NGS method for detecting donor proportion in recipient

Abstract

The invention relates to an NGS method for detecting the proportion of donors in a recipient. The method comprises the following steps: obtaining genotype mixed cell samples of the donors and the recipient; the genotype mixed cell sample is sequenced to obtain a homozygous gene combination and a heterozygous gene combination, so that the detection data can be reduced, and the detection cost is reduced; furthermore, the homozygous gene combination and the heterozygous gene combination can be counted to obtain a plurality of SNP sites, a plurality of InDel sites and a probability density map; respectively analyzing a plurality of SNP loci and a plurality of InDel loci in the homozygous gene combination and the heterozygous gene combination according to the probability density map to obtain an NGS result which comprises the ddcfDNA content of the donor and quantitative information of each InDel locus and is used for detecting the proportion of the donor in the recipient; therefore, SNP site and InDel site analysis can be carried out on a donor and a recipient, so that the obtained donor DNA content information is more accurate.

Description

Technical Field

[0001] The present invention relates to the field of NGS detection technology, and in particular to an NGS method for detecting the donor ratio in a recipient. Background Art ‌dd-cfDNA (donor-derived cell-free DNA)‌ refers to the free DNA fragments from the donor present in the recipient's blood and urine. These fragments carry information about the donor and can be used to assess the health status and damage sites of transplant patients.

[0002] dd-cfDNA technology detects the content of donor-derived free DNA in blood and urine through the second-generation gene sequencing platform, which can predict the damage of the transplant. As a diagnostic indicator of rejection reaction, it has the advantages of non-invasiveness, high sensitivity and real-time monitoring, so it is also called liquid biopsy. Studies at multiple transplant centers around the world have found that dd-cfDNA technology can detect transplant rejection reactions and can replace traditional puncture biopsies. It has even become a routine detection method abroad. Compared with traditional puncture biopsies, dd-cfDNA testing can avoid the risks and inconveniences brought about by invasive examinations. However, the sequencing and analysis of recipient gDNA and cfDNA in the existing technology is cumbersome and costly. If the recipient's genomic information is not required, the accuracy of the test will be insufficient. .

[0003] Therefore, there is an urgent need to propose an NGS method for detecting the donor ratio in the recipient, in order to solve the technical problem that when detecting the ddcfDNA content in the context of the recipient cfDNA, the existing technology requires sequencing and analysis of the recipient gDNA and cfDNA, which is cumbersome and costly. If the recipient's genomic information is not required, the detection accuracy will be insufficient. Summary of the invention

[0004] In view of this, it is necessary to provide an NGS method for detecting the donor ratio in the recipient, so as to solve the technical problem that when detecting the ddcfDNA content in the recipient cfDNA background in the existing technology, it is necessary to sequence and analyze the recipient gDNA and cfDNA, which is cumbersome and costly. If the recipient genome information is not required, the detection accuracy will be not high enough.

[0005] In order to solve the above problems, the present invention provides an NGS method for detecting the donor ratio in a recipient, comprising: Obtain a mixed cell sample of donor and recipient genotypes; Sequencing the genotype mixed cell sample to obtain homozygous gene combinations and heterozygous gene combinations; Performing statistics on the homozygous gene combination and the heterozygous gene combination to obtain multiple SNP sites, multiple InDel sites and a probability density map; According to the probability density map, the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed respectively to obtain the NGS result of the donor ratio in the detection recipient; the NGS result of the donor ratio in the detection recipient includes the ddcfDNA content of the donor and the quantitative information of each InDel site.

[0006] In a possible implementation, statistics are performed on the homozygous gene combination and the heterozygous gene combination to obtain multiple SNP sites, multiple InDel sites and a probability density map, including: Based on the population genetic equilibrium model, the expected loci were obtained; According to the expected site, the homozygous gene combination and the heterozygous gene combination are counted to obtain a statistical result; the statistical result includes multiple SNP sites and multiple InDel sites; Screening the multiple SNP sites in the homozygous gene combination and the heterozygous gene combination to obtain a minor allele frequency data set; The cell samples in the minor allele frequency dataset are plotted to obtain a probability density map.

