A method for preparing a pregnant woman simulated plasma standard that can be enriched for fetal concentration by fragment size

Abstract

The present application belongs to the technical field of non-invasive prenatal testing, and particularly relates to a preparation method of a pregnant woman simulated plasma standard product capable of fetal concentration enrichment through fragment size. The present application provides a preparation method of a pregnant woman plasma free DNA simulation sample. The method combines specific DNA fragmentation processing technology to prepare a simulation sample with high fidelity and stability and capable of accurately simulating the fetal free DNA and pregnant woman free DNA distribution proportion in real pregnant woman plasma, for quality control and evaluation of non-invasive prenatal diagnosis technology. The method can accurately prepare the pregnant woman simulated plasma DNA with the corresponding fetal concentration according to the requirement, and the prepared pregnant woman simulated plasma can meet the requirement of the application of fetal concentration enrichment test, and at the same time, the cfDNA fragment distribution in the pregnant woman plasma is restored in the maximum range.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of non-invasive prenatal testing, and particularly relates to a preparation method of a pregnant woman simulated plasma standard product capable of enriching fetal concentration through fragment size. BACKGROUND

[0002] Prenatal screening is an important means to reduce birth defects and improve the quality of the population. Among them, non-invasive prenatal screening technology can analyze the risk of fetal carrying single gene disease or chromosomal disease by detecting fetal cell-free DNA in the plasma of pregnant women. In 1997, Lo et al. first described the phenomenon of fetal cell-free DNA existing in maternal plasma. In 2011, high-throughput sequencing (NGS) was introduced into the field of NIPT, which significantly improved the sensitivity and specificity of detection. Based on whole genome sequencing or targeted region sequencing, the fetal chromosomal abnormalities can be accurately analyzed, and the current detection accuracy is close to 99%. The initial NIPT mainly targets common chromosomal aneuploidy abnormalities, and in recent years, it has gradually expanded to sex chromosome abnormalities (such as Turner syndrome), microdeletion and microduplication syndrome (such as 22q11.2 deletion syndrome) and detection of single gene genetic diseases.

[0003] Fetal DNA concentration (fetal fraction, FF) is one of the key factors for the success of NIPT detection, which directly affects the sensitivity and specificity of detection. Studies have shown that the detection accuracy of NIPT may be limited in the case of low fetal DNA concentration (<4%) 【1】; and for non-invasive prenatal microdeletion and microduplication detection (NIPT-plus), the detection accuracy is positively correlated with fetal DNA concentration, and the improvement of fetal DNA concentration significantly improves the resolution ability of small genomic abnormalities 【2】. In the case of low fetal DNA concentration, the interference of background maternal DNA increases, and the signal-to-noise ratio decreases, which may cause the signal of chromosomal copy number abnormality to be masked, resulting in false negative results. For the detection of microdeletion and microduplication syndrome, since the range of genomic abnormalities involved is small, the resolution ability of fetal DNA signal is higher, so the concentration level has a more significant impact on the detection result. Therefore, in the non-invasive prenatal screening technology, the accurate quantification and evaluation of fetal DNA concentration are particularly important. The collection or preparation of pregnant woman plasma cell-free samples with different fetal DNA concentrations can be used to evaluate the quantification of fetal concentration and the accuracy of fetal chromosomal or single gene disease abnormality detection, which has important significance.

[0004] However, the incidence of most single gene diseases and chromosomal diseases is low, and positive samples are difficult to obtain. Among the three common aneuploidy abnormalities, 21-trisomy syndrome (Down syndrome), 18-trisomy syndrome (Edward syndrome), and 13-trisomy syndrome (Patau syndrome) are the three most common autosomal aneuploidy diseases, with incidences of 1 / (600-800), 1 / (3500-8000), and 1 / (7000-20000) in newborns, respectively. The plasma of pregnant women with positive samples is limited, and only 5-10 ng of DNA can be extracted per ml of pregnant women's plasma, which meets the demand of one experimental detection.

