatch rate density distribution range of this patent [0, 3.125%]. Therefore, the results support the paternity relationship between C and D.

[0047] Example 6

[0048] E is known to be a single-cell amplification product of an embryo, and F is the sperm donor. DNA was extracted from E and F for paternity testing. The sequencing depth of E was 85X, and that of F was 151X. Sequencing analysis yielded SNP genotyping results for E and F, identifying 1815 valid SNP loci and 1 mismatch locus. Correlation and mismatch rate were calculated. The results showed a correlation of 0.531, consistent with the correlation distribution range of this patent [0.45, 0.60]; and a mismatch rate of 0.055%, consistent with the mismatch rate density distribution range of this patent [0, 3.125%]. Therefore, the results support the paternity relationship between E and F.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining parentage between assisted reproductive embryos and sperm or egg donors, characterized in that, The method includes the following steps: S101: Obtain single-cell amplification product sample S and sperm donor / egg donor sample F / M; S102: Sequencing of the dimorphic sites in the two samples yielded DNA data N(S) and N(F / M); S103: Sites in the statistical site set N(S)∩N(F / M) are considered as valid sites; S104: Calculate the mismatch rate and correlation between two samples based on valid sites; S105: Determine the parent-child relationship between two samples based on the mismatch rate and correlation; if the correlation ∈ [0.45, 0.60] and the mismatch rate ∈ [0, 3.125%], then determine that the two samples are parent-child. Autosomal SNP loci with a mutation frequency between [0.05-0.95] on the sample genome were selected as genetic markers, and the depth of each effective locus was not less than 20X; Calculate the correlation γ according to formula I: (I); Where x is the ratio of the depth of S at the SNP site A to the sum of the depths of A and a, denoted as: x （S） =A （S） / (A+a) （S） ; y is the ratio of the depth of F / M at the SNP site A to the sum of the depths of A and a, denoted as: y （F / M） =A （F / M） / (A+a) （F / M） A represents the wild-type site, and a represents the mutant site.

2. The method for determining parentage between assisted reproductive embryos and sperm or egg donors according to claim 1, characterized in that, Calculate the mismatch rate using Formula II: (Ⅱ)。

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