CN111088303A

CN111088303A - Plasma simulation cfDNA (deoxyribonucleic acid) and preparation method thereof as well as construction method of sequencing library

Info

Publication number: CN111088303A
Application number: CN202010017254.XA
Authority: CN
Inventors: 毛瑞芳; 王量; 闫慧慧
Original assignee: Hangzhou Repugene Technology Co ltd
Current assignee: Hangzhou Repugene Technology Co ltd
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2020-05-01

Abstract

The invention relates to a simulated plasma cfDNA, a preparation method and a construction method of a sequencing library. The preparation method of the simulated plasma cfDNA comprises the following steps: providing cultured cells, crushing the cells by using digestive juice, and separating to obtain a sample containing genome DNA; performing enzyme digestion treatment on the sample containing the genomic DNA by using a double-stranded DNAseLancese to obtain the simulated plasma cfDNA. The simulated plasma cfDNA obtained by the method has the similar fragment size, distribution and sequence complexity as the natural plasma cfDNA, and the preparation method is simple, does not need a fragmentation instrument and saves the instrument cost.

Description

Plasma simulation cfDNA (deoxyribonucleic acid) and preparation method thereof as well as construction method of sequencing library

Technical Field

The invention relates to the technical field of biology, in particular to a simulated plasma cfDNA, a preparation method and a construction method of a sequencing library.

Background

According to global cancer statistics, new cancer cases worldwide are 1810 thousands in 2018, new death cases are 960 thousands, and the cancer becomes the first killer of human beings. With the increase of the incidence and mortality of cancer, the development of technological innovation and gene detection level, molecular screening and gene-assisted therapy of cancer are becoming important auxiliary means for research hotspots and precise medication of modern scientists and various scientific research institutions.

Liquid biopsy technology is becoming the main means for detecting cancer molecules in laboratories, and compared with the problems of insufficient samples, sampling heterogeneity and the like often existing in tissue biopsy, the advantages of convenience and non-invasiveness of liquid biopsy of plasma ctDNA and the like are also gaining favor of cancer patients and medical researchers. However, plasma ctDNA liquid biopsy also has some technical challenges, and most importantly, a complete set of experimental system and biological information analysis need to be established, and strict performance verification is performed, so that stable and repeatable detection of mutation information with lower frequency can be ensured. There is also a need for a mock plasma cfDNA that can be stably prepared, stored for a long period of time, has stable performance, and has a high mutation coverage.

Further improvements are needed to obtain stable performance, performance similar to natural cfDNA, of mock plasma cfDNA.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a plasma cfDNA simulator, a preparation method and a sequencing library construction method.

Human plasma cfDNA quality simulation is mainly dependent on the following three properties: firstly, the size and the distribution of DNA, the very important attribute of the plasma DNA is that the average size is 170bp, and the simulated plasma must have the size and the distribution similar to those of the natural plasma; secondly, stability and function, the commonly used method for simulating the plasma cfDNA is to use the genome DNA of a cancer cell line to directly dilute the genome DNA into the plasma DNA of a normal person, or dilute the genome DNA into the plasma of the normal person after ultrasonic disruption, the simulated cfDNA obtained by the method is often wider in DNA distribution range than the natural cfDNA, so that a plurality of DNAs cannot be subjected to subsequent connection amplification and cannot be integrated into a built library, the service life of the natural cfDNA is shorter, because of the proteins and other molecules existing in the plasma, the DNA and the plasma cannot be simply mixed to obtain a stable mixture, the DNA is degraded quickly and cannot be used for long-term and stable experimental performance detection, and the simulated plasma DNA needs to be stably prepared and stored for a long time and has the connection performance, the amplification performance and the like which are close to the natural cfDNA; third, the mutation coverage rate, the number of mutation types and mutation points detected clinically for liquid biopsy is increasing, and laboratories must have the ability and confidence to detect the widest range of mutation sites and mutation types, so that samples containing more mutation sites also need to be constructed for performance verification.