[0005] In the free DNA in the plasma of pregnant women, the fragment length of fetal free DNA (main peak 148 bp) is significantly lower than that of maternal free DNA (main peak 168 bp). Therefore, a series of algorithms have been developed to predict fetal concentration or improve the accuracy of fetal chromosomal abnormality detection by taking advantage of the length difference between fetal and maternal free DNA. CN 110373458 A (A kit and analysis system for thalassemia detection) found that by screening short fragment DNA with a length of ≤158 bp to enrich fetal free DNA, combined with a multi-dimensional Bayesian algorithm, fetal severe thalassemia non-invasive prenatal screening was achieved. In addition, NIPT-plus requires detection of common microdeletion / microduplication syndromes as low as 3 Mb, which poses a great challenge to the sensitivity and specificity of the algorithm. CN119007804A (An analysis method and system for non-invasive prenatal screening) uses computer methods to screen short fragment free DNA to enrich fetal DNA, and develops a corresponding model to compare the fetal concentration and copy number values before and after enrichment to improve detection accuracy. The existing method for preparing simulated plasma standard of pregnant women is generally to fragment the genomic DNA from the mother and the fetus using ultrasonic or enzymatic methods (CN109112209B), and then mix or mix with plasma. Although the simulated plasma of pregnant women prepared by this method can simulate clinical samples, it cannot be applied when fetal concentration enrichment is required, and there is also a huge difference in fragment distribution from naturally occurring pregnant plasma. Therefore, it is necessary to study a method for preparing simulated plasma of pregnant women that can enrich fetal concentration, simulate the main peak and length difference of fetal and maternal free DNA, and be used for downstream wet experiments or computer methods to enrich fetal free DNA concentration.

[0006] [1] Liao C, Yin AH, Peng CF, et al. Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing. Proc Natl Acad Sci U S A. 2014; 111(20): 7415-7420. doi: 10.1073 / pnas.1321997111.

[0007] [2] Yin AH, Peng CF, Zhao X, et al. Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA. Proc Natl Acad Sci U S A. 2015; 112(47): 14670-14675. doi: 10.1073 / pnas.1518151112. SUMMARY

[0008] The first aspect of the present application aims to provide a preparation method of a simulated maternal plasma DNA standard product for enriching fetal DNA concentration.

[0009] The second aspect of the present application aims to provide a simulated maternal plasma DNA standard product.

[0010] The third aspect of the present application aims to provide the use of the preparation method of the first aspect of the present application and the simulated maternal plasma DNA standard product of the second aspect of the present application.

[0011] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows:

[0012] The first aspect of the present application provides a preparation method of a simulated maternal plasma DNA standard product for enriching fetal DNA concentration, comprising the following steps:

[0013] Preparation of simulated fetal DNA 1: Take the offspring genomic DNA 1 and perform fragmentation treatment using fragmentation condition 1 to obtain fragmented DNA 1, and purify the fragmented DNA 1 using fragment purification condition 1 to obtain;

[0014] Preparation of simulated maternal DNA 2: Take the maternal genomic DNA 2 and perform fragmentation treatment using fragmentation condition 2 to obtain fragmented DNA 2, and purify the fragmented DNA 2 using fragment purification condition 2 to obtain;

[0015] Mixing the simulated fetal DNA 1 and the simulated maternal DNA 2 to obtain the simulated pregnant woman plasma DNA standard.

[0016] In some embodiments of the present application, the fragmentation method is enzyme cleavage.

[0017] In some embodiments of the present application, the fragmentation condition 1 is a condition for breaking DNA to a main peak of 145-180 bp.

[0018] In some embodiments of the present application, the fragmentation condition 2 is a condition for breaking DNA to a main peak of 180-210 bp.

[0019] In some embodiments of the present application, the enzyme cleavage condition of the fragmentation condition 1 is 4°C for 1 min, 32°C for 32 min, and 65°C for 10 min.

[0020] In some embodiments of the present application, the enzyme cleavage condition of the fragmentation condition 2 is 4°C for 1 min, 32°C for 24 min, and 65°C for 10 min.

[0021] In some embodiments of the present application, the fragment purification condition 1 comprises the following steps:

[0022] 1) The fragmented DNA 1 is mixed with the magnetic bead suspension for the first time, and the final concentration of polyethylene glycol in the mixed solution of the magnetic bead suspension and the fragmented DNA 1 solution is 8% to 9.5%; preferably, 9%.