[0007] In a possible implementation, the screening of the multiple SNP sites in the homozygous gene combination and the heterozygous gene combination to obtain a minor allele frequency data set includes: According to the statistical results, the sequencing depth of each SNP site is obtained; According to the sequencing depth, the base frequency of each SNP site is obtained; According to the base frequencies, the minor allele frequencies are obtained; The multiple SNP sites are screened according to the minor allele frequency to obtain a minor allele frequency data set.

[0008] In a possible implementation, the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed respectively according to the probability density map to obtain an NGS result for detecting the donor ratio in the recipient, including: Perform Gaussian distribution on the homozygous gene combination and the heterozygous gene combination according to the probability density graph and a preset Gaussian mixture model to obtain the ddcfDNA content of the donor; The homozygous gene combination and the heterozygous gene combination are analyzed according to the probability density map to obtain quantitative information of each InDel site.

[0009] In a possible implementation, performing Gaussian distribution on the homozygous gene combination and the heterozygous gene combination according to the probability density map and a preset Gaussian mixture model to obtain the ddcfDNA content of the donor includes: According to the independent assortment rule and the minor allele frequency, the initial admixture ratio is obtained; According to the probability density graph, an initial first mean, an initial second mean, an initial first standard deviation, and an initial second standard deviation are obtained; Iteratively optimizing the initial first mean, the initial second mean, the initial mixing ratio, the initial first standard deviation, and the initial second standard deviation according to the maximum expectation algorithm to obtain the target first mean, the target second mean, the target mixing ratio, the target first standard deviation, and the target second standard deviation; Replacing the parameters in the preset Gaussian mixture model according to the target first mean, the target second mean, the minor allele frequency, the target mixing ratio, the target first standard deviation, and the target second standard deviation to obtain an updated Gaussian mixture model; The homozygous gene combination and the heterozygous gene combination are Gaussian distributed according to the updated Gaussian mixture model to obtain the ddcfDNA content of the donor.

[0010] In a possible implementation, analyzing the homozygous gene combination and the heterozygous gene combination according to the probability density map to obtain quantitative information of each InDel site includes: According to the probability density map, the mutation abundance of each InDel site is obtained; Analyze each InDel site according to the mutation abundance to obtain the corresponding donor content; The corresponding InDel sites on the probability density map are marked according to the donor content to obtain quantitative information of each InDel site.

[0011] In a possible implementation, sequencing the genotype mixed cell sample to obtain a homozygous gene combination and a heterozygous gene combination includes: Set the preset sequencing depth and preset cfDNA content; Under the condition that the preset conditions are met, determining the target SNP site in the genotype mixed cell sample; Obtaining an actual value according to the target SNP site and the preset cfDNA content; Obtaining an observed value according to the target SNP site and the preset sequencing depth; According to the actual value and the observed value, a minimum sequencing depth under the preset cfDNA content is obtained; The genotype mixed cell sample is identified according to the minimum sequencing depth to obtain a homozygous gene combination and a heterozygous gene combination.

[0012] In a possible implementation, the quantitative information includes a theoretical value of the fragment analysis of each InDel site; after the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed respectively according to the probability density map to obtain an NGS result for detecting the donor ratio in the recipient, the method further includes: Obtaining the deviation of each InDel site according to the donor content and the fragment analysis theoretical value; Determining at least one target deviation that is smaller than a preset deviation based on all deviations of the plurality of InDel sites; Subsequent monitoring is performed based on the site corresponding to the at least one target deviation.

[0013] In a possible implementation, the density function formula of the Gaussian mixture model is:

[0014] In the formula, x is the minor allele frequency, p is the target mixing ratio, μ 1 is the target first mean, μ 2 is the target second mean, σ 1 is the target first standard deviation, σ 2 is the target second standard deviation.