The existing method for preparing the simulated cfDNA product mainly comprises the methods of mechanical breaking, artificial plasmid synthesis, non-specific enzyme cutting breaking and the like. Mechanical disruption is a method for simulating cfDNA which is used at present, and is prepared by mechanical vibration and ultrasonic disruption, and the experimental principle is that a mechanical vibration or sound wave vibration mode is adopted to break complete genome DNA, so that the genome DNA is fragmented, and the method is quite different from the mode of forming the cfDNA in a human body through the action of related enzymes. The method has the defects that the difference between the tail end of a DNA fragment formed by mechanical breaking and a natural cfDNA fragment is large, and the following experiments have the defects of low detection lower limit, high background noise, high false positive rate, inconvenience for automation and the like. Artificially synthesizing a plasmid: the disadvantage is that the sequence complexity is much lower than that of natural cfDNA, and the nucleic acid characteristics of natural cfDNA cannot be simulated. And by adopting non-specific enzyme cutting interruption, the gDNA is processed by using non-specific endonuclease to generate fragmented DNA similar to the cfDNA, the defect that most nucleases have preference to base sites, the DNA fragmentation degree depends on the quality of a sample, and the deviation of the plasma cfDNA obtained by simulation and the natural plasma cfDNA is large.

The inventors of the present invention found through research that: a novel mimic cfDNase enzyme preparation can be used to specifically cleave the phosphodiester bond of double stranded DNA molecules to produce short chain DNA with 5 'phosphate and 3' hydroxyl termini, and without the cleavage preference of AU or AT rich regions. Thereby, the approximately natural plasma cfDNA with high complexity and high sensitivity can be prepared.

Specifically, the invention provides the following technical scheme:

in a first aspect of the invention, the invention provides a method of preparing a mock plasma cfDNA, comprising: providing cultured cells, crushing the cells by using digestive juice, and separating to obtain a sample containing genome DNA; performing enzyme digestion treatment on the sample containing the genomic DNA by using a double-stranded DNAseLancese to obtain the simulated plasma cfDNA.

The enzyme Atlantis dsDNase is a specific double-stranded DNA endonuclease that specifically cleaves the phosphodiester bond of double-stranded DNA molecules to produce short-stranded DNA with 5 'phosphate and 3' hydroxyl termini, and has no cleavage preference for AU or AT rich regions. The method can be used for bringing a large amount of simulated plasma cfDNA which can be prepared in a stable manner and has high performance to medical laboratories and scientific research institutions, reducing the research and development cost of laboratories and shortening the research and development period of experiments. And the preparation method is simple, has low cost and is suitable for automation and industrial production.

According to an embodiment of the present invention, the method for preparing a mock plasma cfDNA described above may further include the following technical features:

in some embodiments of the present invention, the temperature of the enzyme cutting treatment is 37 to 42 degrees celsius, and preferably 42 degrees celsius. The enzyme digestion treatment is carried out at the temperature, so that the enzyme digestion efficiency can be improved, and the yield of the target simulated cfDNA can be increased.

In some embodiments of the invention, every 10⁶The amount of the double-stranded DNase for each cell is 1U or more. Therefore, the enzyme digestion efficiency can be improved, and the yield of the target simulated cfDNA can be increased.

In some embodiments of the present invention, the time of the enzyme digestion treatment is 20 to 60 minutes.

In some embodiments of the invention, the nucleic acid with the size of 80-220 bp in the simulated plasma cfDNA accounts for more than 70%.

In some embodiments of the invention, the cell is a cell line carrying a genetic mutation or a wild-type cell line.

In some embodiments of the invention, further comprising: mock plasma cfDNA obtained by a cell line carrying a gene mutation and mock plasma cfDNA obtained by a cell line carrying a wild type were mixed in a predetermined ratio to obtain a cfDNA standard.

In a second aspect of the invention, the invention provides a mock plasma cfDNA obtained by the method according to any of the examples of the first aspect of the invention.

In some embodiments of the invention, the nucleic acid with the size of 80-220 bp in the simulated plasma cfDNA accounts for more than 70%. Specifically, within 500bp, the nucleic acid of 160-180bp in the natural cfDNA accounts for 80-90%; the proportion of gDNA broken by ultrasonic is about 50 percent; the percentage of the simulated plasma cfDNA obtained by using the double-stranded DNAse of the methylene blue is between 70 and 80 percent.