[0023] 2) Magnetic bead separation, the supernatant is mixed with the new magnetic bead suspension for the second time, and the final concentration of polyethylene glycol in the mixed solution of the magnetic bead suspension and the supernatant is 12% to 15%; preferably, 13.5%;

[0024] 3) Magnetic bead separation, washing and eluting the magnetic beads to obtain the simulated fetal DNA 1.

[0025] In some embodiments of the present application, the fragment purification condition 2 comprises the following steps:

[0026] 1) The fragmented DNA 2 is mixed with the magnetic bead suspension for the first time, and the final concentration of polyethylene glycol in the mixed solution of the magnetic bead suspension and the fragmented DNA 2 solution is 7%-9%; preferably, 8%.

[0027] 2) Magnetic bead separation, the supernatant is mixed with the new magnetic bead suspension for the second time, and the final concentration of polyethylene glycol in the mixed solution of the magnetic bead suspension and the supernatant is 10% to 12%; preferably, 11%.

[0028] 3) Magnetic bead separation, washing and eluting the magnetic beads to obtain the simulated fetal DNA 2.

[0029] In some embodiments of the present application, the main peak of the fragmented DNA 1 is 145-180 bp; preferably, 145-155 bp.

[0030] In some embodiments of the present application, the main peak of the fragmented DNA 2 is 180-210 bp; preferably, 180-200 bp.

[0031] In some embodiments of the present application, the simulated fetal DNA and the simulated maternal DNA can be mixed according to the predetermined fetal concentration. For example, if a simulated pregnant woman plasma standard with a fetal concentration of 10% is desired, the simulated fetal DNA and the simulated maternal DNA can be mixed in a mass ratio of 1:9 to obtain a simulated pregnant woman plasma standard with an expected fetal concentration of 10%.

[0032] In one embodiment, genomic DNA 1 and genomic DNA 2 are respectively used to obtain simulated fetal DNA and simulated maternal DNA by the same fragmentation and screening method, mixed into a pregnant woman plasma standard with an expected fetal concentration according to the mass ratio, and then the pregnant woman plasma standard is subjected to library construction, hybrid capture and high-throughput sequencing, and the sequencing results are analyzed by bioinformatics. Based on the SNP site, fetal concentration analysis is performed, and based on the reads length, fetal concentration enrichment is performed. The results show that the calculated fetal concentration is basically close to the expected fetal concentration, but the fetal concentration is not improved when the fetal concentration is enriched.

[0033] In one embodiment, genomic DNA 1 and genomic DNA 2 are respectively fragmented by different fragmentation conditions, and then purified by the same fragmentation and screening method to obtain simulated fetal DNA and simulated maternal DNA, mixed into a pregnant woman plasma standard with an expected fetal concentration according to the mass ratio, and then the pregnant woman plasma standard is subjected to library construction, hybrid capture and high-throughput sequencing, and the sequencing results are analyzed by bioinformatics. Based on the SNP site, fetal concentration analysis is performed, and based on the reads length, fetal concentration enrichment is performed. The results show that the calculated fetal concentration is basically close to the expected fetal concentration, but the fetal concentration is not improved when the fetal concentration is enriched, and the PPV remains unchanged or decreases, and the sensitivity decreases.

[0034] In an embodiment, genomic DNA 1 and genomic DNA 2 are fragmented respectively using different fragmentation conditions, and then purified using different size selection conditions, to obtain simulated fetal DNA and simulated maternal DNA respectively, which are mixed into a pregnant woman plasma standard with an expected fetal concentration, and then the pregnant woman plasma standard is subjected to library construction, hybrid capture and high-throughput sequencing, and the sequencing results are analyzed by bioinformatics. Based on the SNP site, the fetal concentration is analyzed, and based on the reads length, the fetal concentration is enriched. The results show that the calculated fetal concentration is basically close to the expected fetal concentration, and when the fetal concentration is enriched, the fetal concentration is increased by about 2 times, and the PPV is increased by at least 50% under the premise that the sensitivity is basically unchanged.