[0015] The beneficial effects of the present invention are: obtaining a mixed cell sample of the genotype of the donor and the recipient; sequencing the mixed cell sample of the genotype to obtain a homozygous gene combination and a heterozygous gene combination, thereby reducing the detection data and reducing the detection cost; further, the homozygous gene combination and the heterozygous gene combination can be statistically analyzed to obtain multiple SNP sites, multiple InDel sites and a probability density map; according to the probability density map, the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed respectively, including the NGS result of the detection of the ddcfDNA content of the donor and the quantitative information of each InDel site and the proportion of the donor in the recipient; thereby, the individual SNP sites and multiple InDel sites of the donor and the recipient can be analyzed, so that the obtained donor DNA content information is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic flow chart of an embodiment of the NGS method for detecting the donor ratio in a recipient provided by the present invention; Figure 2 For the present invention Figure 1A schematic flow chart of an embodiment of step S102; Figure 3 For the present invention Figure 1 A schematic flow chart of an embodiment of step S103; Figure 4 A schematic diagram of a coordinate system of an embodiment of a probability density map provided by the present invention; Figure 5 A schematic diagram of an embodiment of the process for determining the ddcfDNA content of a donor provided by the present invention; Figure 6 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 1 provided by the present invention; Figure 7 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 2 provided by the present invention; Figure 8 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 3 provided by the present invention; Fig. 9 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 4 provided by the present invention; Fig.10 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 5 provided by the present invention; Fig.11 A schematic diagram of a coordinate system of an embodiment of a density distribution diagram of sample No. 6 provided by the present invention; Fig.12 A schematic diagram of an embodiment of a flow chart for determining quantitative information provided by the present invention. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0018] like Figure 1 As shown, a specific embodiment of the present invention discloses an NGS method for detecting the donor ratio in a recipient, comprising: S101. Obtain a mixed cell sample of donor and recipient genotypes; S102, sequencing the genotype mixed cell sample to obtain homozygous gene combinations and heterozygous gene combinations; S103, performing ascending statistics on homozygous gene combinations and heterozygous gene combinations to obtain multiple SNP sites, multiple InDel sites and probability density maps; S104. Analyze multiple SNP sites and multiple InDel sites in the homozygous gene combination and the heterozygous gene combination according to the probability density map, and obtain the NGS result of the donor ratio in the detection recipient; the NGS result of the donor ratio in the detection recipient includes the ddcfDNA content of the donor and the quantitative information of each InDel site.

[0019] It should be understood that in biomedical research and clinical practice, accurate identification and quantification of different components in genotype mixed cell samples are crucial for understanding disease mechanisms, evaluating treatment effects, and monitoring transplant rejection reactions. The embodiments of the present invention are intended to solve the problem of identifying the proportions of two different human cell components after mixing. It can be widely used in the field of cell immunotherapy such as evaluation of chimerism after allogeneic stem cell transplantation, monitoring of dd-cfDNA after organ transplantation, and allogeneic CAR-T or CAR-NK.

[0020] In a specific embodiment of the present invention, a mixed genotype cell sample of the donor and the recipient can be obtained. Specifically, the genomic DNA samples of the donor and the recipient can be mixed in a certain ratio, and the concentrations can be calculated and mixed with references to form a reference ratio gradient of 0.10%, 0.50%, 1.00%, 1.25%, 2.50% and 5.00% as a benchmark, which are recorded as sample numbers 1-6. The specific settings can be made according to actual conditions, and the embodiments of the present invention are not limited here. The genotype mixed cell samples can then be grouped. Specifically, the Hardy-Weinberg law is a basic population genetic equilibrium model. According to this law, the probability of a specific genotype in a population can be calculated. Let m be the probability of the "A" genotype in the population, and 1-m be the probability of the "a" genotype. Based on this, the following probabilities can be obtained: the probability of type AA is m 2 , the probability of type aa is (1−m) 2 , the probability of type Aa is 2m-2m 2 For the genotype mixed cell samples of donor D and recipient R, they can be divided into three situations: P1: The probability of a homozygous gene combination where the recipient is AA and the donor is aa, or the recipient is aa and the donor is AA is as shown in formula (1): (1) P2: The probability of a heterozygous gene combination where the recipient is AA and the donor is Aa, or the recipient is aa and the donor is Aa is as shown in formula (2): (2) P3: The remaining probability is as shown in formula (3), that is, the case other than P1 and P2: (3) Then, the cell samples in the homozygous gene combination and the heterozygous gene combination can be sequenced to obtain multiple SNP sites, multiple InDel sites and probability density maps. Then, the cell samples of multiple SNP sites and multiple InDel sites in the homozygous gene combination and the heterozygous gene combination can be analyzed separately according to the probability density map to obtain the NGS results of the donor ratio in the detection recipient; the NGS results of the donor ratio in the detection recipient include the ddcfDNA content of the donor and the quantitative information of each InDel site; and then the NGS results of the donor ratio in the detection recipient can be analyzed by a comprehensive analysis method, so that more accurate donor DNA content information can be extracted from the SNP and InDel sites, providing more comprehensive molecular genetic evidence for clinical diagnosis and research.