In a third aspect of the invention, a method of preparing a mock cfDNA, comprising: performing enzyme digestion treatment on the sample containing the genome DNA by using a double-stranded DNAse to obtain the simulated plasma cfDNA;

in some embodiments of the present invention, the temperature of the enzyme cutting treatment is 37-42 ℃.

In a fourth aspect of the invention, the invention provides a method for constructing a sequencing library, comprising: preparing a mock cfDNA according to the method of any embodiment of the first aspect of the invention or according to the method of the third aspect of the invention; building a library based on the mock cfDNA to obtain the sequencing library.

The invention researches and discovers that the double-stranded DNA enzyme of the methylene blue can specifically cut the phosphodiester bond of the double-stranded DNA molecule to generate the short-chain DNA with 5 'phosphate group and 3' hydroxyl end, and can improve the enzyme cutting efficiency and increase the yield of the DNA of the mononucleosomes by properly increasing the enzyme dosage and the incubation time. The method can bring a large amount of prepared and stable-performance simulated plasma cfDNA for medical laboratories and scientific research institutions, and can reduce the research and development cost of the laboratories and shorten the research and development period of experiments.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

fig. 1 is a technical roadmap provided according to an embodiment of the present invention.

Fig. 2 is a graph of a comparison of nucleic acid distribution of natural, digested, and sonicated cfDNA provided in accordance with an embodiment of the present invention.

Fig. 3 is a graph comparing the results of library pre-amplification library concentrations for native, digested and sonicated cfDNA provided in accordance with an embodiment of the present invention.

Fig. 4 is a graph of library complexity comparisons of native, enzymatically cleaved, and sonicated cfDNA provided in accordance with an embodiment of the present invention.

Fig. 5 is a graph of the average sequencing depth comparison results of native, digested and sonicated cfDNA provided in accordance with an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. It should be noted that the described embodiments are exemplary and are intended to be illustrative of the invention, and should not be construed as limiting the invention.

Herein, the term "atlas double-stranded dnase" refers to Atlantis dsDNase, which is a commercially available enzyme.

The invention provides a method for preparing a plasma cfDNA simulator, as shown in figure 1, comprising the following steps: providing cultured cells, crushing the cells by using digestive juice, and separating to obtain a sample containing genome DNA; and (3) carrying out enzyme digestion treatment on the sample containing the genome DNA by using a double-stranded DNAse, and purifying to obtain the simulated plasma cfDNA. The simulated plasma cfDNA obtained by the method has the similar fragment size, distribution and sequence complexity as the natural plasma cfDNA, and the preparation method is simple, does not need an intended instrument and saves the instrument cost. Can be used for preparing enterprise reference materials and quality control materials required by medical institutions and detection institutions in large quantities. In preparing a reference or quality control, a cfDNA standard can be obtained by mixing a mock plasma cfDNA obtained by a cell line carrying a gene mutation and a mock plasma cfDNA obtained by a cell line carrying a wild type in a predetermined ratio.

In the purification, the purification may be performed using magnetic beads.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Example 1 provides a method for preparing mock plasma cfDNA by a streetzia double-stranded dnase, comprising the steps of:

(1) GM24631 cell line (purchased from Coriell organization, and obtained from human cell line subjected to whole genome sequencing) was revived and proliferated in FBS medium, and 1 × 10 cells were taken for each experiment⁶A cell;

(2) washing the cells with phosphate buffer, centrifuging at 200 Xg, and collecting the cells; cleaning is repeated for one time;

(3) discarding the supernatant, resuspending the cells with 1 ml of phosphate buffer, and transferring to a 1.5ml centrifuge tube;

(4) the supernatant was discarded by centrifugation, and 100. mu.l of a nucleic acid preparation (containing 0.25% trypsin as a main component and NaHCO as a main component) was added thereto₃Adjusting the pH to 8.0), blowing or inverting the tube with a pipette to resuspend the cell pellet, and incubating on ice for 5 minutes;