[0035] The beneficial effects of the present application are:

[0036] The present application provides a preparation method of a pregnant woman plasma free DNA simulation sample. The method combines specific DNA fragmentation processing technology to prepare a simulation sample with high fidelity and stability, which can accurately simulate the distribution ratio of fetal free DNA and pregnant woman free DNA in real pregnant woman plasma, for quality control and evaluation of non-invasive prenatal diagnosis technology. The method can accurately prepare pregnant woman simulation plasma DNA with corresponding fetal concentration according to the needs, and the prepared pregnant woman simulation plasma can meet the needs of fetal concentration enrichment test applications, and at the same time, the cfDNA fragment distribution in the pregnant woman plasma is reduced to the maximum extent. BRIEF DESCRIPTION OF DRAWINGS

[0037] The present application will be further described below in combination with the drawings and examples, in which:

[0038] Figure 1 The technical roadmap of the present application.

[0039] Figure 2 The genomic DNA 1 simulation fetal plasma free DNA fragment distribution graph under the same fragmentation and size selection conditions.

[0040] Figure 3 The genomic DNA 2 simulation maternal plasma free DNA size distribution graph under the same fragmentation and size selection conditions.

[0041] Figure 4 The genomic DNA 1 simulation fetal plasma free DNA fragment distribution graph under the same fragmentation condition and different size selection conditions.

[0042] Figure 5 The genomic DNA 2 simulation maternal plasma free DNA size distribution graph under the same fragmentation condition and different size selection conditions.

[0043] Figure 6Figure 1 shows the genomic DNA 1 simulated fetal plasma free DNA fragment distribution map under different fragmentation conditions and different size selection conditions.

[0044] Figure 7 Figure 2 shows the genomic DNA 2 simulated maternal plasma free DNA size selection distribution map under different fragmentation conditions and different size selection conditions. DETAILED DESCRIPTION

[0045] The concept and technical effects of the present application will be described below in conjunction with the embodiments so as to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0046] Example 1: Effect of the maternal simulated plasma standard obtained under the same fragmentation and size selection conditions

[0047] 1. Sample selection: The sample contains two groups of genomic DNA 1 and genomic DNA 2, wherein the genomic DNA 1 and the genomic DNA 2 are parent-child paired DNA. The specific information of the two groups of genomic DNA used in this embodiment is shown in Table 1.

[0048] Table 1

[0049]

[0050] 2. DNA fragmentation: 150 ng of genomic DNA 1 and 400 ng of genomic DNA 2 were taken respectively and fragmented by DNA Shearing Enzyme Mix (purchased from Kaygen, item number CM0162) according to the recommended system of the kit. The fragmentation conditions were 4°C for 1 min, 32°C for 30 min and 65°C for 10 min.

[0051] 3. Size selection purification of fragmented DNA (the size selection purification coefficient used for genomic DNA 1 and genomic DNA 2 in this embodiment is consistent): The fragmented genomic DNA 1 and the fragmented genomic DNA 2 were subjected to size selection purification by SPRI magnetic beads (purchased from Bechman-Coulter), and the steps were as follows:

[0052] (1) The fragmented genomic DNA was added to the SPRI magnetic bead suspension, and after mixing uniformly, it was incubated for 5 min. The volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA was 0.8:1, wherein the final concentration of polyethylene glycol was 8%;

[0053] (2) After placing on the magnetic stand for 3 min, the supernatant was transferred to a centrifuge tube containing a new SPRI magnetic bead suspension, mixed evenly, and incubated for 5 min, the volume ratio of the SPRI magnetic bead suspension to the fragmented genomic DNA solution was 1.2:1, and the final concentration of polyethylene glycol was 12%;

[0054] (3) After placing on the magnetic stand for 3 min, the supernatant was discarded, the magnetic beads were washed twice with 80% ethanol, and after the ethanol evaporated, the magnetic beads were eluted with TE buffer, i.e. simulated maternal DNA and simulated fetal DNA were obtained.

[0055] 4. DNA quality control: The Qubit fluorometer was used to quantitate the simulated maternal free DNA and simulated fetal free DNA, and the Agilent 2100 bioanalyzer was used to detect the size of the simulated maternal free DNA and simulated fetal free DNA.

[0056] The specific fragment recovery quantitative results are shown in Table 2. The 2100 analysis results are shown in Tables 3 and 4, respectively. Figure 2 、 3 As can be seen from the 2100 graph, the DNA main peaks of Genomic 1 and Genomic 2 after fragment recovery differ by about 20 bp, but there is no significant difference in the distribution width.