[0021] Compared with the prior art, the present embodiment provides a method for obtaining a mixed cell sample of the genotype of the donor and the recipient; sequencing the mixed cell sample of the genotype to obtain a homozygous gene combination and a heterozygous gene combination, thereby reducing the detection data and reducing the detection cost; further, the homozygous gene combination and the heterozygous gene combination can be statistically analyzed to obtain multiple SNP sites, multiple InDel sites and a probability density map; according to the probability density map, the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed separately, including the NGS result of the detection of the ddcfDNA content of the donor and the quantitative information of each InDel site and the proportion of the donor in the recipient; thereby, the individual SNP sites and multiple InDel sites of the donor and the recipient can be analyzed, so that the obtained donor DNA content information is more accurate.

[0022] In some embodiments of the present invention, Figure 2 As shown, step S102 includes: S201, setting a preset sequencing depth and a preset cfDNA content; S202, determining the target SNP site in the genotype mixed cell sample when the preset conditions are met; S203, obtaining an actual value according to the target SNP site and the preset cfDNA content; S204, obtaining an observed value according to the target SNP site and a preset sequencing depth; S205. Obtaining a minimum sequencing depth under a preset cfDNA content according to the actual value and the observed value; S206. Identify the genotype mixed cell sample according to the minimum sequencing depth to obtain homozygous gene combinations and heterozygous gene combinations.

[0023] In a specific embodiment of the present invention, the processing can be performed by estimating the sequencing depth. Specifically, a preset sequencing depth D1 and a preset cfDNA content can be set. The preset cfDNA content can be 1%. The target SNP site in the genotype mixed cell sample can be determined when the preset conditions are met. The preset conditions can be to determine the target SNP site under the above-mentioned two conditions P1 and P2. The actual value of the cfDNA content of the target SNP site is 1% represented by p, and the observed donor depth is represented by d indicates that, and d follows a binomial distribution, which describes the D 1 The observed proportion of donor alleles, the expected observed value p′=d′ / D 1 falls on the actual value p The probability of being within the relative error range of 1% is at least 92%, so the minimum sequencing depth at the preset cfDNA content of 1% can be determined. D As shown in formula (4): (4) When the actual cfDNA content is 0.1%, the minimum sequencing depth required is 478,037, and the relative error is 8%. As the actual ddcfDNA content increases, the minimum sequencing depth required gradually decreases. For example: in liver transplant patients, the actual cfDNA content of 10% is used as the threshold of cfDNA content, and the AUC for rejection diagnosis is as high as 96.5%, sensitivity 90.3%, and specificity 92.9%; assuming that P1 occurs i times and P2 occurs a small sequencing depth of 4,307; in lung transplantation, the cfDNA level of 1% is used as the threshold of cfDNA content, showing that the sensitivity of severe acute rejection reaction is 100%, and the minimum sequencing depth required is 47,373; in heart transplantation, the cfDNA level of 0.25% is used as the threshold of cfDNA content, and the negative predictive value for AR is 99%, which can safely eliminate 81% of endomyocardial biopsies, and the minimum sequencing depth required is 190,928. However, as the sequencing depth increases, high-throughput targeted sequencing will also have a certain proportion of off-target effects, leading to an increase in detection costs. Therefore, the number of SNPs can be increased to reduce costs. This method can be used to identify homozygous gene combination P1 events and heterozygous gene combination P2 events.

[0024] In some embodiments of the present invention, Figure 3 As shown, step S103 includes: S301. Obtain the expected loci based on the population genetic equilibrium model; S302, performing statistics on homozygous gene combinations and heterozygous gene combinations according to expected sites to obtain statistical results; the statistical results include multiple SNP sites and multiple InDel sites; S303, screening multiple SNP sites in the homozygous gene combination and the heterozygous gene combination to obtain a minor allele frequency data set; S304. Plot the cell samples in the minor allele frequency data set to obtain a probability density map.

[0025] In a specific embodiment of the present invention, it is assumed that the expected number of P1 occurrences i times and P2 occurrences j times is , as shown in formula (5): (5) In the formula, is the expected number of P1 occurring i-1 times and P2 occurring j times; is the expected number of times P1 occurs i times and P2 occurs j-1 times.

