Preparation method of pregnant woman simulated plasma standard substance capable of enriching fetal concentration through fragment size

Abstract

The invention belongs to the technical field of noninvasive prenatal examination, and particularly relates to a preparation method of a pregnant woman simulated plasma standard substance capable of enriching fetal concentration through fragment size. The invention provides a preparation method of a pregnant woman plasma free DNA simulation sample, which is combined with a specific DNA fragmentation treatment technology to prepare the simulation sample which has high fidelity and stability and can accurately simulate the distribution proportion of fetal free DNA and pregnant woman free DNA in real pregnant woman plasma, and is used for quality control and evaluation of a noninvasive prenatal diagnosis technology. According to the method, the pregnant woman simulated plasma DNA with the corresponding fetal concentration can be accurately prepared according to requirements, the prepared pregnant woman simulated plasma can meet the application requirements of fetal concentration enrichment testing, and meanwhile the distribution condition of cfDNA fragments in the pregnant woman plasma is reduced to the maximum extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of non-invasive prenatal examination, and in particular relates to a method for preparing a standard product of simulated plasma of pregnant women capable of enriching fetal concentration by fragment size. Background Art

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

[0003] Fetal DNA concentration (FF) is one of the key factors for the success of NIPT testing, which directly affects the sensitivity and specificity of the test. Studies have shown that the accuracy of NIPT testing may be limited when the fetal DNA concentration is low (<4%) [1]. However, for non-invasive prenatal microdeletion and microduplication testing (NIPT-plus), its detection accuracy is positively correlated with the fetal DNA concentration. The increase in fetal DNA concentration significantly improves the detection ability to distinguish small genomic abnormalities [2]. At low fetal DNA concentration, the interference of background maternal DNA increases and the signal-to-noise ratio decreases, which may cause the signal of chromosome copy number abnormalities to be masked, resulting in false negative results. For the detection of microdeletion and microduplication syndrome, since the range of genomic abnormalities involved is smaller, the resolution ability of fetal DNA signals is required to be higher, so the concentration level has a more significant impact on the test results. For this reason, accurate quantification and evaluation of fetal DNA concentration is particularly important in non-invasive prenatal screening technology. Collecting or preparing maternal plasma free samples with different fetal DNA concentrations can be used to evaluate the accuracy of fetal concentration quantification and fetal chromosome or single gene disease abnormality detection, which is of great significance.

[0004] However, most monogenic and chromosomal diseases have a low incidence rate in the population, and positive samples are difficult to obtain. Among the three common aneuploidy abnormalities, trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) are the three most common autosomal aneuploidy diseases, with incidence rates of 1 / (600-800), 1 / (3500-8000) and 1 / (7000-20000) in newborns, respectively. The amount of maternal plasma for positive samples is limited, and on average only 5-10 ng of DNA can be extracted from each ml of maternal plasma to complete one experimental test requirement.

[0005] In the plasma free DNA of pregnant women, both the free DNA of the fetus and the free DNA of the mother are included. The fragment length of the fetal free DNA (main peak 148bp) is significantly lower than the fragment of the maternal free DNA (main peak 168bp). Therefore, a series of algorithms have been developed to predict the fetal concentration or improve the accuracy of fetal chromosome abnormality detection using the difference in the length of the fetal and maternal free DNA. CN 110373458 A (a kit and analysis system for thalassemia detection) found that the fetal free DNA was enriched by screening short fragments of DNA ≤158bp in length, and the non-invasive prenatal screening of fetal severe thalassemia was achieved by combining a multidimensional Bayesian algorithm. In addition, NIPT-plus needs to detect common microdeletion / microduplication syndromes as low as about 3Mb, which brings great challenges to the algorithm detection sensitivity and specificity. CN119007804A (a non-invasive prenatal screening analysis method and system) uses a computer method to screen short fragments of free DNA to enrich fetal DNA, and develops a corresponding model to compare the fetal concentration and copy number before and after enrichment to improve the detection accuracy. The existing method for preparing a standard simulated plasma of pregnant women generally involves fragmenting the genomic DNA from the mother and the fetus by ultrasound or enzyme digestion (CN109112209B), and then mixing or mixing with plasma. Although the simulated plasma of pregnant women prepared by this method can simulate clinical samples, it cannot be used when the fetal concentration is required to be enriched, and the fragment distribution is also greatly different from that of naturally existing pregnant plasma. Therefore, it is necessary to study a method for preparing simulated plasma of pregnant women that can be enriched in fetal concentration, simulate the main peak and length difference of fetal and maternal free DNA, and can be used for downstream wet experiments or computer methods to enrich the concentration of fetal free DNA.