[0057] Table 2

[0058] Sample No. Recovery concentration (ng / μL) Reconstitution volume (μL) Recovery rate (%) E5100015 2.9 25 48.3％ E9100028Y 14.3 25 89.4％ E5100082 1.94 25 32.3％ E9100220Y 11.1 25 69.4％

[0059] 5. Sample mixing: Prepare simulated samples with expected fetal concentration of 5%, 10% and 20% by mass ratio.

[0060] According to the mass ratio of fetal simulated cfDNA: maternal simulated cfDNA = 5:95, the progeny fragmented DNA and parent fragmented DNA were mixed to obtain a simulated sample with a fetal concentration of 5%; according to the mass ratio of fetal simulated cfDNA: maternal simulated cfDNA = 10:90, the progeny fragmented DNA and parent fragmented DNA were mixed to obtain a simulated sample with a fetal concentration of 10%; according to the mass ratio of fetal simulated cfDNA: maternal simulated cfDNA = 20:80, the progeny fragmented DNA and parent fragmented DNA were mixed to obtain a simulated sample with a fetal concentration of 20%. After preparation, 12 μL TE buffer was added for re-quantification. The preparation and sampling process is shown in Table 3.

[0061] Table 3

[0062]

[0063] 6. Sequencing library construction: The expected 5%, 10%, 20% of the fetal simulation samples obtained in step 5 were re-quantified using a Qubit fluorometer, and then 10 ng of mixed DNA was taken for library construction and hybrid capture using a non-invasive dominant single disease library kit according to the instructions.

[0064] 7. Sequencing: The constructed library was sequenced using Novaseq6000, and the sequencing results were analyzed using bioinformatics.

[0065] 8. Result analysis: The fetal concentration was calculated based on the SNP site, and the reads with a fragment length of ≤158 bp were further screened for fetal concentration enrichment. The results are shown in Table 4.

[0066] The fetal concentration calculated based on the SNP site is basically close to the expected fetal concentration, but the fetal concentration is not improved based on the reads length for fetal concentration enrichment, i.e., the fetal concentration enrichment effect is not achieved, indicating that the simulation plasma obtained by this method cannot meet the quality control requirements of fetal concentration detection applications that require fetal concentration enrichment. The detailed results are shown in Table 4.

[0067] Table 4

[0068]

[0069] Example 2: Effect of simulation plasma standard obtained under different fragmentation conditions and the same screening conditions

[0070] 1. Sample selection: The samples contain two groups of genomic DNA1 and genomic DNA2, wherein the genomic DNA1 and genomic DNA2 are parent-child paired DNA. The specific information of the two groups of genomic DNA in this embodiment is shown in Table 5.

[0071] Table 5

[0072]

[0073] 2. DNA fragmentation: 100 ng of genomic DNA1 and 400 ng of genomic DNA2 were taken and fragmented using DNA Shearing Enzyme Mix according to the recommended system of the kit (purchased from Kaygen, item number CM0162), wherein the fragmentation conditions for genomic DNA1 were 4°C for 1 min, 32°C for 32 min, and 65°C for 10 min, and the fragmentation conditions for genomic DNA2 were 4°C for 1 min, 32°C for 24 min, and 65°C for 10 min.

[0074] 3. Size selection of fragmented DNA (the same size selection coefficient was used for genomic DNA 1 and genomic DNA 2 in this example): The fragmented genomic DNA 1 and genomic DNA 2 were subjected to size selection using SPRI magnetic beads (purchased from Bechman-Coulter) according to the following steps:

[0075] (1) The fragmented genomic DNA was added to the SPRI magnetic bead suspension, mixed well and incubated for 5 min, the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA was 0.8:1, and the final concentration of polyethylene glycol was 8%;

[0076] (2) After placing on the magnetic stand for 3 min, the supernatant was transferred to a centrifuge tube containing a new SPRI magnetic bead suspension, mixed well and incubated for 5 min, the volume ratio of the SPRI magnetic bead suspension to the fragmented genomic DNA solution was 1.2:1, and the final concentration of polyethylene glycol was 12%;

[0077] (3) After placing on the magnetic stand for 3 min, the supernatant was discarded, the magnetic beads were washed twice with 80% ethanol, and then eluted with TE buffer after the ethanol evaporated, thus obtaining the simulated maternal DNA and the simulated fetal DNA.