[0026] In the binomial distribution, 1 SNP has a probability of 500,000×, which is equivalent to 50 SNPs with a probability of 10,000×. As shown in formula (5), N 100,200,0 is about 800. Therefore, using 930 sites (901 SNPs, 29 InDel sites) with an average depth of 5,000X can meet expectations, wherein 901 SNP sites and 29 InDel sites can be screened from population frequency databases such as gnomad, 1000 Genomes 1000G, UCSC and literature, and polymorphic sites with a preset mutation frequency and only two alleles in Asians. When the homozygous gene combination and the heterozygous gene combination are statistically analyzed according to the expected sites by the sequencing depth estimation method, the statistical results can be obtained; 901 SNP sites and 29 InDel sites are distributed in the three combinations of P1, P2 and P3, so that the number of SNP sites and the number of InDel sites included in the homozygous gene combination P1 and the heterozygous gene combination P2 can be determined according to the statistical results, that is, multiple SNP sites and multiple InDel sites, P3 is not P1 and P2, and the embodiment of the present invention does not process it.

[0027] In some embodiments of the present invention, step S303 includes: According to the statistical results, the sequencing depth of each SNP site was obtained; According to the sequencing depth, the base frequency of each SNP site is obtained; Based on the base frequency, the minor allele frequency is obtained; Multiple SNP sites were screened according to the minor allele frequency to obtain the minor allele frequency data set.

[0028] In a specific embodiment of the present invention, after obtaining the statistical results, bioinformatics analysis can be performed on the statistical results, and the sequencing depth of each SNP site in the detection kit and the sequencing depth of the four bases A / T / C / G are statistically analyzed. Then, the base frequencies of the four bases A / T / C / G of each SNP site are calculated, and compared with the reference alleles and alternative alleles in the population frequency database to determine the minor allele frequency, and other allele frequencies inconsistent with the reference alleles and alternative alleles are used as the error rate caused by PCR or sequencing, and the uncovered SNP sites are filtered, and then the error rates are sorted, and the error rate value with a quantile of 0.95 is calculated as the cutoff, and SNP sites with minor allele frequencies greater than the cutoff and less than or equal to 0.1 are screened out, and outliers are filtered out. The retained SNP SNPs are called valid SNP sites, that is, a minor allele frequency data set composed of minor allele frequencies of valid SNP sites is obtained.

[0029] Furthermore, the cell samples in the minor allele frequency data set can be plotted to obtain a probability density map, such as Figure 4 As shown, the x-axis is the minor allele frequency, the y-axis is the density, Peak1 is the first peak, and Peak2 is the second peak.

[0030] In some embodiments of the present invention, step S104 includes: Gaussian distribution of homozygous gene combinations and heterozygous gene combinations was performed according to the probability density graph and the preset Gaussian mixture model to obtain the ddcfDNA content of the donor; Homozygous and heterozygous gene combinations were analyzed according to the probability density plot to obtain quantitative information of each InDel site.

[0031] In a specific embodiment of the present invention, the homozygous gene combination and the heterozygous gene combination can be Gaussian distributed according to the probability density map and the preset Gaussian mixture model to obtain the ddcfDNA content of the donor; the homozygous gene combination and the heterozygous gene combination can also be analyzed according to the probability density map to obtain quantitative information of each InDel site.

[0032] In some embodiments of the present invention, Figure 5 As shown, according to the probability density diagram and the preset Gaussian mixture model, the homozygous gene combination and the heterozygous gene combination are Gaussian distributed to obtain the ddcfDNA content of the donor, including: S501, according to the independent assortment rule and the minor allele frequency, the initial mixing ratio is obtained; S502, obtaining an initial first mean, an initial second mean, an initial first standard deviation, and an initial second standard deviation according to the probability density graph; S503, iteratively optimizing the initial first mean, the initial second mean, the initial mixing ratio, the initial first standard deviation, and the initial second standard deviation according to the maximum expectation algorithm to obtain a target first mean, a target second mean, a target mixing ratio, a target first standard deviation, and a target second standard deviation; S504, replacing the parameters in the preset Gaussian mixture model according to the target first mean, the target second mean, the minor allele frequency, the target mixing ratio, the target first standard deviation, and the target second standard deviation to obtain an updated Gaussian mixture model; S505. Perform Gaussian distribution on the homozygous gene combination and the heterozygous gene combination according to the updated Gaussian mixture model to obtain the ddcfDNA content of the donor.