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

[0007] [2] Yin AH, Peng CF, Zhao Summary of the invention

[0008] The purpose of the first aspect of the present invention is to provide a method for preparing a simulated maternal plasma DNA standard for enriching fetal DNA concentration.

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

[0010] The purpose of the third aspect of the present invention is to provide the preparation method of the first aspect of the present invention and the application of the simulated pregnant woman plasma DNA standard product of the second aspect of the present invention.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

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

[0013] Preparation of simulated fetal DNA 1: taking offspring genomic DNA 1 and fragmenting it using fragmentation condition 1 to obtain fragmented DNA 1, and purifying the fragmented DNA 1 using sheet-screen purification condition 1 to obtain;

[0014] Simulate the preparation of maternal DNA 2: take maternal genomic DNA 2 and fragment it using fragmentation condition 2 to obtain fragmented DNA 2, and purify the fragmented DNA 2 using sheet sieve purification condition 2 to obtain;

[0015] The simulated fetal DNA 1 and the simulated maternal DNA 2 were mixed to obtain a simulated maternal plasma DNA standard.

[0016] In some embodiments of the present invention, the fragmentation method is enzymatic cleavage.

[0017] In some embodiments of the present invention, the fragmentation condition 1 is a condition in which the DNA is fragmented to a main peak of 145 to 180 bp.

[0018] In some embodiments of the present invention, the fragmentation condition 2 is a condition in which the DNA is fragmented to a main peak of 180 to 210 bp.

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

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

[0021] In some embodiments of the present invention, the sheet-screen purification condition 1 comprises the following steps:

[0022] 1) The fragmented DNA 1 is mixed and incubated 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, mixing and incubating the supernatant with a new magnetic bead suspension for a second time, wherein the final concentration of polyethylene glycol in the mixture 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 simulated fetal DNA 1.

[0025] In some embodiments of the present invention, the sheet-screen purification condition 2 comprises the following steps:

[0026] 1) The fragmented DNA2 is mixed and incubated 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 DNA2 solution is 7%-9%; preferably, 8%.

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

[0028] 3) Magnetic bead separation, washing and elution of the magnetic beads to obtain simulated fetal DNA2.

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

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

[0031] In some embodiments of the present invention, the simulated fetal DNA and the simulated maternal DNA can be mixed at a mass ratio according to a predetermined fetal concentration. For example, if one wants to obtain a simulated maternal plasma standard product with a fetal concentration of 10%, the simulated fetal DNA and the simulated maternal DNA are mixed at a mass ratio of 1:9 to obtain a simulated maternal plasma standard product with an expected concentration of 10%.

[0032] In one embodiment, genomic DNA 1 and genomic DNA 2 are respectively fragmented and screened by the same method to obtain simulated fetal DNA and simulated maternal DNA, which are mixed into a maternal plasma standard with an expected fetal concentration according to a mass ratio, and then the maternal plasma standard is subjected to library construction, hybridization capture and high-throughput sequencing, and the sequencing results are analyzed by bioinformatics. Fetal concentration analysis is performed based on SNP sites, and fetal concentration enrichment is performed based on reads length. The results show that the calculated fetal concentration is basically close to the expected fetal concentration, but the fetal concentration does not increase when the fetal concentration is enriched.