[0078] 4. DNA quality control: The simulated maternal free DNA and the simulated fetal free DNA were quantified using a Qubit fluorometer, and the size of the simulated maternal free DNA and the simulated fetal free DNA was detected using an Agilent 2100 bioanalyzer. The specific fragment recovery and quantification results are shown in Table 6. The 2100 analysis results are shown in Table 6 and Table 7, respectively. As can be seen from the 2100 graph, the DNA main peaks of genomic 1 and genomic 2 after size selection and recovery differ by about 20 bp, but there is no obvious difference in the distribution width. Figure 4 、 5

[0079] Table 6

[0080] Sample No. Recovery concentration (ng / μL) Reconstitution volume (μL) Recovery rate (%) E6100224 0.546 100 54.6％ E6100223 1.85 100 46.3％ E6100084 0.608 100 60.8％ E9100602 1.81 100 45.3％

[0081] 5. Sample mixing: The simulated samples with expected fetal concentration of 1%, 3%, 5%, and 10% were prepared according to the mass ratio. The preparation process is shown in Table 7.

[0082] Table 7

[0083]

[0084] 6. Sequencing library construction: The simulated samples with expected fetal concentration of 1%, 3%, 5%, and 10% obtained in step 5 were re-quantified using a Qubit fluorometer, and then 10 ng of the mixed DNA was taken for library construction and hybridization capture. ​

[0085] 7. Sequencing: The constructed library was sequenced by Novaseq6000, and the sequencing results were analyzed by bioinformatics.

[0086] 8. Result analysis: The fetal concentration was calculated based on SNP sites, and the reads with fragment length ≤158bp were further screened for fetal concentration enrichment. The results showed that the fetal concentration calculated based on SNP sites was basically close to the expected fetal concentration, but the fetal concentration did not increase based on reads length for fetal concentration enrichment, i.e., the fetal concentration enrichment effect was not achieved, indicating that the simulated plasma obtained by this method cannot meet the quality control requirements of fetal concentration enrichment detection application. The detailed results are shown in Table 8.

[0087] Table 8

[0088]

[0089] Since a single sample of family mother-child pairs was used to prepare the pregnant woman's plasma standard, according to the principle of predicting fetal free DNA concentration based on SNP, the mutant sites with genotype AA (wild-type homozygous) in the mother and genotype AB (heterozygous) in the offspring were selected as the gold standard set for analyzing the detection performance of the prepared pregnant woman's plasma standard before and after fetal concentration enrichment for low-frequency sites.

[0090] Table 9 is the detection performance results of the pregnant woman's plasma standard before and after short fragment enrichment. According to the process of calculating fetal concentration based on SNP, the mean and standard deviation of the mutation frequency of low-frequency mutant sites from the offspring in the pregnant woman's plasma standard can be obtained, and the frequency range for collecting low-frequency sites is further confirmed to be greater than the mean frequency minus 3 times the standard deviation. The results show that the PPV decreases or has no obvious change after short fragment enrichment, and the sensitivity decreases.

[0091] Table 9

[0092]

[0093] Example 3: Effect of pregnant woman's simulated plasma standard obtained under different fragmentation conditions and different fragment screening conditions

[0094] 1. Sample selection: Four groups of mother-child paired genomic DNA purchased from Corriell Institute were selected, in which the offspring were carriers containing different genetic mutations, and the mothers were wild types of the corresponding genetic mutations. The specific information is shown in Table 10.