(5) centrifuging to collect cell nucleic acid, and discarding supernatant;

(6) washing cells with 100 μ l of methylene blue digest (mainly containing 20mg/mL proteinase K and 50mM Tris-HCl, pH 8.0), mixing by up-down inversion for 2-3 times, centrifuging, and discarding the supernatant;

(7) repeating the step (6) once;

(8) gently resuspending the cellular nucleic acids with 100 μ l of methylene blue digest;

(9) adding 10 μ l of methylene Lantius double-stranded DNA enzyme (purchased from ZYMO RESEARCH), flicking the tube wall, and mixing;

(10) digesting and incubating for 40 minutes at 42 ℃;

(11) 20. mu.l of a reaction-terminating solution (0.5M EDTANA as a main component) was added thereto₂Adjusting the pH value to 8.0 by NaOH), uniformly mixing by vortex, and terminating the reaction;

(12) adding 600 microliters of DNA binding solution (containing 5M hydrochloride, 20% dipropyl acrylate, and 0.12M vinegar as main ingredients), and mixing by vortex;

(13) transferring to a centrifugal column (installed in a collecting tube), centrifuging for 30 seconds at 10000- & 13000 multiplied by g, and discarding the filtrate;

(14) adding 300 microliters of DNA cleaning solution (the main component is 80% ethylene propylene), centrifuging for 30 seconds at 10000-;

(15) repeating the step (14) once;

(16) centrifuging the empty tube for 1 minute, removing residual cleaning solution, and transferring the centrifugal column to a new 1.5ml centrifuge tube;

(17) adding 30 μ l DNA eluate (main ingredient is 0.01M Tris-HCl, 0.01M EDTA) into the centrifugal column, standing at room temperature for 3 min;

(18) centrifuging for 30 seconds to collect purified DNA (abbreviated as enzyme-cleaved cfDNA in FIGS. 2 to 5); meanwhile, the concentration detection and the fragment distribution detection are carried out on the purified DNA by utilizing the Qubit and the Qsep 400.

In addition, comparisons were made with native cfDNA, as well as ultrasound cfDNA obtained by ultrasound.

The natural cfDNA is obtained by performing fractional differential centrifugation on a real blood sample of a clinical patient to obtain plasma, and then extracting by using a QIAamp Circulating Nucleic Acid Kit.

Ultrasound cfDNA, purchased from Horizon, cat # HD 780.

The results of comparing the nucleic acid distribution of the obtained natural cfDNA, the digested cfDNA, and the ultrasonic cfDNA are shown in fig. 2. From fig. 2, it can be found that the nucleic acid distribution of the digested cfDNA and the natural cfDNA is very similar, when the nucleic acids of the three are around 170, the digested cfDNA and the natural cfDNA are narrow and steep, while the ultrasonic cfDNA is wide and flat.

Example 2

Example 2 the natural cfDNA, the enzyme-cleaved cfDNA, and the ultrasonic cfDNA prepared in example 1 were subjected to library construction and sequencing, including the following steps:

(1) respectively taking 30ng of natural cfDNA, enzyme-digested cfDNA and ultrasonic cfDNA (Horizon standard, HD780) for library construction;

(2) according to Agilent SureSelect XT^HSEstablishing a library by a standard process (the kit is Agilent XT HS, and the product number is G9706A);

(3) the kit is adopted for the detection of the Agencour AMPure XP Reagent with the instruction content of A63882 (purchased from BECKMAN), and AMPure magnetic beads are adopted for purification:

a. adding 80 mu L of AMPure XP magnetic beads (0.8 times of volume) into each tube of sample, and uniformly mixing for 8-10 times by blowing and sucking;

b. standing at room temperature for 5 min;

c. placing the sample tube into a magnetic frame, and standing for 5-10 min; after the liquid is clarified, the supernatant is carefully removed to avoid being absorbed into magnetic beads;

d. adding 200 μ L of 80% ethylene-propylene into the sample tube, standing for 1min, and removing the supernatant;

e. adding 21 mu L of clean-free water into the sample tube, and uniformly mixing by blowing, sucking and blowing;

f. placing the sample tube into a magnetic frame, and standing for 1-2min until the liquid is clear;

g. transfer 21. mu.L of supernatant to a new PCR tube.