[0033] In a specific embodiment of the present invention, the initial mixing ratio can be calculated according to the independent assortment rule and the minor allele frequency. p , that is, the probability of homozygous and heterozygous genotypes of the donor SNP, and then calculate all peaks based on the plotted probability density graph, and use the first peak as the initial first mean of the first Gaussian model μ 1, twice its value is used as the initial second mean of the second Gaussian model μ 2. And determine the standard deviation corresponding to the two Gaussian distributions, that is, the initial first standard deviation σ 1 and the initial second standard deviation σ 2. Then, the expectation-maximization algorithm (EM) is used to iterate the optimization process. p , μ 1. μ 2. σ 1 and σ 2 Make an accurate estimate, so that the target first mean can be obtained μ 1. Target second mean μ 2. Target Mixing Ratio p , Target first standard deviation σ 1 and target second standard deviation σ 2, then the target first mean can be μ 1. Target second mean μ 2. Minor allele frequency and target mixing ratio p , Target first standard deviation σ 1 and target second standard deviation σ2. Substitute into the preset Gaussian mixture model, update and replace the parameters in the preset Gaussian mixture model, so that the homozygous gene combination and the heterozygous gene combination can be Gaussian distributed according to the updated Gaussian mixture model, and the ddcfDNA content of the donor can be obtained through the density function of the Gaussian mixture model, where the density function formula of the Gaussian mixture model is: (6) In the formula, x is the minor allele frequency, p is the target mixing ratio, μ 1 is the target first mean, μ 2 is the target second mean, σ 1 is the target first standard deviation, σ 2 is the target second standard deviation.

[0034] Samples numbered 1-6 were 0.10%, 0.50%, 1.00%, 1.25%, 2.50% and 5.00% standard samples, respectively, and were processed by the embodiments of the present invention, gender internal standard reads and STR (Short Tandem Repeat) method, and the final ddcfDNA results were shown in Table 1: Table 1. Test results of ddcfDNA content of sample numbers 1-6

[0035] Among them, the STR method did not detect the ddcfDNA content of samples 1-3 because its detection limit was higher than 1%. The detection results of the gender internal standard reads were too different from those of the standard. However, the ddcfDNA content obtained by the method in the embodiment of the present invention was closest to the theoretical dilution, indicating that the embodiment of the present invention can detect a more accurate ddcfDNA content. Subsequently, the density distribution diagram of the minor allele frequency after screening was drawn to form a curve in the figure, in which the abscissa is the minor allele frequency distribution and the ordinate is the number of candidate sites. The abscissa corresponding to the first vertical line is used as the initial mean, and the abscissa of the second vertical line represents the mean of the Gaussian distribution after fitting, which is finally used as the relative content of ddcfDNA. The results are as follows Figure 6-11 As shown, Figure 6 This is the density distribution diagram of sample No. 1. Figure 7 This is the density distribution diagram of sample No. 2. Figure 8 This is the density distribution diagram of sample No. 3. Fig. 9 This is the density distribution diagram of sample No. 4. Fig.10 This is the density distribution diagram of sample No. 5. Fig.11 This is the density distribution diagram of sample No. 6, Peak1 represents peak 1, and Peak2 represents peak 2.

[0036] In some embodiments of the present invention, Fig.12 As shown in the figure, the homozygous gene combination and the heterozygous gene combination are analyzed according to the probability density map to obtain the quantitative information of each InDel site, including: S1201, obtaining the mutation abundance of each InDel site according to the probability density map; S1202, analyzing each InDel site according to the mutation abundance to obtain the corresponding donor content; S1203. Annotate the corresponding InDel sites on the probability density map according to the donor content to obtain quantitative information of each InDel site.

[0037] In a specific embodiment of the present invention, for SNP sites, the alleles are represented as A / a, and in InDel sites, they are simplified to In / Del (or abbreviated as + / -). Assume that 901 SNP sites are analyzed by the next generation sequencing (NGS) panel, and the probability distribution density curve of the donor has been fitted (such as Figure 4 ).

[0038] The first peak (Peak1) corresponds to the P2 event (i.e., R=+ / +, D=+ / - or R=- / -, D=+ / -), and the second peak (Peak2) corresponds to the P1 event (R=+ / +, D=- / - or R=- / -, D=+ / +).