[0095] Table 10

[0096] Sample No. Genotype Type NA17413 COL1A2 positive DNA Offspring DNA (Genomic DNA 1) NA17415 COL1A2 paired negative DNA NA17415 paired mother DNA (Genomic DNA 2) NA11301 MECP2 positive DNA Offspring DNA (Genomic DNA 1) NA11302 MECP2 paired negative DNA NA11301 paired mother DNA (Genomic DNA 2) NA19977 NSD1 positive DNA Offspring DNA (Genomic DNA 1) NA19975 NSD1 paired negative DNA NA19977 paired mother DNA (Genomic DNA 2) NA19978 NSD1 positive DNA Offspring DNA (Genomic DNA 1) NA19976 NSD1 paired negative DNA NA19976 paired mother DNA (Genomic DNA 2)

[0097] 2. DNA fragmentation: 100 ng of genomic DNA 1 and 400 ng of genomic DNA 2 were fragmented with DNA Shearing Enzyme Mix (purchased from KJ Biotech, Cat# CM0162) according to the recommended protocol, wherein the fragmentation condition for genomic DNA 1 was 4°C for 1 min, 32°C for 32 min, 65°C for 10 min, and the fragmentation condition for genomic DNA 2 was 4°C for 1 min, 32°C for 24 min, 65°C for 10 min.

[0098] 3. Size selection and purification of the fragmented DNA (genomic DNA 1 and genomic DNA 2 were purified with different size selection and purification coefficients in this example):

[0099] Size selection and purification of the fragmented genomic DNA 1, the steps were as follows:

[0100] (1) The fragmented genomic DNA 1 was added to the SPRI magnetic bead suspension, mixed uniformly, and then incubated for 5 min, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA was 1:1, and the final concentration of polyethylene glycol was 9%;

[0101] (2) After placing on the magnetic stand for 3 min, the supernatant was transferred to a centrifuge tube containing new SPRI magnetic bead suspension, mixed uniformly, and then incubated for 5 min, wherein the volume ratio of the SPRI magnetic bead suspension to the solution of the fragmented genomic DNA was 2:1, and the final concentration of polyethylene glycol was 13.5%; (3) After placing on the magnetic stand for 3 min, the supernatant was aspirated, the magnetic beads were washed with 80% ethanol for two times, and then the magnetic beads were eluted with TE buffer after the ethanol was evaporated, thereby obtaining the simulated fetal DNA.

[0102] Size selection and purification of the fragmented genomic DNA 2, the steps were as follows:

[0103] (1) The fragmented genomic DNA 2 was added to the SPRI magnetic bead suspension, mixed uniformly, and then incubated for 5 min, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA was 0.8:1, and the final concentration of polyethylene glycol was 8%;

[0104] (2) After placing on the magnetic stand for 3 min, the supernatant was transferred to a centrifuge tube containing new SPRI magnetic bead suspension, mixed uniformly, and then incubated for 5 min, wherein the volume ratio of the SPRI magnetic bead suspension to the solution of the fragmented genomic DNA was 0.8:1, and the final concentration of polyethylene glycol was 11%;

[0105] (3) After placing on the magnetic stand for 3 min, the supernatant was aspirated, the magnetic beads were washed with 80% ethanol for two times, and then the magnetic beads were eluted with TE buffer after the ethanol was evaporated, thereby obtaining the simulated maternal DNA.

[0106] 4. DNA quality control: Qubit fluorometer was used to quantify the simulated maternal free DNA and simulated fetal free DNA, and Agilent 2100 bioanalyzer was used to detect the size of the simulated maternal free DNA and simulated fetal free DNA. The specific fragment recovery quantitative results are shown in Table 11. The 2100 analysis results are shown in Table 11 and Table 12, respectively. As can be seen from the 2100 diagram, the DNA main peaks of Genomic 1 and Genomic 2 after fragmenting and screening recovery are about 50 bp apart. Figure 6 , 7

[0107] Table 11

[0108] Sample No. Concentration (ng / μL) Reconstitution volume (μL) NA17413 0.29 150 NA17415 1.76 100 NA11301 0.268 150 NA11302 1.79 100 NA19977 0.265 150 NA19975 2.02 100 NA19978 0.232 150 NA19976 1.67 100

[0109] 5. Sample mixing: Prepare simulated samples with expected fetal concentrations of 1%, 3%, 5%, 10%, and 15% by mass ratio. The preparation sampling process is shown in Table 12.

[0110] Table 12

[0111]

[0112]

[0113] 6. Sample fetal concentration detection: The simulated samples with expected fetal concentrations of 1%, 3%, 5%, 10%, and 15% obtained in step 5 were re-quantified using Qubit fluorometer, then 10 ng was taken for library construction using the non-invasive dominant single disease library kit to obtain sequencing library, then high-depth sequencing was performed using Novseq6000, and bioinformatics analysis was performed on the sequencing results.