(4) Agilent SureSelect XT using Agilent^HSPerforming pre-amplification before hybrid capture;

(5) adopting AMPure XP magnetic beads for purification:

a. adding 50 mu L of AMPure XP magnetic beads (1 time volume) into each tube of sample, and blowing, sucking and uniformly mixing for 8-10 times;

b. standing at room temperature for 5 min;

e. repeating the step d once;

f. adding 15 mu L of nucleic-free water into the sample tube, and uniformly mixing by blowing and sucking;

g. placing the sample tube into a magnetic frame, and standing for 1-2min until the liquid is clear;

h. transfer 15 μ L of supernatant to a new 1.5mL EP tube.

(6) Using the Qubit to detect the DNA concentration and Agilent 4150TapeStation to analyze the nucleic acid fragment;

(7) adopting Agilent SureSelect XT^HSCarrying out subsequent library building on the standard flow of the kit;

(8) and (3) amplifying to obtain a library, and purifying by adopting AMPure XP magnetic beads:

b. standing at room temperature for 5 min;

e. repeating the step d once;

f. adding 25 mu L of nucleic-free water into the sample tube, and uniformly mixing by blowing and sucking;

(9) placing the sample tube into a magnetic frame, and standing for 2min until the liquid is clear;

(10) transfer 25 μ L of supernatant to a new 1.5mL EP tube.

(11) Using the Qubit to detect the DNA concentration, and using Agilent 4150HS D1000 Screen Tape to detect the library quality;

(12) performing on-machine sequencing according to an Illumina Hiseq X standard on-machine process;

(13) and analyzing the sequencing result on the computer by adopting a standard letter generation analysis process.

The experimental results are as follows:

the results of comparing the concentrations of the pre-amplification libraries of the natural cfDNA, the enzyme-cleaved cfDNA and the ultrasonic cfDNA are shown in figure 3, wherein the ordinate in figure 3 represents the concentration of the pre-amplification library, and the concentration of the pre-amplification library reflects the yield of the pre-amplification library, which is a necessary condition for ensuring the smooth performance of the subsequent hybridization capture experiment. From figure 3, it can be found that the yield of the pre-amplified library of the enzyme digestion simulation cfDNA is relatively close to that of the natural cfDNA, and is significantly better than that of the ultrasonic disruption simulation cfDNA. Without being limited by theory, the yield of the enzyme-cleaved cfDNA pre-amplification library is high due to the fact that the ligation efficiency of the short-fragment nucleic acid and the adaptor generated by the enzyme-cleavage method is superior to that of the short-fragment nucleic acid generated by ultrasonic disruption.

The results of comparing the library complexity of native cfDNA, digested cfDNA and sonicated cfDNA are shown in figure 4. The ordinate in fig. 4 represents library complexity, which is an important index for measuring the validity of one sample data, and the higher the library complexity, the higher the validity of the sequencing data is, so that the success rate of the experiment can be effectively improved. From figure 4, it can be found that enzyme digestion simulation cfDNA is very close to natural cfDNA and is significantly better than ultrasonic simulation cfDNA.

The results of the average sequencing depth comparisons of native cfDNA, digested cfDNA and sonicated cfDNA are shown in figure 5. The ordinate in fig. 5 represents the average sequencing depth, and the high average sequencing depth can improve the effectiveness of sequencing data, thereby improving the success rate of the experiment and reducing the experiment cost by reducing the data volume. From fig. 5, it can be found that the average sequencing depth of the enzyme-cleaved cfDNA is between that of the natural cfDNA and that of the ultrasonic cfDNA, and is significantly better than that of the ultrasonic cfDNA.

The above examples and experimental data show that the enzyme digestion simulated cfDNA is very similar to the natural cfDNA, and the nucleic acid distribution and sequencing performance are both significantly better than those of ultrasonic disruption.

Example 3

Example 3 based on example 1, the influence of the change in reaction conditions on the mock plasma cfDNA obtained by the enzymatic cleavage with asiatllis double-stranded dnase was examined. Specific reaction conditions are shown in table 1 below.