[0039] For the 29 InDel-type mutation sites, the mutation abundance F of each InDel site can be obtained, and the data of the InDel site can be integrated into the analysis framework, as shown in formula (7): (7) Then, according to the mutation abundance F of each InDel site, the corresponding donor content X can be obtained through formula (7), so that the corresponding InDel sites on the probability density map can be annotated according to the donor content, and the quantitative information of each InDel site can be obtained, so as to more comprehensively evaluate the content and distribution of donor DNA. This comprehensive analysis method can extract more accurate donor DNA content information from the NGS results of the donor ratio in the test recipient from SNP and InDel sites, providing more comprehensive molecular genetic evidence for clinical diagnosis and research.

[0040] In some embodiments of the present invention, the quantitative information includes the theoretical value of the fragment analysis of each InDel site; after step S105, it also includes: According to the donor content and the theoretical value of fragment analysis, the deviation of each InDel site was obtained; Determining at least one target deviation that is smaller than a preset deviation based on all deviations of the plurality of InDel sites; Subsequent monitoring is performed based on the sites corresponding to at least one target deviation.

[0041] In a specific embodiment of the present invention, the quantitative information is shown in Table 2: Table 2. Quantitative information of InDel sites

[0042]

[0043] The ID column in Table 1 represents the rsID information of the locus, and the "Fragment Analysis Theoretical Value" column shows the results of digital PCR fragment analysis. The locus with the donor content X value in the Peak2±20% area is judged as having a P1 event, and the locus with the X value in the Peak1±20% area is judged as having a P2 event, as shown in Formula 8 in the "Donor D Typing / Recipient R Typing" column: (8) The typing results of the donor and the recipient are provided, and the deviation between the InDel site and the theoretical value of the fragment analysis is evaluated to obtain the deviation of each InDel site, that is, the donor content X of each InDel site is calculated with the theoretical value of the fragment analysis to obtain the deviation of the corresponding InDel site. The calculation process can be set according to the actual situation, and the embodiment of the present invention is not limited here. In order to ensure the accuracy of the monitoring results, at least one target deviation less than the preset deviation is determined according to all deviations of multiple InDel sites; for example, the preset deviation can be 5%, so as to obtain at least one target deviation less than 5%, and then digital PCR (dPCR) or quantitative PCR (qPCR) technology can be used to perform subsequent monitoring of the site corresponding to at least one target deviation. This method not only improves the accuracy of the analysis, but also enhances the sensitivity and reliability of changes in the donor DNA content, and provides a solid data foundation for subsequent clinical tracking and research. Through this comprehensive analysis and precise monitoring strategy, it is possible to more effectively evaluate and track microchimerism or the presence of ddcfDNA after transplantation, thereby providing strong molecular genetic support for clinical decision-making.

[0044] The NGS method for detecting the donor ratio in the recipient provided by the present invention is introduced in detail above. Specific examples are used herein to illustrate the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation mode and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. An NGS method for detecting the donor ratio in a recipient, characterized in that: include: Obtain a mixed cell sample of donor and recipient genotypes; Sequencing the genotype mixed cell sample to obtain homozygous gene combinations and heterozygous gene combinations; Performing statistics on the homozygous gene combination and the heterozygous gene combination to obtain multiple SNP sites, multiple InDel sites and a probability density map; According to the probability density graph, the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed respectively to obtain an NGS result for detecting the donor ratio in the recipient; The NGS result for detecting the donor ratio in the recipient includes the ddcfDNA content of the donor and the quantitative information of each InDel site.

2. The NGS method for detecting the donor ratio in a recipient according to claim 1, characterized in that: The statistics of the homozygous gene combination and the heterozygous gene combination are performed to obtain multiple SNP sites, multiple InDel sites and a probability density map, including: Based on the population genetic equilibrium model, the expected loci were obtained; According to the expected site, the homozygous gene combination and the heterozygous gene combination are counted to obtain a statistical result; the statistical result includes multiple SNP sites and multiple InDel sites; Screening the multiple SNP sites in the homozygous gene combination and the heterozygous gene combination to obtain a minor allele frequency data set; The cell samples in the minor allele frequency dataset are plotted to obtain a probability density map.