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

[0034] In one embodiment, genomic DNA 1 and genomic DNA 2 are fragmented using different fragmentation conditions, and then purified using different sieving conditions to obtain simulated fetal DNA and simulated maternal DNA, respectively, which are mixed into a maternal plasma standard with an expected fetal concentration according to a mass ratio, and then the maternal plasma standard is subjected to library construction, hybrid capture and high-throughput sequencing, and the sequencing results are analyzed by bioinformatics. Fetal concentration analysis is performed based on SNP sites, and fetal concentration enrichment is performed based on reads length. The results show that the calculated fetal concentration is basically close to the expected fetal concentration. When fetal concentration enrichment is performed, the fetal concentration is increased by about 2 times, and the PPV is increased by at least 50% under the premise that the sensitivity remains basically unchanged.

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

[0036] The present invention provides a method for preparing a simulated sample of free DNA in maternal plasma. The method combines a specific DNA fragmentation processing technology to prepare a simulated sample with high fidelity and stability and can accurately simulate the distribution ratio of fetal free DNA and maternal free DNA in real maternal plasma, which is used for quality control and evaluation of non-invasive prenatal diagnosis technology. The method can accurately prepare simulated plasma DNA of maternal plasma with corresponding fetal concentration according to demand, and the prepared simulated plasma of maternal plasma can meet the needs of applications that require fetal concentration enrichment testing, while restoring the distribution of cfDNA fragments in maternal plasma to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 It is the technical roadmap of the present invention.

[0039] Figure 2 This is the distribution diagram of genomic DNA1 simulating fetal plasma free DNA fragments under the same fragment screening conditions.

[0040] Figure 3 This is the fragmentation distribution diagram of genomic DNA2 simulating maternal plasma free DNA under the same fragmentation conditions.

[0041] Figure 4 The fragment distribution diagram of genomic DNA1 simulating fetal plasma free DNA under the same shearing conditions and different screening conditions.

[0042] Figure 5 This is the fragmentation distribution diagram of genomic DNA2 simulating maternal plasma free DNA under different fragmentation conditions for the same fragmentation conditions.

[0043] Figure 6The fragment distribution diagrams of genomic DNA1 simulating fetal plasma free DNA under different shearing conditions and different screening conditions.

[0044] Figure 7 The sieving distribution diagram of genomic DNA2 simulating maternal plasma free DNA under different shearing conditions and different sieving conditions. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0046] Example 1 Effect of the standard sample of simulated plasma of pregnant women obtained under the same screening conditions

[0047] 1. Sample selection: The sample includes two sets of genomic DNA 1 and genomic DNA 2, wherein genomic DNA 1 and genomic DNA 2 are mother-child paired DNA. The specific information of the two sets of genomes 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 respectively sheared with DNA Shearing Enzyme Mix according to the recommended system of the kit (purchased from Qiagen, catalog number CM0162). The shearing conditions were 4°C for 1 min, 32°C for 30 min, and 65°C for 10 min.

[0051] 3. Purification of fragmented DNA by sheet sieve (the sheet sieve purification coefficients used for genomic DNA 1 and genomic DNA 2 in this example are the same): Fragmented genomic DNA 1 and genomic DNA 2 are purified by sheet sieve using SPRI magnetic beads (purchased from Bechman-Coulter) respectively, and the steps are as follows:

[0052] (1) adding the fragmented genomic DNA to the SPRI magnetic bead suspension, mixing evenly and incubating for 5 minutes, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA is 0.8:1, wherein the final concentration of polyethylene glycol is 8%;

[0053] (2) After placing on the magnetic stand for 3 minutes, the supernatant was transferred to a centrifuge tube containing a new SPRI magnetic bead suspension, mixed evenly and incubated for 5 minutes. 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 minutes, the supernatant was discarded, and the magnetic beads were washed twice with 80% ethanol. After the ethanol evaporated, the magnetic beads were eluted with TE buffer to obtain simulated maternal DNA and simulated fetal DNA, respectively.

[0055] 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 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 Figure 2 , 3 As shown in Figure 2100, the main peaks of DNA in genome 1 and genome 2 recovered by fragmentation screening 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) Redissolution 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 5%, 10% and 20% of the expected fetus respectively according to the mass ratio.