TABLE 1 different reaction conditions

	Experimental group A	Experimental group B	Experimental group C	Experimental group D
					Number of cells	1.5×10⁶	1.5×10⁶	1.0×10⁶	1.0×10⁶
Enzyme dosage	10μL	10μL	5μL	10μL
					Incubation time
	50 minutes	60 minutes	50 minutes	60 minutes
					Average fragment size	597.4	280.5	554.3	306.2
Peak value of enzyme digestion fragment	160-180	150-170	160-180	150-170

In the experimental process, the number of GM24631 cells is 1.5X 10⁶And moreover, the reaction conditions are optimized. The experimental results show that: the dosage of the enzyme is 10 mu L, when the incubation time is set to be 50 minutes, the enzyme contains more large fragments, and the average molecular fragment size of repeated experiments is 597.4; when the dosage of the enzyme of the Sperlaties is 10 mu L and the incubation time is set to be 60 minutes, the large fragment is less, the average molecular fragment size of the.

Similarly, the number of GM24631 cells was 1.0X 10⁶And moreover, the reaction conditions are optimized. The experimental results show that: when the dosage of the enzyme is 5 mu L and the incubation time is set to be 50 minutes, the enzyme contains more large fragments, and the average molecular fragment size of repeated experiments is 554.3; when the dosage of the enzyme of the Sperlaties is 10 mu L and the incubation time is set to be 60 minutes, the large fragment is less, the average molecular fragment size of the.

The experimental results show that different cell numbers and different dosages of the enzyme, which is the enzyme, have important influence on the results. When the number of cells is 1.0X 10⁶1.5 x 10⁶In one time, the dosage of the enzyme of the Sarlanstase is 10 mu L, the incubation time is 60 minutes, and the effect is better.

Meanwhile, experiments were carried out with reference to examples 1 and 2 for other cells, for example, dozens of cell lines such as NCI-H1650, NCI-1975, H2228, H1299, and the like. The result shows that the simulated plasma cfDNA with excellent performances in all aspects can be obtained by adopting the enzyme digestion treatment of the enzyme.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A method of preparing a mock plasma cfDNA, comprising:

providing cultured cells, crushing the cells by using digestive juice, and separating to obtain a sample containing genome DNA;

performing enzyme digestion treatment on the sample containing the genomic DNA by using a double-stranded DNAseLancese to obtain the simulated plasma cfDNA.

2. The method according to claim 1, wherein the temperature of the enzyme cutting treatment is 37-42 ℃, preferably 42 ℃.

3. The method of claim 1, wherein every 10 is⁶The dosage of the double-stranded DNA enzyme of the Portland needs to be more than 1U for each cell;

optionally, the enzyme digestion treatment time is 20-60 minutes.

4. The method according to claim 1, wherein the ratio of nucleic acids with a size of 80-220 bp in the mock plasma cfDNA is more than 70%.

5. The method of claim 1, wherein the cell is a cell line carrying a genetic mutation or a wild-type cell line.

6. The method of claim 5, further comprising:

mock plasma cfDNA obtained by a cell line carrying a gene mutation and mock plasma cfDNA obtained by a cell line carrying a wild type were mixed in a predetermined ratio to obtain a cfDNA standard.

7. A mock plasma cfDNA obtained by the method of any one of claims 1 to 6.

8. The mock plasma cfDNA according to claim 7, wherein the nucleic acid with a size of 80-220 bp in the mock plasma cfDNA is present in an amount of more than 70%.

9. A method of preparing a mock cfDNA, comprising:

performing enzyme digestion treatment on the sample containing the genome DNA by using a double-stranded DNAse to obtain the simulated plasma cfDNA;

optionally, the temperature of the enzyme digestion treatment is 37-42 ℃;

the enzyme digestion treatment time is 20-60 minutes.

10. A method of constructing a sequencing library, comprising:

preparing a mock cfDNA according to the method of any one of claims 1-6 or according to the method of claim 9;

based on the mock cfDNA, a library was created in order to obtain the sequencing library.