3. The NGS method for detecting the donor ratio in a recipient according to claim 2, characterized in that: The step of screening the multiple SNP sites in the homozygous gene combination and the heterozygous gene combination to obtain a minor allele frequency data set includes: According to the statistical results, the sequencing depth of each SNP site is obtained; According to the sequencing depth, the base frequency of each SNP site is obtained; According to the base frequencies, the minor allele frequencies are obtained; The multiple SNP sites are screened according to the minor allele frequency to obtain a minor allele frequency data set.

4. The NGS method for detecting the donor ratio in a recipient according to claim 3, characterized in that: The method of analyzing the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination according to the probability density map to obtain an NGS result for detecting the donor ratio in the recipient includes: Perform Gaussian distribution on the homozygous gene combination and the heterozygous gene combination according to the probability density graph and a preset Gaussian mixture model to obtain the ddcfDNA content of the donor; The homozygous gene combination and the heterozygous gene combination are analyzed according to the probability density map to obtain quantitative information of each InDel site.

5. The NGS method for detecting the donor ratio in a recipient according to claim 4, characterized in that: The step of performing Gaussian distribution on the homozygous gene combination and the heterozygous gene combination according to the probability density graph and a preset Gaussian mixture model to obtain the ddcfDNA content of the donor includes: According to the independent assortment rule and the minor allele frequency, the initial admixture ratio is obtained; According to the probability density graph, an initial first mean, an initial second mean, an initial first standard deviation, and an initial second standard deviation are obtained; Iteratively optimizing the initial first mean, the initial second mean, the initial mixing ratio, the initial first standard deviation, and the initial second standard deviation according to the maximum expectation algorithm to obtain the target first mean, the target second mean, the target mixing ratio, the target first standard deviation, and the target second standard deviation; Replacing the parameters in the preset Gaussian mixture model according to the target first mean, the target second mean, the minor allele frequency, the target mixing ratio, the target first standard deviation, and the target second standard deviation to obtain an updated Gaussian mixture model; The homozygous gene combination and the heterozygous gene combination are Gaussian distributed according to the updated Gaussian mixture model to obtain the ddcfDNA content of the donor.

6. The NGS method for detecting the donor ratio in a recipient according to claim 4, characterized in that: The step of analyzing the homozygous gene combination and the heterozygous gene combination according to the probability density map to obtain quantitative information of each InDel site includes: According to the probability density map, the mutation abundance of each InDel site is obtained; Analyze each InDel site according to the mutation abundance to obtain the corresponding donor content; The corresponding InDel sites on the probability density map are marked according to the donor content to obtain quantitative information of each InDel site.

7. The NGS method for detecting the donor ratio in a recipient according to claim 1, characterized in that: The step of sequencing the genotype mixed cell sample to obtain a homozygous gene combination and a heterozygous gene combination comprises: Set the preset sequencing depth and preset cfDNA content; Under the condition that the preset conditions are met, determining the target SNP site in the genotype mixed cell sample; Obtaining an actual value according to the target SNP site and the preset cfDNA content; Obtaining an observed value according to the target SNP site and the preset sequencing depth; According to the actual value and the observed value, a minimum sequencing depth under the preset cfDNA content is obtained; The genotype mixed cell sample is identified according to the minimum sequencing depth to obtain a homozygous gene combination and a heterozygous gene combination.

8. The NGS method for detecting the donor ratio in a recipient according to claim 6, characterized in that: The quantitative information includes the theoretical value of the fragment analysis of each InDel site; after the multiple SNP sites and the multiple InDel sites in the homozygous gene combination and the heterozygous gene combination are analyzed according to the probability density map, and the NGS result of detecting the donor ratio in the recipient is obtained, it also includes: Obtaining the deviation of each InDel site according to the donor content and the fragment analysis theoretical value; Determining at least one target deviation that is smaller than a preset deviation based on all deviations of the plurality of InDel sites; Subsequent monitoring is performed based on the site corresponding to the at least one target deviation.

9. The NGS method for detecting the donor ratio in a recipient according to claim 5, characterized in that: The density function formula of the Gaussian mixture model is: In the formula, x is the minor allele frequency, p is the target mixing ratio, μ 1 is the target first mean, μ 2 is the second mean. σ 1 is the target first standard deviation, σ 2 is the target second standard deviation.

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