[0060] The offspring fragmented DNA was mixed with the parental fragmented DNA at a mass ratio of fetal simulated cfDNA: mother simulated cfDNA = 5:95 to obtain a simulated sample with a fetal concentration of 5%; the offspring fragmented DNA was mixed with the parental fragmented DNA at a mass ratio of fetal simulated cfDNA: mother simulated cfDNA = 10:90 to obtain a simulated sample with a fetal concentration of 10%; the offspring fragmented DNA was mixed with the parental fragmented DNA at a mass ratio of fetal simulated cfDNA: mother simulated cfDNA = 20:80 to obtain a simulated sample with a fetal concentration of 20%. After the preparation was completed, 12 μL TE buffer was added and re-quantified. The preparation and sampling process is shown in Table 3 below.

[0061] Table 3

[0062]

[0063] 6. Sequencing library construction: The expected fetal 5%, 10%, and 20% simulated samples 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 using the non-invasive dominant single disease library construction kit according to the instructions.

[0064] 7. Sequencing: The constructed library was sequenced using Novasek6000, and the sequencing results were analyzed using bioinformatics.

[0065] 8. Result analysis: The fetal concentration of the library was calculated based on the SNP site, and reads with a fragment length of ≤158bp 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 did not increase when the fetal concentration was enriched based on the reads length, that is, the effect of enriching the fetal concentration was not achieved, indicating that the simulated plasma obtained by this method cannot meet the quality control requirements of the application that requires enrichment of the fetal concentration. The detailed results are shown in Table 4.

[0067] Table 4

[0068]

[0069] Example 2 Effects of pregnant women simulated plasma standards obtained under different fragmentation conditions and the same screening conditions

[0070] 1. Sample selection: The sample includes two sets of genomic DNA 1 and genomic DNA 2, wherein genomic DNA 1 and genomic DNA 2 are mother-child paired DNA. The specific information of the two sets of genomes 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 respectively sheared with DNA Shearing Enzyme Mix according to the system recommended by the kit (purchased from Qiagen, cat. no. CM0162), wherein the shearing conditions for genomic DNA1 were 4°C for 1 min, 32°C for 32 min, and 65°C for 10 min, and the shearing conditions for genomic DNA2 were 4°C for 1 min, 32°C for 24 min, and 65°C for 10 min.

[0074] 3. Purification of fragmented DNA by sheet sieve (the sheet sieve purification coefficients used for genomic DNA 1 and genomic DNA 2 in this example are the same): Fragmented genomic DNA 1 and genomic DNA 2 are purified by sheet sieve using SPRI magnetic beads (purchased from Bechman-Coulter) respectively, and the steps are as follows:

[0075] (1) adding the fragmented genomic DNA to the SPRI magnetic bead suspension, mixing evenly and incubating for 5 minutes, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA is 0.8:1, wherein the final concentration of polyethylene glycol is 8%;

[0076] (2) After placing on the magnetic stand for 3 minutes, the supernatant was transferred to a centrifuge tube containing a new SPRI magnetic bead suspension, mixed evenly and incubated for 5 minutes. 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 minutes, the supernatant was discarded, and the magnetic beads were washed twice with 80% ethanol. After the ethanol evaporated, the magnetic beads were eluted with TE buffer to obtain simulated maternal DNA and simulated fetal DNA, respectively.

[0078] 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 simulated maternal free DNA and simulated fetal free DNA. The specific fragment recovery quantitative results are shown in Table 6. The 2100 analysis results are shown in Table 6. Figure 4 , 5 As shown in Figure 2100, the main peaks of DNA in genome 1 and genome 2 recovered by fragmentation screening differ by about 20 bp, but there is no significant difference in the distribution width.

[0079] Table 6

[0080] Sample No. Recovery concentration (ng / μL) Redissolution 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: Prepare 1%, 3%, 5%, and 10% simulated samples of expected fetus according to the mass ratio. The sampling process is shown in Table 7.

[0082] Table 7

[0083]

[0084] 6. Sequencing library construction: The simulated samples with expected fetal concentrations 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 using Novasek6000, and the sequencing results were analyzed using bioinformatics.