[0114] The results are shown in Table 13. The fetal concentration calculated based on SNP sites is basically close to the expected fetal concentration. When the fetal concentration is enriched based on reads length, the fetal concentration can be increased by 2 times, indicating that the simulated plasma obtained by this method can meet the quality control requirements of fetal concentration detection applications that require fetal concentration enrichment.

[0115] Table 13

[0116]

[0117]

[0118] Since a single sample of a family mother-child pair was used to prepare the pregnant woman's plasma standard, according to the principle of predicting fetal free DNA concentration based on SNP, a mutant site with genotype AA (wild-type homozygous) in the mother and genotype AB (heterozygous) in the offspring was selected as the gold standard set for analyzing the detection performance of the prepared pregnant woman's plasma standard for low-frequency sites before and after fetal concentration enrichment.​

[0119] Table 14 shows the performance of the detection of maternal plasma standards before and after short fragment enrichment. The mean and standard deviation of the mutation frequency of the low frequency mutation sites from the fetus in the maternal plasma standards can be obtained according to the process of calculating the fetal concentration of the SNP, and the frequency range for collecting low frequency sites is further confirmed to be greater than the mean frequency-3*standard deviation. The results show that after short fragment enrichment, the PPV is at least increased by 50% while the sensitivity is basically maintained.

[0120] Table 14

[0121]

[0122]

Claims

1. A method for preparing a simulated pregnant woman's plasma DNA standard for enriching fetal DNA concentration, comprising the following steps: Preparation of simulated fetal DNA1: Fragmentation of progeny genomic DNA1 was performed using fragmentation condition 1 to obtain fragmented DNA1. Fragmented DNA1 was then purified using sieve purification condition 1 to obtain the final product. Preparation of simulated maternal DNA2: Maternal genomic DNA2 was fragmented using fragmentation condition 2 to obtain fragmented DNA2. The fragmented DNA2 was then purified using sieve purification condition 2 to obtain the final product. By mixing simulated fetal DNA1 and simulated maternal DNA2, a simulated pregnant woman's plasma DNA standard was obtained. The fragmentation method is ultrasonic or enzymatic digestion. The fragmentation condition 1 breaks the DNA down to a peak of 145-180 bp. The fragmentation condition 2 breaks the DNA down to a peak of 180–210 bp. The purification conditions 1 for the sieve include the following steps: 1) The fragmented DNA1 was mixed and incubated with the magnetic bead suspension for the first time. The final concentration of polyethylene glycol in the mixture of the magnetic bead suspension and the fragmented DNA1 solution was 8.5% to 9.5%. 2) Magnetic bead separation: The supernatant is mixed and incubated with a new magnetic bead suspension for a second time. The final concentration of polyethylene glycol in the mixture of magnetic bead suspension and supernatant is 12% to 15%. 3) Separate magnetic beads, wash and elute the magnetic beads to obtain simulated fetal DNA1; The purification conditions 2 for the sieve filter include the following steps: 1) The fragmented DNA2 was mixed and incubated with the magnetic bead suspension for the first time. The final concentration of polyethylene glycol in the mixture of the magnetic bead suspension and the fragmented DNA2 solution was 7% to 9%. 2) Magnetic bead separation: The supernatant is mixed and incubated with a new magnetic bead suspension for a second time. The final concentration of polyethylene glycol in the mixture of magnetic bead suspension and supernatant is 10% to 12%. 3) Separate the magnetic beads, wash and elute the magnetic beads to obtain simulated fetal DNA2.

2. The method according to claim 1, characterized in that: The main peak of the fragmented DNA1 is 145–180 bp.

3. The method according to claim 1, characterized in that: The main peak of the fragmented DNA2 is 180–210 bp.

4. A standard for simulating pregnant women's plasma DNA, prepared by the method described in any one of claims 1 to 3.

5. The preparation method according to any one of claims 1 to 3, and the application of the simulated pregnant woman plasma DNA standard according to claim 4 in the enrichment of fetal concentration.

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