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

[0087] Table 8

[0088]

[0089] Since a single sample of a family mother-child pair is used to prepare the maternal plasma standard, based on the principle of SNP predicting the fetal free DNA concentration, mutation sites with a genotype of AA (wild homozygous) in the mother and a genotype of AB (heterozygous) in the offspring can be screened as the gold standard set to analyze the detection performance of the prepared maternal plasma standard for low-frequency sites before and after fetal concentration enrichment.

[0090] Table 9 shows the test performance results of maternal plasma standards before and after short fragment enrichment. The process of calculating fetal concentration based on SNP can obtain the mutation frequency mean and standard deviation of low-frequency mutation sites from offspring in maternal plasma standards, and further confirm that the frequency range used to collect low-frequency sites is greater than the frequency mean -3*standard deviation. The results show that after short fragment enrichment, PPV decreases or has no significant change, and sensitivity decreases.

[0091] Table 9

[0092]

[0093] Example 3 Effects of pregnant women simulated plasma standards obtained under different fragmentation conditions and different slice screening conditions

[0094] 1. Sample selection: Four sets of mother-child paired genomic DNA purchased from the Corriell Institute were selected, where the offspring were carriers of different gene mutations and the mothers were wild-type with the corresponding gene mutations. Specific information is shown in Table 10.

[0095] Table 10

[0096] Sample No. genotype type NA17413 COL1A2 positive DNA Progeny DNA (genomic DNA1) NA17415 COL1A2 paired negative DNA NA17415 matched maternal DNA (genomic DNA2) NA11301 MECP2-positive DNA Progeny DNA (genomic DNA1) NA11302 MECP2 paired negative DNA NA11301 matched maternal DNA (genomic DNA2) NA19977 NSD1-positive DNA Progeny DNA (genomic DNA1) NA19975 NSD1 paired negative DNA NA19977 matched maternal DNA (genomic DNA2) NA19978 NSD1-positive DNA Progeny DNA (genomic DNA1) NA19976 NSD1 paired negative DNA NA19976 matched maternal DNA (genomic DNA2)

[0097] 2. DNA fragmentation: 100 ng of genomic DNA1 and 400 ng of genomic DNA2 were respectively sheared with DNA Shearing Enzyme Mix according to the system recommended by the kit (purchased from Qiagen, cat. no. CM0162), wherein the shearing conditions for genomic DNA1 were 4°C for 1 min, 32°C for 32 min, and 65°C for 10 min, and the shearing conditions for genomic DNA2 were 4°C for 1 min, 32°C for 24 min, and 65°C for 10 min.

[0098] 3. Purification of fragmented DNA by sieve-slice (in this example, genomic DNA 1 and genomic DNA 2 were purified using different sieve-slice purification coefficients):

[0099] Fragmented genomic DNA 1 piece screen purification, the steps are as follows:

[0100] (1) adding the fragmented genomic DNA 1 to the SPRI magnetic bead suspension, mixing evenly and incubating for 5 minutes, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA is 1:1, wherein the final concentration of polyethylene glycol is 9%;

[0101] (2) After placing on the magnetic stand for 3 minutes, the supernatant was transferred to a centrifuge tube containing a new SPRI magnetic bead suspension, mixed evenly and incubated for 5 minutes. The volume ratio of the SPRI magnetic bead suspension to the fragmented genomic DNA solution was 2:1, and the final concentration of polyethylene glycol was 13.5%; (3) After placing on the magnetic stand for 3 minutes, the supernatant was discarded, and the magnetic beads were washed twice with 80% ethanol. After the ethanol evaporated, the magnetic beads were eluted with TE buffer to obtain simulated fetal DNA.

[0102] Fragmented genomic DNA 2-piece screen purification, the steps are as follows:

[0103] (1) adding the fragmented genomic DNA 2 to the SPRI magnetic bead suspension, mixing evenly and incubating for 5 minutes, wherein the volume ratio of the SPRI magnetic bead suspension to the solution containing the fragmented genomic DNA is 0.8:1, wherein the final concentration of polyethylene glycol is 8%;

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

[0105] (3) After placing on the magnetic stand for 3 minutes, discard the supernatant, wash the magnetic beads twice with 80% ethanol, and after the ethanol evaporates, elute the magnetic beads with TE buffer to obtain 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 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. Figure 6 , 7 As shown in Figure 2100, it can be seen that the main DNA peaks of genome 1 and genome 2 recovered by fragmentation screening differ by about 50 bp.

[0107] Table 11

[0108] Sample No. Concentration (ng / μL) Redissolution 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% respectively according to the mass ratio. The 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 a Qubit fluorometer, and then 10 ng was taken to construct a sequencing library using a non-invasive dominant single disease library construction kit, and then high-depth sequencing was performed using Novseq6000, and the sequencing results were analyzed using bioinformatics.

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

[0115] Table 13

[0116]

[0117]

[0118] Since a single sample of a family mother-child pair is used to prepare the maternal plasma standard, based on the principle of SNP predicting the fetal free DNA concentration, mutation sites with a genotype of AA (wild homozygous) in the mother and a genotype of AB (heterozygous) in the offspring can be screened as the gold standard set to analyze the detection performance of the prepared maternal plasma standard for low-frequency sites before and after fetal concentration enrichment.

[0119] Table 14 shows the test performance results of maternal plasma standards before and after short fragment enrichment. The process of calculating fetal concentration based on SNP can obtain the mutation frequency mean and standard deviation of low-frequency mutation sites from offspring in maternal plasma standards, and further confirm that the frequency range used to collect low-frequency sites is greater than the frequency mean -3*standard deviation. The results show that after short fragment enrichment, PPV is increased by at least 50% while the sensitivity remains basically unchanged.

[0120] Table 14

[0121]

[0122]

Claims

1. A method for preparing a simulated maternal plasma DNA standard for enriching fetal DNA concentration, comprising the following steps: Preparation of simulated fetal DNA 1: taking offspring genomic DNA 1 and fragmenting it using fragmentation condition 1 to obtain fragmented DNA 1, and purifying the fragmented DNA 1 using sheet-screen purification condition 1 to obtain; Simulate the preparation of maternal DNA 2: take maternal genomic DNA 2 and fragment it using fragmentation condition 2 to obtain fragmented DNA 2, and purify the fragmented DNA 2 using sheet sieve purification condition 2 to obtain; The simulated fetal DNA 1 and the simulated maternal DNA 2 were mixed to obtain a simulated maternal plasma DNA standard.

2. The preparation method according to claim 1, characterized in that: The fragmentation method is ultrasound or enzyme cutting.

3. The preparation method according to claim 2, characterized in that: The fragmentation condition 1 is a condition for fragmenting DNA to a main peak of 145 to 180 bp.

4. The preparation method according to claim 2, characterized in that: The fragmentation condition 2 is a condition for fragmenting DNA to a main peak of 180 to 210 bp.

5. The preparation method according to claim 1, characterized in that: The sheet sieve purification condition 1 comprises the following steps: 1) The fragmented DNA1 is mixed and incubated 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 DNA1 solution is 8.5% to 9.5%; 2) Magnetic bead separation, mixing the supernatant with a new magnetic bead suspension for a second incubation, wherein the final concentration of polyethylene glycol in the mixture of the magnetic bead suspension and the supernatant is 12% to 15%; 3) Magnetic bead separation, washing and eluting the magnetic beads to obtain simulated fetal DNA 1.

6. The preparation method according to claim 1, characterized in that: The sheet sieve purification condition 2 comprises the following steps: 1) The fragmented DNA2 is mixed and incubated 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 DNA2 solution is 7% to 9%; 2) Magnetic bead separation, mixing the supernatant with a new magnetic bead suspension for a second incubation, wherein the final concentration of polyethylene glycol in the mixture of the magnetic bead suspension and the supernatant is 10% to 12%; 3) Magnetic bead separation, washing and elution of the magnetic beads to obtain simulated fetal DNA2.

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

8. The method according to claim 1, characterized in that: The main peak of the fragmented DNA 2 is 180 to 210 bp.

9. A simulated maternal plasma DNA standard prepared by the method according to any one of claims 1 to 8.

10. Use of the preparation method according to any one of claims 1 to 8 and the simulated maternal plasma DNA standard according to claim 9 in fetal concentration enrichment.

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